JP7186470B2 - Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof - Google Patents
Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof Download PDFInfo
- Publication number
- JP7186470B2 JP7186470B2 JP2021532852A JP2021532852A JP7186470B2 JP 7186470 B2 JP7186470 B2 JP 7186470B2 JP 2021532852 A JP2021532852 A JP 2021532852A JP 2021532852 A JP2021532852 A JP 2021532852A JP 7186470 B2 JP7186470 B2 JP 7186470B2
- Authority
- JP
- Japan
- Prior art keywords
- vesicles
- derived
- present
- bacteria
- corynebacterium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 241000894006 Bacteria Species 0.000 title description 75
- 241000186216 Corynebacterium Species 0.000 title description 60
- 241000186226 Corynebacterium glutamicum Species 0.000 claims description 43
- 239000000203 mixture Substances 0.000 claims description 42
- 208000027866 inflammatory disease Diseases 0.000 claims description 30
- 238000011282 treatment Methods 0.000 claims description 21
- 239000004480 active ingredient Substances 0.000 claims description 16
- 235000013305 food Nutrition 0.000 claims description 12
- 230000002265 prevention Effects 0.000 claims description 11
- 239000008194 pharmaceutical composition Substances 0.000 claims description 10
- 239000002537 cosmetic Substances 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 210000004369 blood Anatomy 0.000 description 57
- 239000008280 blood Substances 0.000 description 57
- 230000001580 bacterial effect Effects 0.000 description 36
- 238000004458 analytical method Methods 0.000 description 35
- 241000282412 Homo Species 0.000 description 29
- 238000000034 method Methods 0.000 description 26
- 201000010099 disease Diseases 0.000 description 22
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 22
- 238000009826 distribution Methods 0.000 description 21
- 206010012601 diabetes mellitus Diseases 0.000 description 20
- 108090000623 proteins and genes Proteins 0.000 description 20
- 206010006187 Breast cancer Diseases 0.000 description 18
- 208000026310 Breast neoplasm Diseases 0.000 description 18
- 208000000592 Nasal Polyps Diseases 0.000 description 18
- 208000006011 Stroke Diseases 0.000 description 18
- 208000006673 asthma Diseases 0.000 description 18
- 208000019425 cirrhosis of liver Diseases 0.000 description 18
- 206010012289 Dementia Diseases 0.000 description 17
- 206010012438 Dermatitis atopic Diseases 0.000 description 17
- 201000008937 atopic dermatitis Diseases 0.000 description 17
- 230000028327 secretion Effects 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 12
- 241000588724 Escherichia coli Species 0.000 description 12
- 230000002757 inflammatory effect Effects 0.000 description 12
- 238000003752 polymerase chain reaction Methods 0.000 description 12
- 206010061218 Inflammation Diseases 0.000 description 11
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 11
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 11
- 230000004054 inflammatory process Effects 0.000 description 11
- 239000006210 lotion Substances 0.000 description 11
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 10
- 239000012528 membrane Substances 0.000 description 10
- 230000001681 protective effect Effects 0.000 description 10
- 210000004379 membrane Anatomy 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 8
- 230000001717 pathogenic effect Effects 0.000 description 8
- 210000004400 mucous membrane Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 108020004465 16S ribosomal RNA Proteins 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 230000000172 allergic effect Effects 0.000 description 6
- 208000010668 atopic eczema Diseases 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000008595 infiltration Effects 0.000 description 6
- 238000001764 infiltration Methods 0.000 description 6
- 239000004615 ingredient Substances 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 210000004185 liver Anatomy 0.000 description 6
- 210000002540 macrophage Anatomy 0.000 description 6
- 210000002850 nasal mucosa Anatomy 0.000 description 6
- 241000894007 species Species 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 239000006071 cream Substances 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 229940079593 drug Drugs 0.000 description 5
- 239000000796 flavoring agent Substances 0.000 description 5
- 230000036541 health Effects 0.000 description 5
- 210000004969 inflammatory cell Anatomy 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 210000004877 mucosa Anatomy 0.000 description 5
- -1 pH adjusters Substances 0.000 description 5
- 239000002953 phosphate buffered saline Substances 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000002965 ELISA Methods 0.000 description 4
- 206010016654 Fibrosis Diseases 0.000 description 4
- 241000699670 Mus sp. Species 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 230000003110 anti-inflammatory effect Effects 0.000 description 4
- 230000007882 cirrhosis Effects 0.000 description 4
- 230000000994 depressogenic effect Effects 0.000 description 4
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 4
- 238000003745 diagnosis Methods 0.000 description 4
- 210000002919 epithelial cell Anatomy 0.000 description 4
- 235000013355 food flavoring agent Nutrition 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 235000016709 nutrition Nutrition 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000699666 Mus <mouse, genus> Species 0.000 description 3
- 208000008589 Obesity Diseases 0.000 description 3
- 230000030429 T-helper 17 type immune response Effects 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 201000011510 cancer Diseases 0.000 description 3
- 208000037976 chronic inflammation Diseases 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000002552 dosage form Substances 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 239000000686 essence Substances 0.000 description 3
- 230000029142 excretion Effects 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 208000016366 nasal cavity polyp Diseases 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 235000020824 obesity Nutrition 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 150000003839 salts Chemical class 0.000 description 3
- 230000008685 targeting Effects 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000001225 therapeutic effect Effects 0.000 description 3
- MEZZCSHVIGVWFI-UHFFFAOYSA-N 2,2'-Dihydroxy-4-methoxybenzophenone Chemical compound OC1=CC(OC)=CC=C1C(=O)C1=CC=CC=C1O MEZZCSHVIGVWFI-UHFFFAOYSA-N 0.000 description 2
- ALYNCZNDIQEVRV-UHFFFAOYSA-N 4-aminobenzoic acid Chemical compound NC1=CC=C(C(O)=O)C=C1 ALYNCZNDIQEVRV-UHFFFAOYSA-N 0.000 description 2
- 241000203069 Archaea Species 0.000 description 2
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 102000004127 Cytokines Human genes 0.000 description 2
- 108090000695 Cytokines Proteins 0.000 description 2
- 241000206602 Eukaryota Species 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 2
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- AFYNADDZULBEJA-UHFFFAOYSA-N bicinchoninic acid Chemical compound C1=CC=CC2=NC(C=3C=C(C4=CC=CC=C4N=3)C(=O)O)=CC(C(O)=O)=C21 AFYNADDZULBEJA-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000030833 cell death Effects 0.000 description 2
- 230000022534 cell killing Effects 0.000 description 2
- 208000037893 chronic inflammatory disorder Diseases 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- RXKJFZQQPQGTFL-UHFFFAOYSA-N dihydroxyacetone Chemical compound OCC(=O)CO RXKJFZQQPQGTFL-UHFFFAOYSA-N 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000005090 green fluorescent protein Substances 0.000 description 2
- 210000002216 heart Anatomy 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 238000000703 high-speed centrifugation Methods 0.000 description 2
- 210000005260 human cell Anatomy 0.000 description 2
- 230000036737 immune function Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- DXGLGDHPHMLXJC-UHFFFAOYSA-N oxybenzone Chemical compound OC1=CC(OC)=CC=C1C(=O)C1=CC=CC=C1 DXGLGDHPHMLXJC-UHFFFAOYSA-N 0.000 description 2
- 229960001173 oxybenzone Drugs 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000003449 preventive effect Effects 0.000 description 2
- HELXLJCILKEWJH-NCGAPWICSA-N rebaudioside A Chemical compound O([C@H]1[C@H](O)[C@@H](CO)O[C@H]([C@@H]1O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)O[C@]12C(=C)C[C@@]3(C1)CC[C@@H]1[C@@](C)(CCC[C@]1([C@@H]3CC2)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O HELXLJCILKEWJH-NCGAPWICSA-N 0.000 description 2
- 210000003296 saliva Anatomy 0.000 description 2
- 239000000344 soap Substances 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 210000002700 urine Anatomy 0.000 description 2
- 239000011782 vitamin Substances 0.000 description 2
- 235000013343 vitamin Nutrition 0.000 description 2
- 229940088594 vitamin Drugs 0.000 description 2
- 229930003231 vitamin Natural products 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- HEOCBCNFKCOKBX-RELGSGGGSA-N (1s,2e,4r)-4,7,7-trimethyl-2-[(4-methylphenyl)methylidene]bicyclo[2.2.1]heptan-3-one Chemical compound C1=CC(C)=CC=C1\C=C/1C(=O)[C@]2(C)CC[C@H]\1C2(C)C HEOCBCNFKCOKBX-RELGSGGGSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- WHQOKFZWSDOTQP-UHFFFAOYSA-N 2,3-dihydroxypropyl 4-aminobenzoate Chemical compound NC1=CC=C(C(=O)OCC(O)CO)C=C1 WHQOKFZWSDOTQP-UHFFFAOYSA-N 0.000 description 1
- TYYHDKOVFSVWON-UHFFFAOYSA-N 2-butyl-2-methoxy-1,3-diphenylpropane-1,3-dione Chemical compound C=1C=CC=CC=1C(=O)C(OC)(CCCC)C(=O)C1=CC=CC=C1 TYYHDKOVFSVWON-UHFFFAOYSA-N 0.000 description 1
- JGUMTYWKIBJSTN-UHFFFAOYSA-N 2-ethylhexyl 4-[[4,6-bis[4-(2-ethylhexoxycarbonyl)anilino]-1,3,5-triazin-2-yl]amino]benzoate Chemical compound C1=CC(C(=O)OCC(CC)CCCC)=CC=C1NC1=NC(NC=2C=CC(=CC=2)C(=O)OCC(CC)CCCC)=NC(NC=2C=CC(=CC=2)C(=O)OCC(CC)CCCC)=N1 JGUMTYWKIBJSTN-UHFFFAOYSA-N 0.000 description 1
- WVCHIGAIXREVNS-UHFFFAOYSA-N 2-hydroxy-1,4-naphthoquinone Chemical compound C1=CC=C2C(O)=CC(=O)C(=O)C2=C1 WVCHIGAIXREVNS-UHFFFAOYSA-N 0.000 description 1
- BUOYTFVLNZIELF-UHFFFAOYSA-N 2-phenyl-1h-indole-4,6-dicarboximidamide Chemical compound N1C2=CC(C(=N)N)=CC(C(N)=N)=C2C=C1C1=CC=CC=C1 BUOYTFVLNZIELF-UHFFFAOYSA-N 0.000 description 1
- UAIUNKRWKOVEES-UHFFFAOYSA-N 3,3',5,5'-tetramethylbenzidine Chemical compound CC1=C(N)C(C)=CC(C=2C=C(C)C(N)=C(C)C=2)=C1 UAIUNKRWKOVEES-UHFFFAOYSA-N 0.000 description 1
- HMKKFLSUPRUBOO-IUPFWZBJSA-N 3,4-dihydroxy-5-[3,4,5-tris[[(z)-octadec-9-enoyl]oxy]benzoyl]oxybenzoic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC1=C(OC(=O)CCCCCCC\C=C/CCCCCCCC)C(OC(=O)CCCCCCC\C=C/CCCCCCCC)=CC(C(=O)OC=2C(=C(O)C=C(C=2)C(O)=O)O)=C1 HMKKFLSUPRUBOO-IUPFWZBJSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108010011485 Aspartame Proteins 0.000 description 1
- 206010003645 Atopy Diseases 0.000 description 1
- 208000017667 Chronic Disease Diseases 0.000 description 1
- 208000035473 Communicable disease Diseases 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- 239000004386 Erythritol Substances 0.000 description 1
- UNXHWFMMPAWVPI-UHFFFAOYSA-N Erythritol Natural products OCC(O)C(O)CO UNXHWFMMPAWVPI-UHFFFAOYSA-N 0.000 description 1
- FMRHJJZUHUTGKE-UHFFFAOYSA-N Ethylhexyl salicylate Chemical compound CCCCC(CC)COC(=O)C1=CC=CC=C1O FMRHJJZUHUTGKE-UHFFFAOYSA-N 0.000 description 1
- 239000001512 FEMA 4601 Substances 0.000 description 1
- 229930091371 Fructose Natural products 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 239000004378 Glycyrrhizin Substances 0.000 description 1
- 239000006391 Luria-Bertani Medium Substances 0.000 description 1
- 239000005913 Maltodextrin Substances 0.000 description 1
- 229920002774 Maltodextrin Polymers 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 108700005443 Microbial Genes Proteins 0.000 description 1
- 241000736262 Microbiota Species 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- YBGZDTIWKVFICR-JLHYYAGUSA-N Octyl 4-methoxycinnamic acid Chemical compound CCCCC(CC)COC(=O)\C=C\C1=CC=C(OC)C=C1 YBGZDTIWKVFICR-JLHYYAGUSA-N 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- WYWZRNAHINYAEF-UHFFFAOYSA-N Padimate O Chemical compound CCCCC(CC)COC(=O)C1=CC=C(N(C)C)C=C1 WYWZRNAHINYAEF-UHFFFAOYSA-N 0.000 description 1
- 229920002230 Pectic acid Polymers 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- HELXLJCILKEWJH-SEAGSNCFSA-N Rebaudioside A Natural products O=C(O[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@@]1(C)[C@@H]2[C@](C)([C@H]3[C@@]4(CC(=C)[C@@](O[C@H]5[C@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@@H](O[C@H]6[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O6)[C@H](O)[C@@H](CO)O5)(C4)CC3)CC2)CCC1 HELXLJCILKEWJH-SEAGSNCFSA-N 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 244000228451 Stevia rebaudiana Species 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 210000000068 Th17 cell Anatomy 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- UBNYRXMKIIGMKK-RMKNXTFCSA-N amiloxate Chemical compound COC1=CC=C(\C=C\C(=O)OCCC(C)C)C=C1 UBNYRXMKIIGMKK-RMKNXTFCSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229960004050 aminobenzoic acid Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229960005193 avobenzone Drugs 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- XVAMCHGMPYWHNL-UHFFFAOYSA-N bemotrizinol Chemical compound OC1=CC(OCC(CC)CCCC)=CC=C1C1=NC(C=2C=CC(OC)=CC=2)=NC(C=2C(=CC(OCC(CC)CCCC)=CC=2)O)=N1 XVAMCHGMPYWHNL-UHFFFAOYSA-N 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 description 1
- 235000013361 beverage Nutrition 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- YEAYGXLRPMKZBP-KQGICBIGSA-N bis(2-hydroxyethyl)azanium;(e)-3-(4-methoxyphenyl)prop-2-enoate Chemical compound OCCNCCO.COC1=CC=C(\C=C\C(O)=O)C=C1 YEAYGXLRPMKZBP-KQGICBIGSA-N 0.000 description 1
- FQUNFJULCYSSOP-UHFFFAOYSA-N bisoctrizole Chemical compound N1=C2C=CC=CC2=NN1C1=CC(C(C)(C)CC(C)(C)C)=CC(CC=2C(=C(C=C(C=2)C(C)(C)CC(C)(C)C)N2N=C3C=CC=CC3=N2)O)=C1O FQUNFJULCYSSOP-UHFFFAOYSA-N 0.000 description 1
- 239000002981 blocking agent Substances 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007975 buffered saline Substances 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 235000014171 carbonated beverage Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 235000013351 cheese Nutrition 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000019219 chocolate Nutrition 0.000 description 1
- 230000006020 chronic inflammation Effects 0.000 description 1
- CMDKPGRTAQVGFQ-RMKNXTFCSA-N cinoxate Chemical compound CCOCCOC(=O)\C=C\C1=CC=C(OC)C=C1 CMDKPGRTAQVGFQ-RMKNXTFCSA-N 0.000 description 1
- 229960001063 cinoxate Drugs 0.000 description 1
- 230000019771 cognition Effects 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003599 detergent Substances 0.000 description 1
- 238000002405 diagnostic procedure Methods 0.000 description 1
- FDATWRLUYRHCJE-UHFFFAOYSA-N diethylamino hydroxybenzoyl hexyl benzoate Chemical compound CCCCCCOC(=O)C1=CC=CC=C1C(=O)C1=CC=C(N(CC)CC)C=C1O FDATWRLUYRHCJE-UHFFFAOYSA-N 0.000 description 1
- 229960001630 diethylamino hydroxybenzoyl hexyl benzoate Drugs 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 229940120503 dihydroxyacetone Drugs 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 229960004960 dioxybenzone Drugs 0.000 description 1
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- 239000002270 dispersing agent Substances 0.000 description 1
- 235000006694 eating habits Nutrition 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- HELXLJCILKEWJH-UHFFFAOYSA-N entered according to Sigma 01432 Natural products C1CC2C3(C)CCCC(C)(C(=O)OC4C(C(O)C(O)C(CO)O4)O)C3CCC2(C2)CC(=C)C21OC(C1OC2C(C(O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OC(CO)C(O)C(O)C1O HELXLJCILKEWJH-UHFFFAOYSA-N 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 229960004697 enzacamene Drugs 0.000 description 1
- 210000004920 epithelial cell of skin Anatomy 0.000 description 1
- 235000019414 erythritol Nutrition 0.000 description 1
- UNXHWFMMPAWVPI-ZXZARUISSA-N erythritol Chemical compound OC[C@H](O)[C@H](O)CO UNXHWFMMPAWVPI-ZXZARUISSA-N 0.000 description 1
- 229940009714 erythritol Drugs 0.000 description 1
- CBZHHQOZZQEZNJ-UHFFFAOYSA-N ethyl 4-[bis(2-hydroxypropyl)amino]benzoate Chemical compound CCOC(=O)C1=CC=C(N(CC(C)O)CC(C)O)C=C1 CBZHHQOZZQEZNJ-UHFFFAOYSA-N 0.000 description 1
- 229940068171 ethyl hexyl salicylate Drugs 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 230000001815 facial effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000012041 food component Nutrition 0.000 description 1
- 239000005417 food ingredient Substances 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 238000001997 free-flow electrophoresis Methods 0.000 description 1
- 235000013376 functional food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000001641 gel filtration chromatography Methods 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- LPLVUJXQOOQHMX-UHFFFAOYSA-N glycyrrhetinic acid glycoside Natural products C1CC(C2C(C3(CCC4(C)CCC(C)(CC4C3=CC2=O)C(O)=O)C)(C)CC2)(C)C2C(C)(C)C1OC1OC(C(O)=O)C(O)C(O)C1OC1OC(C(O)=O)C(O)C(O)C1O LPLVUJXQOOQHMX-UHFFFAOYSA-N 0.000 description 1
- 229960004949 glycyrrhizic acid Drugs 0.000 description 1
- UYRUBYNTXSDKQT-UHFFFAOYSA-N glycyrrhizic acid Natural products CC1(C)C(CCC2(C)C1CCC3(C)C2C(=O)C=C4C5CC(C)(CCC5(C)CCC34C)C(=O)O)OC6OC(C(O)C(O)C6OC7OC(O)C(O)C(O)C7C(=O)O)C(=O)O UYRUBYNTXSDKQT-UHFFFAOYSA-N 0.000 description 1
- 235000019410 glycyrrhizin Nutrition 0.000 description 1
- LPLVUJXQOOQHMX-QWBHMCJMSA-N glycyrrhizinic acid Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@@H]1O[C@@H]1C([C@H]2[C@]([C@@H]3[C@@]([C@@]4(CC[C@@]5(C)CC[C@@](C)(C[C@H]5C4=CC3=O)C(O)=O)C)(C)CC2)(C)CC1)(C)C)C(O)=O)[C@@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O LPLVUJXQOOQHMX-QWBHMCJMSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 244000005709 gut microbiome Species 0.000 description 1
- 239000008269 hand cream Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- CSFWPUWCSPOLJW-UHFFFAOYSA-N hydroxynaphthoquinone Natural products C1=CC=C2C(=O)C(O)=CC(=O)C2=C1 CSFWPUWCSPOLJW-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000003905 indoor air pollution Methods 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 201000007270 liver cancer Diseases 0.000 description 1
- 208000014018 liver neoplasm Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 229940035034 maltodextrin Drugs 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- SOXAGEOHPCXXIO-DVOMOZLQSA-N menthyl anthranilate Chemical compound CC(C)[C@@H]1CC[C@@H](C)C[C@H]1OC(=O)C1=CC=CC=C1N SOXAGEOHPCXXIO-DVOMOZLQSA-N 0.000 description 1
- 229960002248 meradimate Drugs 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 238000001471 micro-filtration Methods 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000007758 minimum essential medium Substances 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229960001679 octinoxate Drugs 0.000 description 1
- FMJSMJQBSVNSBF-UHFFFAOYSA-N octocrylene Chemical group C=1C=CC=CC=1C(=C(C#N)C(=O)OCC(CC)CCCC)C1=CC=CC=C1 FMJSMJQBSVNSBF-UHFFFAOYSA-N 0.000 description 1
- 229960000601 octocrylene Drugs 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- LCLHHZYHLXDRQG-ZNKJPWOQSA-N pectic acid Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)O[C@H](C(O)=O)[C@@H]1OC1[C@H](O)[C@@H](O)[C@@H](OC2[C@@H]([C@@H](O)[C@@H](O)[C@H](O2)C(O)=O)O)[C@@H](C(O)=O)O1 LCLHHZYHLXDRQG-ZNKJPWOQSA-N 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 239000010318 polygalacturonic acid Substances 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 235000019203 rebaudioside A Nutrition 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 210000002345 respiratory system Anatomy 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 239000012679 serum free medium Substances 0.000 description 1
- 239000002453 shampoo Substances 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000000527 sonication Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- CXVGEDCSTKKODG-UHFFFAOYSA-N sulisobenzone Chemical compound C1=C(S(O)(=O)=O)C(OC)=CC(O)=C1C(=O)C1=CC=CC=C1 CXVGEDCSTKKODG-UHFFFAOYSA-N 0.000 description 1
- 229960000368 sulisobenzone Drugs 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000031068 symbiosis, encompassing mutualism through parasitism Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 239000000892 thaumatin Substances 0.000 description 1
- 235000010436 thaumatin Nutrition 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- ZQTYRTSKQFQYPQ-UHFFFAOYSA-N trisiloxane Chemical compound [SiH3]O[SiH2]O[SiH3] ZQTYRTSKQFQYPQ-UHFFFAOYSA-N 0.000 description 1
- UEVAMYPIMMOEFW-UHFFFAOYSA-N trolamine salicylate Chemical compound OCCN(CCO)CCO.OC(=O)C1=CC=CC=C1O UEVAMYPIMMOEFW-UHFFFAOYSA-N 0.000 description 1
- 230000002485 urinary effect Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Epidemiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Birds (AREA)
- Food Science & Technology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Dermatology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Nutrition Science (AREA)
- Immunology (AREA)
- Polymers & Plastics (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Cosmetics (AREA)
Description
本発明は、コリネバクテリウム属細菌由来のナノ小胞及びその用途に関し、より具体的には、コリネバクテリウム属細菌に由来するナノ小胞を用いた肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープの診断方法、及び前記小胞を含む前記疾患に対する予防、改善又は治療用組成物などに関する。 TECHNICAL FIELD The present invention relates to nanovesicles derived from Corynebacterium and uses thereof, and more specifically, liver cirrhosis, stroke, diabetes, asthma, and atopic skin using nanovesicles derived from Corynebacterium. The present invention relates to methods for diagnosing inflammation, depression, breast cancer, dementia and nasal polyps, and preventive, ameliorating or therapeutic compositions containing the vesicles.
本出願は、２０１８年１２月１０日に出願された大韓民国特許出願第１０－２０１８－０１５８６２２号及び２０１９年１０月２３日に出願された大韓民国特許出願第１０－２０１９－０１３２１３７号に基づく優先権の利益を主張し、当該出願の明細書及び図面に開示されたすべての内容は本出願に援用される。 This application is based on Korean Patent Application No. 10-2018-0158622 filed on December 10, 2018 and Korean Patent Application No. 10-2019-0132137 filed on October 23, 2019. All content disclosed in the specification and drawings of that application to which benefit is claimed is hereby incorporated by reference into this application.
２１世紀に入ってから、過去には伝染病と認識されていた急性感染性疾患の重要性が減る一方、ヒトとマイクロバイオーム（Ｍｉｃｒｏｂｉｏｍｅ）との不調和により発生する免疫機能の異常を伴った慢性炎症疾患が主要疾患になり疾病パターンが変わった。特に、食習慣の西欧化による肥満、糖尿病、心血管疾患、神経精神疾患及び癌などの慢性炎症疾患と家屋構造の変化による室内大気汚染と室内での生活時間の増加によって皮膚及び呼吸器炎症疾患が国民保健において大きな問題となっている。 Since the beginning of the 21st century, while the importance of acute infectious diseases, which were recognized as epidemics in the past, has decreased, chronic diseases associated with immune function abnormalities caused by the disharmony between humans and the microbiome have become common. Inflammatory diseases have become major diseases and disease patterns have changed. In particular, chronic inflammatory diseases such as obesity, diabetes, cardiovascular disease, neuropsychiatric disease, and cancer due to westernization of eating habits, and skin and respiratory inflammatory diseases due to indoor air pollution and increased indoor living time due to changes in house structures. is a major problem in national health.
前記炎症疾患の発生には、外部の原因因子に対する免疫機能に異常を伴っている。細菌に由来する原因因子に対する免疫反応は、インターロイキン（Ｉｎｔｅｒｌｅｕｋｉｎ、以下、ＩＬ）－１７サイトカインを分泌するＴｈ１７免疫反応が重要であり、細菌性原因因子に露出するとき、Ｔｈ１７免疫反応による好中球性炎症が発生する。また、炎症が発生する過程で細菌性原因因子により分泌される腫瘍壊死因子－α（Ｔｕｍｏｒ Ｎｅｃｒｏｓｉｓ Ｆａｃｔｏｒ－ａｌｐｈａ、以下、ＴＮＦ－α）のような炎症性メディエーターが炎症及び癌発生において重要な役割を担う。炎症性メディエーターのうち細菌性原因因子により分泌されるＩＬ－６は、Ｔｈ１７細胞への分化に重要な役割を担い、Ｔｈ１７免疫反応による慢性炎症は、慢性炎症疾患だけでなく癌発生とも密接な関連があると最近報告されている。 The development of said inflammatory diseases is accompanied by abnormal immune function against external causative factors. Th17 immune response that secretes interleukin (IL)-17 cytokine is important for immune response to causative factors derived from bacteria, and when exposed to bacterial causative factors, neutrophils by Th17 immune response inflammation occurs. In addition, inflammatory mediators such as Tumor Necrosis Factor-alpha (hereinafter referred to as TNF-α) secreted by bacterial causative factors during the process of inflammation play an important role in inflammation and cancer development. bear. Among inflammatory mediators, IL-6, which is secreted by bacterial causative factors, plays an important role in differentiation into Th17 cells, and chronic inflammation caused by Th17 immune response is closely related not only to chronic inflammatory diseases but also to cancer development. It has recently been reported that there are
人体に共生する微生物の個数は、１００兆個に達し、ヒト細胞より約１０倍多く、微生物の遺伝子数は、ヒト遺伝子数の１００倍を超えることが知られている。微生物叢（ｍｉｃｒｏｂｉｏｔａあるいはｍｉｃｒｏｂｉｏｍｅ）は、与えられた居住地に存在する真正細菌、古細菌、真核生物を含む微生物群集（ｍｉｃｒｏｂｉａｌ ｃｏｍｍｕｎｉｔｙ）を言い、腸内微生物叢は、ヒトの生理現象に重要な役割をし、人体細胞と相互作用を通じてヒトの健康と疾病に大きい影響を及ぼすものと知られている。 It is known that the number of symbiotic microorganisms in the human body has reached 100 trillion, which is about ten times more than human cells, and the number of microbial genes is more than 100 times that of human genes. Microbiota or microbiome refers to a microbial community that includes eubacteria, archaea, and eukaryotes present in a given habitat, and the intestinal microbiota is important to human physiology. It is known to have a great influence on human health and disease through interactions with human cells.
人体に共生する真正細菌及び古細菌は、他の細胞への遺伝子、タンパク質などの情報を交換するためにナノメートルサイズの小胞（ｖｅｓｉｃｌｅ）を分泌する。粘膜は、２００ナノメートル（ｎｍ）サイズ以上の粒子は通過できない物理的な防御膜を形成して、粘膜に共生する細菌の場合には、粘膜を通過できないが、細菌由来の小胞は、サイズが１００ナノメートルサイズ以下であるので、比較的自由に粘膜を通じて上皮細胞を通過して人体に吸収される。人体に吸収される病原性細菌由来の小胞は、最近、糖尿病、肥満など代謝疾患の病因において重要な役割を担うことが明らかにされた。 Eubacteria and archaea, which live symbiotically in the human body, secrete nanometer-sized vesicles to exchange information such as genes and proteins with other cells. Mucous membranes form a physical barrier that prevents passage of particles with a size of 200 nanometers (nm) or larger. is less than 100 nanometers in size, it is relatively freely absorbed by the human body through epithelial cells through mucous membranes. Vesicles derived from pathogenic bacteria that are absorbed by the human body have recently been shown to play an important role in the pathogenesis of metabolic diseases such as diabetes and obesity.
コリネバクテリウム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ）属細菌は、好気性グラム陽性細菌であって、自然界に広がって真核生物と共生して生きて行く細菌として知られている。特に、コリネバクテリウム・グルタミクム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ）菌は、産業的にアミノ酸、核酸などの生産に広く用いられている菌である。しかし、現在までコリネバクテリウム属細菌の細胞外小胞を用いた治療技術については報告されたことがない。 Bacteria belonging to the genus Corynebacterium are aerobic Gram-positive bacteria, and are known as bacteria that spread in nature and live in symbiosis with eukaryotes. In particular, Corynebacterium glutamicum is a bacterium that is widely used industrially for the production of amino acids, nucleic acids and the like. However, until now, there have been no reports on therapeutic techniques using extracellular vesicles of bacteria belonging to the genus Corynebacterium.
そこで、本発明では、コリネバクテリウム属細菌から小胞を最初に分離し、その特性を確認することで、前記小胞を多様な炎症疾患の診断及び炎症疾患の予防、改善又は治療用組成物として用いられることを確認した。 Therefore, in the present invention, vesicles are first isolated from bacteria belonging to the genus Corynebacterium, and their properties are confirmed. Confirmed to be used as
本発明者らは、上記のような従来の問題点を解決するために鋭意研究した結果、メタゲノム分析を通じて正常ヒトに比べて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者由来のサンプルからコリネバクテリウム属細菌由来の小胞の含量が有意に減少していることを確認した。また、コリネバクテリウム属細菌由来の小胞が病原性小胞による炎症反応を効率的に抑制することを確認し、これに基づいて本発明を完成した。 As a result of intensive research to solve the above-mentioned conventional problems, the present inventors found that, through metagenomic analysis, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, and cognition compared to normal humans. It was confirmed that the content of vesicles derived from Corynebacterium spp. They also confirmed that vesicles derived from bacteria belonging to the genus Corynebacterium efficiently suppress inflammatory reactions caused by pathogenic vesicles, and completed the present invention based on this finding.
そこで、本発明は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法又は診断のための情報提供方法を提供することを目的とする。 Therefore, the present invention provides diagnostic methods or diagnostic information for one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. The purpose is to provide a method.
また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供することを他の目的とする。 Another object of the present invention is to provide a composition for preventing, improving or treating inflammatory diseases, which contains vesicles derived from bacteria belonging to the genus Corynebacterium as an active ingredient.
しかし、本発明が達成しようとする技術的課題は、上記で言及した課題に制限されず、言及しなかったまた他の課題は、下の記載から当業者に明確に理解されるべきである。 However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned should be clearly understood by those skilled in the art from the following description.
上記のような本発明の目的を達成するために、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断のための情報提供方法を提供する： In order to achieve the objects of the present invention as described above, the present invention provides the following steps: provide informational methods for the diagnosis of one or more of the diseases identified by:
（ａ）正常ヒト及び被検者のサンプルから分離した小胞からＤＮＡを抽出するステップ； (a) extracting DNA from vesicles isolated from normal human and subject samples;
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）を行った後、それぞれのＰＣＲ産物を収得するステップ；及び (b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product;
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。 (c) when the content of Corynebacterium bacterium-derived vesicles is low compared to normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and Determining one or more diseases selected from the group consisting of nasal polyps.
また、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法を提供する： The present invention also provides a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, comprising the steps of: do:
（ａ）正常ヒト及び被検者のサンプルから分離した小胞からＤＮＡを抽出するステップ； (a) extracting DNA from vesicles isolated from normal human and subject samples;
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲを行った後、それぞれのＰＣＲ産物を収得するステップ；及び (b) performing PCR on the extracted DNA using a primer pair prepared based on the gene sequence present in the 16S rDNA, and then obtaining each PCR product; and
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。 (c) when the content of Corynebacterium bacterium-derived vesicles is low compared to normal humans through quantitative analysis of the PCR product, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and Determining one or more diseases selected from the group consisting of nasal polyps.
本発明の一具現例で、前記ステップ（ａ）でのサンプルは、血液、小便、大便、唾液又は鼻粘膜であってもよい。 In one embodiment of the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa.
本発明の他の具現例で、前記ステップ（ｂ）でのプライマー対は、配列番号１及び配列番号２で表示される塩基配列を含むプライマー対であってもよい。 In another embodiment of the present invention, the primer pair in step (b) may be a primer pair comprising nucleotide sequences represented by SEQ ID NO:1 and SEQ ID NO:2.
また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供する。 The present invention also provides a composition for preventing, improving or treating inflammatory diseases, which contains vesicles derived from bacteria of the genus Corynebacterium as an active ingredient.
前記組成物は、薬学的組成物、食品組成物、化粧料組成物及び吸入剤組成物を含むことができる。 The compositions can include pharmaceutical compositions, food compositions, cosmetic compositions and inhalant compositions.
また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物を個体に投与するステップを含む炎症疾患の予防又は治療方法を提供する。 The present invention also provides a method for preventing or treating an inflammatory disease, comprising the step of administering to an individual a composition containing vesicles derived from a bacterium belonging to the genus Corynebacterium as an active ingredient.
また、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患の予防又は治療用途を提供する。 The present invention also provides use of Corynebacterium-derived vesicles for the prevention or treatment of inflammatory diseases.
また、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物の炎症疾患の予防又は治療用途を提供する。 The present invention also provides a use of a composition containing vesicles derived from Corynebacterium as an active ingredient for the prevention or treatment of inflammatory diseases.
また、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患に用いられる薬剤を生産するための用途を提供する。 The present invention also provides uses for producing drugs for inflammatory diseases of vesicles derived from bacteria belonging to the genus Corynebacterium.
本発明の一具現例で、前記小胞は、コリネバクテリウム・グルタミクムから分泌されるものであってもよい。 In one embodiment of the present invention, the vesicle may be secreted from Corynebacterium glutamicum.
本発明の他の具現例で、前記小胞は、平均直径が１０～２００ｎｍであってもよい。 In another embodiment of the present invention, the vesicles may have an average diameter of 10-200 nm.
本発明のまた他の具現例で、前記小胞は、コリネバクテリウム属細菌から自然的又は人工的に分泌されるものであってもよい。 In still another embodiment of the present invention, the vesicles may be naturally or artificially secreted from Corynebacterium spp.
本発明のまた他の具現例で、前記人工小胞は、細菌に熱処理、加圧処理などの方法で分泌されるものであってもよい。 In still another embodiment of the present invention, the artificial vesicles may be secreted by bacteria through heat treatment, pressure treatment, or the like.
本発明のまた他の具現例で、前記炎症疾患は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であってもよい。 In still another embodiment of the present invention, the inflammatory disease is one or more selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps. There may be.
本発明者らは、細菌の場合には体内に吸収されないが、細菌由来の小胞の場合には粘膜の防御膜を通過して粘膜の上皮細胞に吸収されて全身的に分布し、腎臓、肝臓、肺を通じて体外に排泄されることを確認した。また、患者の血液に存在する細菌由来の小胞のメタゲノム分析を通じて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者の血液又は鼻粘膜に存在するコリネバクテリウム属細菌由来の小胞が正常ヒトに比べて有意に減少していることを確認した。また、コリネバクテリウム属細菌の一種であるコリネバクテリウム・グルタミクムを体外で培養して小胞を分離して炎症細胞に投与したとき、病原性小胞によるＴＮＦ－αなどの炎症メディエーターの分泌を有意に抑制したことを確認したところ、本発明によるコリネバクテリウム属細菌由来の小胞は、前記炎症性疾患の予防用あるいは治療用組成物に有用であり得ると期待される。 The inventors have found that, in the case of bacteria, they are not absorbed into the body, but in the case of bacterial-derived vesicles, they pass through the protective membrane of the mucosa and are absorbed by the epithelial cells of the mucosa and are distributed systemically, kidney, It was confirmed that it was excreted outside the body through the liver and lungs. Corynebacteria present in the blood or nasal mucosa of patients with liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps through metagenomic analysis of vesicles derived from bacteria present in patient blood. It was confirmed that the number of vesicles derived from bacteria belonging to the genus Porporium was significantly reduced compared to normal humans. In addition, when Corynebacterium glutamicum, a type of Corynebacterium bacterium, was cultured in vitro and the vesicles were separated and administered to inflammatory cells, the secretion of inflammatory mediators such as TNF-α by pathogenic vesicles was suppressed. As a result of confirming the significant suppression, the vesicles derived from bacteria belonging to the genus Corynebacterium according to the present invention are expected to be useful in preventive or therapeutic compositions for the aforementioned inflammatory diseases.
本発明は、コリネバクテリウム属細菌由来の小胞及びその用途に関する。 The present invention relates to vesicles derived from bacteria belonging to the genus Corynebacterium and uses thereof.
本発明者らは、メタゲノム分析を通じて正常ヒトに比べて肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者由来のサンプルでコリネバクテリウム属由来の小胞の含量が顕著に減少していることを確認した。また、本発明者らは、病原性原因因子を投与する前にコリネバクテリウム・グルタミクム菌由来の小胞で炎症細胞に処理すると、病原性原因因子による炎症反応を効率的に抑制することを確認し、これに基づいて本発明を完成した。 Through metagenomic analysis, we found that vesicles from the genus Corynebacterium spp. It was confirmed that the content was significantly reduced. In addition, the present inventors confirmed that treatment of inflammatory cells with vesicles derived from Corynebacterium glutamicum before administration of a virulence-causing factor efficiently suppresses the inflammatory reaction caused by the virulence-causing factor. Based on this, the present invention was completed.
これにより、本発明は、下記のステップを含む肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の診断方法又は診断のための情報提供方法を提供する： Accordingly, the present invention provides a method for diagnosing one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, or Provide informational methods for diagnosis:
（ａ）正常ヒト及び被検者のサンプルから分離した小胞からＤＮＡを抽出するステップ；
（ｂ）前記抽出したＤＮＡに対して１６Ｓ ｒＤＮＡに存在する遺伝子配列に基づいて製作したプライマー対を用いてＰＣＲ（Ｐｏｌｙｍｅｒａｓｅ Ｃｈａｉｎ Ｒｅａｃｔｉｏｎ）を行った後、それぞれのＰＣＲ産物を収得するステップ；及び
（ｃ）前記ＰＣＲ産物の定量分析を通じて正常ヒトに比べてコリネバクテリウム属細菌由来の小胞の含量が低い場合、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患であると判定するステップ。
(a) extracting DNA from vesicles isolated from normal human and subject samples;
(b) performing PCR (Polymerase Chain Reaction) on the extracted DNA using a primer pair prepared based on the gene sequence present in 16S rDNA, and then obtaining each PCR product; and (c) ) When the content of vesicles derived from Corynebacterium spp. is lower than that of normal humans through quantitative analysis of the PCR products, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps A step of determining one or more diseases selected from the group consisting of
本発明で用いられる用語「診断」とは、広い意味では患者の病気の実態を全ての面にわたって判断することを意味する。判断の内容は、病名、病因、病型、軽重、病状の詳細な様態、合併症の有無及び予後などである。本発明で「診断」は、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープからなる群より選択された一つ以上の疾患の発病有無及び疾患の程度などを判断することである。 The term "diagnosis" used in the present invention means, in a broad sense, judging the actual state of a patient's disease over all aspects. The contents of the judgment include disease name, etiology, disease type, severity, detailed condition of disease, presence or absence of complications, prognosis, and the like. In the present invention, "diagnosis" refers to the presence or absence of one or more diseases selected from the group consisting of liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia, and nasal polyps, and the severity of the disease. is to judge
本発明で用いられる用語「ナノ小胞（Ｎａｎｏｖｅｓｉｃｌｅ）」あるいは「小胞（Ｖｅｓｉｃｌｅ）」とは、多様な細菌から分泌されるナノサイズの膜からなる構造物を意味する。コリネバクテリウムのようなグラム陽性菌（ｇｒａｍ－ｐｏｓｉｔｉｖｅ ｂａｃｔｅｒｉａ）由来の小胞は、タンパク質と核酸外にも細菌の細胞壁の構成成分であるペプチドグリカン（ｐｅｐｔｉｄｏｇｌｙｃａｎ）とリポテイコ酸（ｌｉｐｏｔｅｉｃｈｏｉｃ ａｃｉｄ）、そして小胞内に多様な低分子化合物を有している。本発明において、ナノ小胞あるいは小胞は、コリネバクテリウム属の細菌から自然的に分泌されるか又は細菌に熱処理、加圧処理などを通じて人工的に生産するものであって、１０～２００ｎｍの平均直径を有している。 The term "Nanovesicle" or "Vesicle" used in the present invention means a nano-sized membrane structure secreted by various bacteria. Vesicles derived from Gram-positive bacteria such as Corynebacterium contain not only proteins and nucleic acids but also peptidoglycan and lipoteichoic acid, which are components of bacterial cell walls, and vesicles. It contains various low-molecular-weight compounds. In the present invention, nanovesicles or vesicles are naturally secreted from bacteria of the genus Corynebacterium or artificially produced by bacteria through heat treatment, pressure treatment, etc. have an average diameter.
本発明で用いられる用語「メタゲノム」とは、「群遺伝体」とも言い、土壌、動物の腸など孤立した地域内の全てのウイルス、細菌、カビなどを含む遺伝体の総合を意味するもので、主に培養されない微生物を分析するためにシーケンサーを用いて一度に多くの微生物を同定することを説明する遺伝体の概念として用いられる。特に、「メタゲノム」は、一種のゲノム、遺伝体を言うのではなく、一つの環境単位の全ての種の遺伝体として一種の混合遺伝体を言う。これは、オミックス的に生物学が発展する過程で一つの種を定義するとき、機能的に既存の一種だけではなく、多様な種が互いに相互作用して完全な種を作るという観点から生じた用語である。技術的には、迅速配列分析法を用いて種に関係なく全てのＤＮＡ、ＲＮＡを分析し、一つの環境内での全ての種を同定し、相互作用、代謝作用を解明する技法の対象である。 The term "metagenome" used in the present invention is also called "group genetic material" and means a comprehensive collection of genetic materials including all viruses, bacteria, fungi, etc. in isolated areas such as soil and animal intestines. , is used as a genetic entity concept to describe the identification of many microorganisms at once using a sequencer, primarily for the analysis of uncultivated microorganisms. In particular, "metagenomics" does not refer to one type of genome, genotype, but to a type of mixed genotype as the genotype of all species in one environmental unit. When defining a single species in the process of biology's development from an omics perspective, it arose from the perspective that not only functionally existing species but also diverse species interact with each other to create a complete species. terminology. Technically, it is the subject of techniques that analyze all DNA, RNA, regardless of species, using rapid sequence analysis to identify all species within an environment and elucidate their interactions and metabolism. be.
前記小胞は、コリネバクテリウム属の細菌を含む培養液を遠心分離、超高速遠心分離、押圧、超音波分解、細胞溶解、均質化、冷凍－解凍、電気穿孔、機械的分解、化学物質処理、フィルタによる濾過、ゲル濾過クロマトグラフィー、フリーフロー電気泳動及び毛細管電気泳動からなる群より選択された一つ以上の方法を用いて分離することができる。また、不純物の除去のための洗浄、収得された小胞の濃縮などの過程を追加で含むことができる。 Said vesicles are obtained by centrifugation, ultra-high speed centrifugation, pressing, sonication, cell lysis, homogenization, freeze-thaw, electroporation, mechanical disintegration, chemical treatment of culture medium containing bacteria of the genus Corynebacterium. , filter filtration, gel filtration chromatography, free-flow electrophoresis, and capillary electrophoresis. In addition, additional processes such as washing to remove impurities and concentration of the obtained vesicles may be included.
本発明において、前記ステップ（ａ）でのサンプルは、血液、小便、大便、唾液又は鼻粘膜であってもよいが、これに制限されるものではない。 In the present invention, the sample in step (a) may be blood, urine, stool, saliva or nasal mucosa, but is not limited thereto.
本発明において、前記ステップ（ｂ）でのプライマー対は、配列番号１及び配列番号２で表示される塩基配列を含むプライマー対であってもよいが、これに制限されるものではない。 In the present invention, the primer pair in step (b) may be a primer pair containing the base sequences represented by SEQ ID NO: 1 and SEQ ID NO: 2, but is not limited thereto.
本発明の他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む炎症疾患の予防、改善又は治療用組成物を提供する。 As another aspect of the present invention, the present invention provides a composition for preventing, improving or treating inflammatory diseases, which contains vesicles derived from bacteria belonging to the genus Corynebacterium as an active ingredient.
前記組成物は、薬学的組成物、食品組成物、化粧料組成物及び吸入剤組成物を含む。 The compositions include pharmaceutical compositions, food compositions, cosmetic compositions and inhalant compositions.
本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物を個体に投与するステップを含む炎症疾患の予防又は治療方法を提供する。 As yet another aspect of the present invention, the present invention provides a method for preventing or treating an inflammatory disease comprising the step of administering to an individual a composition containing vesicles derived from bacteria belonging to the genus Corynebacterium as an active ingredient.
本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患の予防又は治療用途を提供する。 As still another aspect of the present invention, the present invention provides use of Corynebacterium-derived vesicles for the prevention or treatment of inflammatory diseases.
本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞を有効成分として含む組成物の炎症疾患の予防又は治療用途を提供する。 As still another aspect of the present invention, the present invention provides a use of a composition containing vesicles derived from bacteria belonging to the genus Corynebacterium as an active ingredient for the prevention or treatment of inflammatory diseases.
本発明のまた他の様態として、本発明は、コリネバクテリウム属細菌由来の小胞の炎症疾患に用いられる薬剤を生産するための用途を提供する。 As still another aspect of the present invention, the present invention provides a use for producing a drug used for inflammatory diseases of vesicles derived from bacteria belonging to the genus Corynebacterium.
本発明で用いられる用語「炎症疾患（Ｉｎｆｌａｍｍａｔｏｒｙ ｄｉｓｅａｓｅ）」とは、炎症を誘発する原因因子に露出されて皮膚あるいは腸の上皮細胞に損傷とその結果として炎症が発生して生ずる疾患を意味し、炎症の結果として発生する代謝疾患、心血管疾患、神経精神疾患、癌を含むが、これに制限されない。 The term "inflammatory disease" used in the present invention means a disease caused by exposure to causative factors that induce inflammation, damage to skin or intestinal epithelial cells, and resulting inflammation, Including, but not limited to, metabolic diseases, cardiovascular diseases, neuropsychiatric diseases, and cancers that develop as a result of inflammation.
本発明による前記炎症疾患の例としては、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症、鼻ポリープなどがあるが、これに制限されるものではない。 Examples of the inflammatory diseases according to the present invention include, but are not limited to, liver cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia, nasal polyps and the like.
本発明で用いられる用語「予防」とは、本発明による組成物の投与によって炎症疾患を抑制させたり発病を遅延させる全ての行為を意味する。 The term "prevention" used in the present invention means all actions of suppressing or delaying the onset of inflammatory diseases by administration of the composition according to the present invention.
本発明で用いられる用語「治療」とは、本発明による組成物の投与によって炎症疾患に対する症状が好転したり有利に変更される全ての行為を意味する。 The term "treatment" as used in the present invention means all actions in which the symptoms of inflammatory diseases are improved or altered favorably by administration of the composition according to the present invention.
本発明で用いられる用語「改善」とは、治療される状態と関連したパラメータ、例えば、症状の程度を少なくとも減少させる全ての行為を意味する。 As used herein, the term "amelioration" means any action that at least reduces the severity of a parameter, eg, symptom, associated with the condition being treated.
本発明の一実施例では、細菌及び細菌由来の小胞をマウスに経口投与して細菌及び小胞の体内吸収、分布及び排泄様相を評価して、細菌の場合には、腸粘膜を介して吸収されないのに比べて、小胞は、投与５分以内に吸収されて全身的に分布し、腎臓、肝臓などを通じて排泄されることを確認した（実施例１参照）。 In one embodiment of the present invention, bacteria and bacterial-derived vesicles are orally administered to mice to assess the absorption, distribution and excretion profile of bacteria and vesicles in the body and, in the case of bacteria, through the intestinal mucosa. It was confirmed that the vesicles were absorbed within 5 minutes of administration, distributed systemically, and excreted through the kidneys, liver, etc., whereas they were not absorbed (see Example 1).
本発明の他の実施例では、細菌と細菌由来の小胞を腸に直接投与して腸粘膜の防御膜を通過するかを評価した結果、細菌の場合には、腸粘膜の防御膜を通過しなかったが、細菌由来の小胞の場合には、粘膜の防御膜を通過することを確認した（実施例２参照）。 In another embodiment of the present invention, bacteria and bacterial-derived vesicles were directly administered to the intestine to assess whether they penetrated the protective membrane of the intestinal mucosa. However, it was confirmed that vesicles derived from bacteria pass through the mucosal protective membrane (see Example 2).
本発明のまた他の実施例では、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者と正常対照群の臨床サンプルからメタゲノム分析を通じてコリネバクテリウム属細菌の分布を比較した結果、正常対照群に比べて前記疾患者の臨床サンプルにコリネバクテリウム属細菌由来の小胞の分布が有意に減少していることを確認した（実施例３～１２参照）。 In yet another embodiment of the present invention, Corynebacterium spp. is detected through metagenomic analysis from clinical samples of patients with cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps and normal controls. As a result of comparing the distribution, it was confirmed that the distribution of vesicles derived from the genus Corynebacterium was significantly reduced in the clinical samples of the patients compared to the normal control group (see Examples 3-12).
本発明のまた他の実施例では、コリネバクテリウム属細菌に属するコリネバクテリウム・グルタミクム菌株を培養し、それから分泌された小胞の炎症誘発効果を評価した。多様な濃度のコリネバクテリウム・グルタミクム由来の小胞をマクロファージに処理した後、代表的病原性小胞である大腸菌由来の小胞を処理して炎症メディエーターの分泌程度を比較した結果、大腸菌由来の小胞によるＩＬ－６及びＴＮＦ－αの分泌と比較してコリネバクテリウム・グルタミクム由来の小胞による分泌能が顕著に減少していた（実施例１４参照）。 In yet another embodiment of the present invention, Corynebacterium glutamicum strains belonging to the genus Corynebacterium were cultured and the pro-inflammatory effects of vesicles secreted therefrom were evaluated. After treating macrophages with various concentrations of Corynebacterium glutamicum-derived vesicles, they treated E. coli-derived vesicles, a representative pathogenic vesicle, and compared the secretion of inflammatory mediators. There was a marked reduction in the ability of vesicles from Corynebacterium glutamicum to secrete IL-6 and TNF-α compared to the secretion of IL-6 and TNF-α by vesicles (see Example 14).
本発明のまた他の実施例では、コリネバクテリウム・グルタミクム菌株由来の小胞の抗炎症効果を評価した。病原性小胞である大腸菌由来の小胞を処理する前に多様な濃度のコリネバクテリウム・グルタミクム由来の小胞をマクロファージに処理した後、炎症メディエーターの分泌を評価した結果、炎症誘発大腸菌由来の小胞によるＴＮＦ－αの分泌をコリネバクテリウム・グルタミクム由来の小胞が効率的に抑制することを確認した（実施例１５参照）。 In yet another example of the present invention, anti-inflammatory effects of vesicles derived from Corynebacterium glutamicum strains were evaluated. After treating macrophages with various concentrations of Corynebacterium glutamicum-derived vesicles before treating them with pathogenic vesicles, E. coli-derived vesicles were evaluated for secretion of inflammatory mediators. It was confirmed that Corynebacterium glutamicum-derived vesicles efficiently inhibited TNF-α secretion by vesicles (see Example 15).
本発明による薬学的組成物は、薬学的に許容可能な担体を含むことができる。前記薬学的に許容可能な担体は、製剤時に通常的に用いられるものであって、食塩水、滅菌水、リンゲル液、緩衝食塩水、シクロデキストリン、デキストロース溶液、マルトデキストリン溶液、グリセロール、エタノール、リポソームなどを含むが、これに限定されず、必要に応じて、抗酸化剤、緩衝液など他の通常の添加剤をさらに含むことができる。また、希釈剤、分散剤、界面活性剤、結合剤、潤滑剤などを付加的に添加して水溶液、懸濁液、乳濁液などのような注射用剤型、丸薬、カプセル、顆粒又は錠剤で製剤化することができる。適合した薬学的に許容される担体及び製剤化についてはレミントンの文献に開示されている方法を用いて各成分によって好ましく製剤化することができる。本発明の薬学的組成物は、剤型に特別な制限はないが、注射剤、吸入剤、皮膚外用剤又は経口摂取剤などに製剤化することができる。 A pharmaceutical composition according to the invention can comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is one commonly used for formulation, and includes saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and the like. including, but not limited to, other conventional additives such as antioxidants, buffers, etc., can be further included as needed. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be additionally added to prepare injectable dosage forms such as aqueous solutions, suspensions, emulsions, pills, capsules, granules or tablets. can be formulated in Suitable pharmaceutically acceptable carriers and formulations are preferably formulated by each component using methods disclosed in the Remington reference. The pharmaceutical composition of the present invention is not particularly limited in dosage form, but can be formulated as an injection, an inhalant, an external skin preparation, or an oral ingestion.
本発明の薬学的組成物は、目的とする方法によって経口投与するか非経口投与（例えば、静脈内、皮下、皮膚、鼻腔、気道に適用）でき、投与量は、患者の状態及び体重、疾病の程度、薬物形態、投与経路及び時間によって異なるが、当業者により適切に選択され得る。 The pharmaceutical composition of the present invention can be administered orally or parenterally (e.g., intravenous, subcutaneous, cutaneous, nasal, respiratory tract application) according to the intended method, and the dosage depends on the patient's condition and body weight, disease. can be appropriately selected by those skilled in the art.
本発明による薬学的組成物は、薬学的に有効な量で投与する。本発明において、薬学的に有効な量は、医学的治療に適用可能な合理的なベネフィット／リスクの割合で疾患を治療するに十分な量を意味し、有効容量のレベルは、患者の疾患の種類、重症度、薬物の活性、薬物に対する敏感度、投与時間、投与経路及び排出の割合、治療期間、同時に用いられる薬物を含んだ要素及びその他医学分野によく知られた要素によって決定され得る。本発明による組成物は、個別治療剤で投与するか他の治療剤と併用して投与され得、従来の治療剤とは順次又は同時に投与され得、単一又は多重投与され得る。上記した要素を全て考慮して副作用なしに最小限の量で最大の効果を得ることができる量を投与することが重要であり、これは、当業者によって容易に決定され得る。 Pharmaceutical compositions according to the invention are administered in a pharmaceutically effective amount. In the context of the present invention, pharmaceutically effective amount means an amount sufficient to treat disease at a reasonable benefit/risk ratio applicable to medical treatment, and effective dose levels refer to levels of disease in patients. It can be determined by factors including type, severity, drug activity, drug sensitivity, administration time, route of administration and excretion rate, duration of treatment, concurrent drugs and other factors well known in the medical field. Compositions according to the present invention can be administered as individual therapeutic agents or in combination with other therapeutic agents, can be administered sequentially or concurrently with conventional therapeutic agents, and can be administered in single or multiple doses. It is important to administer the amount that achieves the maximum effect with the minimum amount without side effects considering all of the above factors, which can be easily determined by those skilled in the art.
具体的に、本発明による薬学的組成物の有効量は、患者の年齢、性別、体重によって変わり得、投与経路、肥満の重症度、性別、体重、年齢などによって増減され得る。 Specifically, the effective amount of the pharmaceutical composition according to the present invention may vary according to the patient's age, sex and weight, and may be increased or decreased according to administration route, severity of obesity, sex, weight, age and the like.
本発明の吸入剤組成物で有効成分を吸入剤にそのまま添加するか他の成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的（予防又は治療用）によって適切に決定され得る。 In the inhalant composition of the present invention, the active ingredient may be added directly to the inhalant or used together with other ingredients, and may be used appropriately by conventional methods. The amount of the active ingredients to be mixed can be appropriately determined depending on the purpose of use (for prevention or treatment).
本発明の食品組成物は、健康機能食品組成物を含む。本発明による食品組成物は、有効成分を食品にそのまま添加するか他の食品又は食品成分とともに用いてもよく、通常的な方法によって適切に用いてもよい。有効成分の混合量は、その使用目的（予防又は改善用）によって適切に決定され得る。一般的に、食品又は飲料の製造時に、本発明の組成物は、原料に対して１５重量％以下、好ましくは、１０重量％以下の量で添加される。しかし、健康及び衛生を目的とするか又は健康調節を目的とする長期間の摂取の場合には、前記量は前記範囲以下であってもよい。 The food composition of the present invention includes health functional food compositions. The food composition according to the present invention may be used by adding the active ingredient directly to the food or together with other food or food ingredients, and may be suitably used by conventional methods. The amount of the active ingredients to be mixed can be appropriately determined depending on the purpose of use (for prevention or improvement). Generally, the composition of the present invention is added in an amount of 15% by weight or less, preferably 10% by weight or less, relative to the raw material during the production of food or beverages. However, in the case of long-term intake for health and hygiene purposes or for health regulation purposes, said amount may be below said range.
本発明の食品組成物は、指示された割合で必須成分として前記有効成分を含有すること以外に他の成分には特別な制限がなく、通常の飲料のように多様な香味剤又は天然炭水化物などを追加成分として含有できる。上述した天然炭水化物の例は、モノサッカライド、例えば、ブドウ糖、果糖など；ジサッカライド、例えば、マルトース、スクロースなど；及びポリサッカライド、例えば、デキストリン、シクロデキストリンなどのような通常的な糖、及びキシリトール、ソルビトール、エリスリトールなどの糖アルコールである。上述したもの以外の香味剤として天然香味剤（ソーマチン、ステビア抽出物、例えば、レバウディオサイドＡ、グリチルリチンなど）及び合成香味剤（サッカリン、アスパルテームなど）を有利に用いることができる。前記天然炭水化物の割合は、当業者の選択によって適切に決定され得る。 The food composition of the present invention contains the above-mentioned active ingredient as an essential ingredient in the indicated ratio, and there is no particular limitation on other ingredients. can be included as an additional ingredient. Examples of the above-mentioned natural carbohydrates are monosaccharides, such as glucose, fructose, etc.; disaccharides, such as maltose, sucrose, etc.; Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (thaumatin, stevia extract, eg rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates can be appropriately determined by the choice of those skilled in the art.
上記外に、本発明の食品組成物は、様々な栄養剤、ビタミン、ミネラル（電解質）、合成風味剤及び天然風味剤などの風味剤、着色剤及び充填剤（チーズ、チョコレートなど）、ペクチン酸及びその塩、アルギン酸及びその塩、有機酸、保護性コロイド増粘剤、ｐＨ調節剤、安定化剤、防腐剤、グリセリン、アルコール、炭酸飲料に用いられる炭酸化剤などを含有することができる。このような成分は、独立的に又は組み合わせて用いることができる。このような添加剤の割合も当業者により適切に選択され得る。 In addition to the above, the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, coloring agents and fillers (cheese, chocolate, etc.), pectic acid. and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages, and the like. Such ingredients can be used individually or in combination. The ratio of such additives can also be appropriately selected by those skilled in the art.
本発明の前記化粧料組成物は、コリネバクテリウム属細菌由来の小胞だけでなく、化粧料組成物に通常的に用いられる成分を含むことができ、例えば、抗酸化剤、安定化剤、溶解化剤、ビタミン、顔料及び香料のような通常的な補助剤、そして担体を含むことができる。 The cosmetic composition of the present invention can contain not only vesicles derived from bacteria belonging to the genus Corynebacterium, but also ingredients commonly used in cosmetic compositions, such as antioxidants, stabilizers, Usual adjuvants such as solubilizers, vitamins, pigments and fragrances, and carriers can be included.
また、本発明の組成物は、コリネバクテリウム属細菌由来の小胞以外に、コリネバクテリウム属細菌由来の小胞と反応して皮膚保護効果を損傷させない限度で従来から用いられてきた有機紫外線遮断剤を混合して用いてもよい。前記有機紫外線遮断剤としては、グリセリルＰＡＢＡ、ドロメトリゾールトリシロキサン、ドロメトリゾール、ジガロイルトリオレエート、フェニルジベンズイミダゾールテトラスルホン酸２Ｎａ、イスコトリジノール、ジエチルアミノヒドロキシベンゾイルヘキシルベンゾエート、ＤＥＡ－メトキシシンナメート、ローソンとジヒドロキシアセトンの混合物、メチレンビス－ベンゾトリアゾリルテトラメチルブチルフェノール、４－メチルベンジリデンカンファ、アントラニル酸メンチル、ベンゾフェノン－３（オキシベンゾン）、ベンゾフェノン－４、ベンゾフェノン－８（ジオキシベンゾン）、ブチルメトキシジベンゾイルメタン、ビスエチルヘキシルオキシフェノールメトキシフェニルトリアジン、シノキサート、エチルジヒドロキシプロピルＰＡＢＡ、オクトクリレン、エチルヘキシルジメチルＰＡＢＡ、エチルヘキシルメトキシシンナメート、エチルヘキシルサリシレート、エチルヘキシルトリアゾン、イソアミル－ｐ－メトキシシンナメート、ポリシリコン－１５（マロン酸ジメチコジエチルベンザル）、テレフタリデンジカンファスルホン酸及びその塩類、ＴＥＡ－サリシレート及びアミノ安息香酸（ＰＡＢＡ）からなる群より選択された１種以上を用いることができる。 In addition to the vesicles derived from bacteria belonging to the genus Corynebacterium, the composition of the present invention also contains organic ultraviolet rays that have been conventionally used as long as they do not react with vesicles derived from bacteria belonging to the genus Corynebacterium to damage the skin protective effect. A mixture of blocking agents may be used. Examples of the organic UV blockers include glyceryl PABA, drometrisol trisiloxane, drometrisol, digalloyl trioleate, phenyldibenzimidazole tetrasulfonate disodium, iscotridinol, diethylaminohydroxybenzoylhexylbenzoate, DEA-methoxycinnamate. , a mixture of Lawsone and dihydroxyacetone, methylenebis-benzotriazolyltetramethylbutylphenol, 4-methylbenzylidenecamphor, menthyl anthranilate, benzophenone-3 (oxybenzone), benzophenone-4, benzophenone-8 (dioxybenzone), butyl methoxydi Benzoylmethane, bisethylhexyloxyphenol methoxyphenyltriazine, cinoxate, ethyldihydroxypropyl PABA, octocrylene, ethylhexyldimethyl PABA, ethylhexyl methoxycinnamate, ethylhexyl salicylate, ethylhexyl triazone, isoamyl-p-methoxycinnamate, polysilicon-15 (malon Acid dimethicodiethylbenzal), terephthalidenedicamphorsulfonic acid and its salts, TEA-salicylate, and one or more selected from the group consisting of aminobenzoic acid (PABA) can be used.
本発明の化粧料組成物を添加できる製品としては、例えば、収れん化粧水、柔軟化粧水、栄養化粧水、各種クリーム、エッセンス、パック、ファンデーションなどのような化粧品類とクレンジング、洗顔剤、石鹸、トリートメント、美容液などがある。本発明の化粧料組成物の具体的な剤型としては、スキンローション、スキンソフナー、スキントナー、アストリンゼント、ローション、ミルクローション、モイスチャーローション、栄養ローション、マッサージクリーム、栄養クリーム、モイスチャークリーム、ハンドクリーム、エッセンス、栄養エッセンス、パック、石鹸、シャンプー、クレンジングフォーム、クレンジングローション、クレンジングクリーム、ボディーローション、ボディークレンザー、乳液、リップスティック、メイクアップベース、ファンデーション、プレスパウダー、ルーズパウダー、アイシャドウなどの剤型を含む。 Examples of products to which the cosmetic composition of the present invention can be added include cosmetics such as astringent lotions, softening lotions, nutritional lotions, various creams, essences, packs, foundations, cleansing agents, facial cleansers, soaps, There are treatments and cosmetics. Specific dosage forms of the cosmetic composition of the present invention include skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nutrition lotion, massage cream, nutrition cream, moisture cream, hand cream, Essence, nutrition essence, mask, soap, shampoo, cleansing foam, cleansing lotion, cleansing cream, body lotion, body cleanser, milky lotion, lipstick, makeup base, foundation, pressed powder, loose powder, eyeshadow, etc. include.
以下、本発明の理解を助けるために好ましい実施例を提示する。しかしながら、下記の実施例は、本発明をより容易に理解するために提供されるものに過ぎず、下記実施例によって本発明の内容が限定されるものではない。 Preferred examples are presented below to aid understanding of the present invention. However, the following examples are merely provided for easier understanding of the present invention, and the content of the present invention is not limited by the following examples.
［実施例１．細菌及び細菌由来の小胞の体内吸収、分布及び排泄様相の分析］
細菌と細菌由来の小胞が粘膜を通じて全身的に吸収されるかを評価するために次のような方法で実験を行った。マウスの胃腸に蛍光で標識した細菌と細菌由来の小胞をそれぞれ５０μｇの用量で胃腸管に投与し、それぞれ０分、５分、３時間、６時間、１２時間後に蛍光を測定した。マウスの全体イメージを観察した結果、図１ａに示したように、細菌の場合には、全身的に吸収されなかったが、細菌由来の小胞の場合には、投与５分後に全身的に吸収され、投与３時間後には、膀胱で蛍光が濃く観察されて、小胞が泌尿器系に排泄されることが分かった。また、小胞は、投与１２時間まで体内に存在することが分かった（図１ａ参照）。
[Example 1. Analysis of body absorption, distribution and excretion of bacteria and bacterial-derived vesicles]
In order to evaluate systemic absorption of bacteria and bacterial-derived vesicles through mucous membranes, the following experiments were carried out. Fluorescently labeled bacteria and bacterial-derived vesicles were administered to the gastrointestinal tract of mice at a dose of 50 μg each, and fluorescence was measured after 0, 5, 3, 6, and 12 hours, respectively. As a result of observing the whole image of the mouse, as shown in Fig. 1a, bacteria were not absorbed systemically, but bacterial-derived vesicles were systemically absorbed 5 minutes after administration. 3 hours after administration, intense fluorescence was observed in the bladder, indicating that the vesicles were excreted into the urinary system. Also, vesicles were found to be present in the body up to 12 hours after administration (see Figure 1a).
細菌と細菌由来の小胞が全身的に吸収された後、多くの臓器に浸潤した様相を評価するために、蛍光で標識した５０μｇの細菌と細菌由来の小胞を上記の方法のように投与した後、投与１２時間後に血液、心臓、肺、肝臓、腎臓、脾臓、脂肪、筋肉を採取した。採取した組織で蛍光を観察した結果、図１ｂに示したように、細菌由来の小胞が血液、心臓、肺、肝臓、腎臓、脾臓、脂肪、筋肉に分布したが、細菌は吸収されないことが分かった（図１ｂ参照）。 To assess the infiltration of many organs after systemic absorption of bacteria and bacterial-derived vesicles, 50 μg of fluorescently labeled bacteria and bacterial-derived vesicles were administered as described above. Then, 12 hours after administration, blood, heart, lung, liver, kidney, spleen, fat and muscle were collected. Observation of fluorescence in the harvested tissues revealed that bacterial-derived vesicles were distributed in blood, heart, lung, liver, kidney, spleen, fat, and muscle, but the bacteria were not absorbed, as shown in Fig. 1b. found (see Fig. 1b).
［実施例２．細菌及び細菌由来の小胞の粘膜防御膜浸透有無の評価］
細菌と細菌由来の小胞が粘膜防御膜を通過して上皮組織に浸潤するかを評価するために、細菌と細菌由来の小胞を腸に直接投与した後、免疫組織染色（Ｉｍｍｕｎｏｈｉｓｔｏｃｈｅｍｉｓｔｒｙ）方法で粘膜防御膜を通過して上皮組織への浸潤を評価した。粘膜組織で細菌と小胞の存在を評価するために、細菌と小胞に対する抗体を作製してＧＦＰ（Ｇｒｅｅｎ ｆｌｕｏｒｅｓｃｅｎｔ ｐｒｏｔｅｉｎ）をつけて用い、ＤＡＰＩ（４，６－ｄｉａｍｉｄｉｎｏ ２－ｐｈｅｎｙｌｉｎｄｏｌｅ）染色をした後、顕微鏡で観察した。
[Example 2. Evaluation of presence or absence of mucosal protective membrane permeation of bacteria and bacterial-derived vesicles]
In order to assess whether bacteria and bacterial-derived vesicles pass through the mucosal protective membrane and infiltrate epithelial tissue, bacteria and bacterial-derived vesicles were administered directly into the intestine, followed by immunohistochemistry. Epithelial invasion across the mucosal protective membrane was assessed. In order to evaluate the presence of bacteria and vesicles in mucosal tissue, antibodies against bacteria and vesicles were prepared and used with GFP (Green Fluorescent Protein), followed by DAPI (4,6-diamidino 2-phenylindole) staining. Afterwards, they were observed under a microscope.
その結果、細菌の場合には、粘膜防御膜を通過しなかった一方、細菌由来の小胞は、粘膜で通過して上皮組織に浸潤することを確認した（図２参照）。 As a result, it was confirmed that bacteria did not penetrate the mucosal protective membrane, whereas bacterial-derived vesicles penetrated the mucosa and infiltrated the epithelial tissue (see FIG. 2).
［実施例３．臨床サンプルに存在する細菌由来の小胞のメタゲノム分析］
血液又は鼻粘膜組織などを先に１０ｍｌチューブに入れ、遠心分離機（３，５００×ｇ、１０ｍｉｎ、４℃）で浮遊物を沈めた後、上澄液のみを新しい１０ｍｌチューブに移した。０．２２μｍフィルタを用いて細菌及び異物を除去した後、セントリプレップチューブ（ｃｅｎｔｒｉｐｒｅｉｇｕｇａｌ ｆｉｌｔｅｒｓ ５０ｋＤ）に移し、１５００×ｇ、４℃で１５分間遠心分離して５０ｋＤより小さい物質は捨て、１０ｍｌまで濃縮させた。もう一度、０．２２μｍフィルタを用いてバクテリア及び異物を除去した後、Ｔｙｐｅ ９０ｔｉローターで１５０，０００×ｇ、４℃で３時間の間超高速遠心分離を行い、上澄液を除去した後、固まったペレットを生理食塩水（Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ＰＢＳ）でとかした。
[Example 3. Metagenomic analysis of bacterial-derived vesicles present in clinical samples]
Blood or nasal mucosal tissue was first placed in a 10 ml tube, centrifuged (3,500×g, 10 min, 4° C.) to sink floating matter, and then only the supernatant was transferred to a new 10 ml tube. After removing bacteria and foreign substances using a 0.22 μm filter, the filter was transferred to a centriprep tube (50 kD) and centrifuged at 1500×g at 4° C. for 15 minutes to discard substances smaller than 50 kD and concentrate to 10 ml. rice field. Bacteria and foreign matter were removed again using a 0.22 μm filter, followed by ultra-high speed centrifugation at 150,000×g and 4° C. for 3 hours in a Type 90ti rotor. The obtained pellet was dissolved with a physiological saline (Phosphate buffered saline, PBS).
上記方法で分離した小胞１００μｌを１００℃でボイルして内部のＤＮＡを脂質外に出るようにし、その後、氷で５分間冷やした。その後、残った浮遊物を除去するために、１０，０００×ｇ、４℃で３０分間遠心分離し、上澄液のみを集めた後、ナノドロップ（Ｎａｎｏｄｒｏｐ）を用いてＤＮＡ量を定量した。その後、前記抽出されたＤＮＡに細菌由来のＤＮＡが存在するかを確認するために、下記表１に示した１６ｓ ｒＤＮＡプライマーでＰＣＲを行って、前記抽出された遺伝子に細菌由来の遺伝子が存在することを確認した。 100 μl of the vesicles separated by the above method were boiled at 100° C. to remove the internal DNA from the lipids, and then chilled on ice for 5 minutes. After that, in order to remove remaining floating substances, the cells were centrifuged at 10,000×g and 4° C. for 30 minutes, and only the supernatant was collected, and the amount of DNA was quantified using Nanodrop. Thereafter, in order to confirm whether the extracted DNA contains DNA derived from bacteria, PCR is performed using the 16s rDNA primers shown in Table 1 below, and the extracted genes contain genes derived from bacteria. It was confirmed.
上記方法で抽出したＤＮＡを上記の１６Ｓ ｒＤＮＡプライマーを用いて増幅した後にシークエンシングを行い（Ｉｌｌｕｍｉｎａ ＭｉＳｅｑ ｓｅｑｕｅｎｃｅｒ）、結果をＳＦＦ（Ｓｔａｎｄａｒｄ Ｆｌｏｗｇｒａｍ Ｆｏｒｍａｔ）ファイルで出力し、ＧＳ ＦＬＸソフトウェア（ｖ２．９）を用いてＳＦＦファイルをｓｅｑｕｅｎｃｅファイル（．ｆａｓｔａ）とｎｕｃｌｅｏｔｉｄｅ ｑｕａｌｉｔｙ ｓｃｏｒｅファイルに変換した後、リードの信用度評価を確認し、ｗｉｎｄｏｗ（２０ ｂｐｓ）平均ｂａｓｅ ｃａｌｌ ａｃｃｕｒａｃｙが９９％未満（Ｐｈｒｅｄ ｓｃｏｒｅ＜２０）である部分を除去した。ＯＵＴ（Ｏｐｅｒａｔｉｏｎａｌ Ｔａｘｏｎｏｍｙ Ｕｎｉｔ）分析のために、ＵＣＬＵＳＴとＵＳＥＡＲＣＨを用いてシーケンス類似度によってクラスタリングを行い、属（ｇｅｎｕｓ）は９４％、科（ｆａｍｉｌｙ）は９０％、目（ｏｒｄｅｒ）は８５％、網（ｃｌａｓｓ）は８０％、門（ｐｈｙｌｕｍ）は７５％のシーケンス類似度を基準にクラスタリングし、各ＯＴＵの門（ｐｈｙｌｕｍ）、網（ｃｌａｓｓ）、目（ｏｒｄｅｒ）、科（ｆａｍｉｌｙ）、属（ｇｅｎｕｓ）レベルの分類を行い、ＢＬＡＳＴＮとＧｒｅｅｎＧｅｎｅｓの１６Ｓ ＲＮＡシーケンスデータベース（１０８，４５３シーケンス）を用いて属レベルで９７％以上のシーケンス類似度を有する細菌をプロファイリングした（ＱＩＩＭＥ）。 After amplifying the DNA extracted by the above method using the above 16S rDNA primers, sequencing was performed (Illumina MiSeq sequencer), the results were output as an SFF (Standard Flowgram Format) file, and the GS FLX software (v2.9) was used. After converting the SFF file into a sequence file (.fasta) and a nucleotide quality score file using removed part. For OUT (Operational Taxonomy Unit) analysis, UCLUST and USEARCH were used to perform clustering according to sequence similarity, with 94% for genus, 90% for family, 85% for order, and 85% for network. (class) is clustered on the basis of sequence similarity of 80% and phylum is 75%. ) level classification was performed and bacteria with sequence similarity greater than 97% at the genus level were profiled using BLASTN and the GreenGenes 16S RNA sequence database (108,453 sequences) (QIIME).
［実施例４．肝硬変患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で肝硬変患者９７人と年齢と性別をマッチングした正常ヒト１７１人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて肝硬変及び肝臓癌患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図３参照）。
[Example 4. Metagenome analysis of bacterial-derived vesicles in the blood of patients with cirrhosis]
Using the method of Example 3, blood from 97 liver cirrhosis patients and 171 age- and sex-matched normal humans was subjected to metagenomic analysis by extracting genes from vesicles present in the blood. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that the vesicles derived from the genus Corynebacterium were significantly reduced in the blood of patients with liver cirrhosis and liver cancer compared to the blood of normal humans (see FIG. 3).
［実施例５．脳卒中患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で脳卒中患者７９人と年齢と性別をマッチングした正常ヒト１５８人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて脳卒中患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図４参照）。
[Example 5. Metagenome analysis of bacterial-derived vesicles in the blood of stroke patients]
Targeting the blood of 79 stroke patients and 158 age- and sex-matched normal humans by the method of Example 3, genes were extracted from vesicles present in the blood and subjected to metagenomic analysis. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that vesicles derived from bacteria of the genus Corynebacterium were significantly reduced in the blood of stroke patients compared to the blood of normal humans (see FIG. 4).
［実施例６．糖尿病患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で糖尿病患者８１人と年齢と性別をマッチングした正常ヒト１２６人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて糖尿病患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図５参照）。
[Example 6. Metagenome analysis of bacterial-derived vesicles in the blood of diabetic patients]
Targeting the blood of 81 diabetic patients and 126 age- and sex-matched normal humans by the method of Example 3, genes were extracted from vesicles present in the blood and subjected to metagenomic analysis. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that vesicles derived from bacteria of the genus Corynebacterium were significantly reduced in the blood of diabetic patients compared to the blood of normal humans (see FIG. 5).
［実施例７．喘息患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で喘息患者１８２人と年齢と性別をマッチングした正常ヒト１８０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて喘息患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図６参照）。
[Example 7. Metagenome analysis of bacterial-derived vesicles in the blood of asthma patients]
The blood of 182 asthma patients and 180 age- and sex-matched normal humans were subjected to metagenomic analysis by extracting genes from vesicles present in the blood by the method of Example 3. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that the vesicles derived from the genus Corynebacterium were significantly reduced in the blood of asthma patients compared to the blood of normal humans (see FIG. 6).
［実施例８．アトピー性皮膚炎患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法でアトピー性皮膚炎患者４２人と年齢と性別をマッチングした正常ヒト４０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べてアトピー性皮膚炎患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図７参照）。
[Example 8. Metagenome analysis of bacterial-derived vesicles in the blood of patients with atopic dermatitis]
The blood of 42 atopic dermatitis patients and 40 age- and sex-matched normal humans was subjected to metagenomic analysis by extracting genes from vesicles present in the blood according to the method of Example 3. , evaluated the distribution of vesicles from the genus Corynebacterium. As a result, it was confirmed that vesicles derived from bacteria belonging to the genus Corynebacterium were significantly reduced in the blood of patients with atopic dermatitis compared to the blood of normal humans (see FIG. 7).
［実施例９．鬱病患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で鬱病患者７２人と年齢と性別をマッチングした正常ヒト８０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて鬱病患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図８参照）。
[Example 9. Metagenomic analysis of bacterial-derived vesicles in the blood of depressed patients]
Using the method of Example 3, the blood of 72 depressed patients and 80 age- and sex-matched normal humans were subjected to metagenomic analysis by extracting genes from vesicles present in the blood. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that the number of vesicles derived from bacteria of the genus Corynebacterium was significantly reduced in the blood of depressed patients compared to the blood of normal humans (see FIG. 8).
［実施例１０．乳癌患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で乳癌患者１０２人と年齢と性別をマッチングした正常ヒト１００人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて乳癌患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図９参照）。
[Example 10. Metagenome analysis of bacterial-derived vesicles in the blood of breast cancer patients]
Using the method of Example 3, blood from 102 breast cancer patients and 100 age- and sex-matched normal humans was subjected to metagenomic analysis by extracting genes from vesicles present in the blood. We evaluated the distribution of vesicles from the bacterium belonging to the genus Porporium. As a result, it was confirmed that vesicles derived from bacteria belonging to the genus Corynebacterium were significantly reduced in breast cancer patient blood compared to normal human blood (see FIG. 9).
［実施例１１．認知症患者の血液で細菌由来の小胞のメタゲノム分析］
前記実施例３の方法で認知症患者７３人と年齢と性別をマッチングした正常ヒト７０人の血液を対象として、血液内に存在する小胞から遺伝子を抽出してメタゲノム分析を行った後、コリネバクテリウム属細菌由来の小胞の分布を評価した。その結果、正常ヒトの血液に比べて認知症患者の血液でコリネバクテリウム属細菌由来の小胞が有意に減少していることを確認した（図１０参照）。
[Example 11. Metagenome analysis of bacterial-derived vesicles in the blood of dementia patients]
Targeting the blood of 73 dementia patients and 70 age- and sex-matched normal humans by the method of Example 3, genes were extracted from vesicles present in the blood and subjected to metagenomic analysis. The distribution of vesicles from the genus Bacterium was evaluated. As a result, it was confirmed that vesicles derived from bacteria belonging to the genus Corynebacterium were significantly reduced in the blood of dementia patients compared to the blood of normal humans (see FIG. 10).
［実施例１２．鼻ポリープ患者及び正常対照群の鼻粘膜のメタゲノム分析を通じたコリネバクテリウム属細菌由来の小胞の浸潤様相］
前記実施例３の方法で鼻ポリープ患者（非アレルギー性患者４３人及びアレルギー性患者４５人）及び正常対照群３９人の鼻粘膜で細菌由来の小胞の浸潤様相を評価するためにメタゲノム分析を行った。その結果、アレルギー性鼻ポリープ患者と非アレルギー性鼻ポリープ患者の鼻腔組織では、正常対照群に比べてコリネバクテリウム属細菌由来の小胞が有意に減少しており、鼻ポリープ患者でアレルギー性の有無とコリネバクテリウム属細菌由来の小胞の浸潤程度には差がなかった（図１１参照）。
[Example 12. Infiltration pattern of vesicles derived from Corynebacterium bacteria through metagenomic analysis of nasal mucosa of nasal polyp patients and normal controls]
Metagenomic analysis was performed to evaluate the infiltration pattern of bacterial-derived vesicles in the nasal mucosa of nasal polyp patients (43 non-allergic patients and 45 allergic patients) and 39 normal controls according to the method of Example 3 above. gone. As a result, vesicles derived from the genus Corynebacterium were significantly reduced in the nasal tissues of patients with allergic nasal polyps and non-allergic nasal polyps compared with normal controls. There was no difference between the presence or absence of Corynebacterium-derived vesicles and the degree of infiltration of vesicles (see FIG. 11).
［実施例１３．コリネバクテリウム・グルタミクム培養液から小胞分離］
コリネバクテリウム・グルタミクム（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ）菌株を培養した後、その小胞を分離して特性を分析した。まず、コリネバクテリウム・グルタミクム菌株を３７℃の培養器で吸光度（ＯＤ ６００）が１．０～１．５になるまでＭＲＳ（ｄｅ Ｍａｎ－Ｒｏｇｏｓａ ａｎｄ Ｓｈａｒｐｅ）培地で培養した後、ＬＢ（Ｌｕｒｉａ－Ｂｅｒｔａｎｉ）培地に継代培養（ｓｕｂ－ｃｕｌｔｕｒｅ）した。その後、菌株が含まれている培養液を回収して１０，０００×ｇ、４℃で２０分間遠心分離して菌体を除去し、０．２２μｍフィルタで濾過した。
[Example 13. Vesicle separation from Corynebacterium glutamicum culture solution]
After culturing the Corynebacterium glutamicum strain, the vesicles were isolated and characterized. First, the Corynebacterium glutamicum strain was cultured in MRS (de Man-Rogosa and Sharpe) medium in an incubator at 37° C. until the absorbance (OD 600) reached 1.0 to 1.5, and then LB (Luria- Bertani medium was sub-cultured. Then, the culture solution containing the strain was recovered, centrifuged at 10,000×g and 4° C. for 20 minutes to remove the cells, and filtered through a 0.22 μm filter.
前記濾過した上澄液を１００ｋＤａ Ｐｅｌｌｉｃｏｎ ２ Ｃａｓｓｅｔｔｅフィルタメンブレイン（Ｍｅｒｃｋ Ｍｉｌｌｉｐｏｒｅ、ＵＳ）でＭａｓｔｅｒＦｌｅｘ ポンプシステム（Ｃｏｌｅ－Ｐａｒｍｅｒ、ＵＳ）を用いて精密濾過（ｍｉｃｒｏｆｉｌｔｒａｔｉｏｎ）を通じて５０ｍｌ以下の体積で濃縮し、濃縮させた上澄液をもう一度０．２２μｍフィルタで濾過した。その後、ＢＣＡ（Ｂｉｃｉｎｃｈｏｎｉｎｉｃ ａｃｉｄ）ａｓｓａｙを用いてタンパク質を定量し、得られた小胞に対して下記実験を実施した。 The filtered supernatant was concentrated through microfiltration with a 100 kDa Pellicon 2 Cassette filter membrane (Merck Millipore, US) using a MasterFlex pump system (Cole-Parmer, US) in a volume of 50 ml or less and concentrated. The supernatant was filtered through a 0.22 μm filter again. After that, the protein was quantified using BCA (Bicinchoninic acid) assay, and the following experiments were performed on the obtained vesicles.
［実施例１４．コリネバクテリウム・グルタミクム由来の小胞の炎症誘発効果］
炎症細胞でコリネバクテリウム・グルタミクム由来の小胞（Ｃｏｒｙｎｅｂａｃｔｅｒｉｕｍ ｇｌｕｔａｍｉｃｕｍ ＥＶ）の炎症メディエーター（ＩＬ－６、ＴＮＦ－α）の分泌に対する影響を確認するために、マウスマクロファージ株であるＲａｗ ２６４．７細胞にコリネバクテリウム・グルタミクム由来の小胞を多様な濃度（０．１、１、１０μｇ／ｍｌ）で処理した後、細胞死滅とＥＬＩＳＡを進行した。
[Example 14. Inflammatory effects of vesicles derived from Corynebacterium glutamicum]
In order to confirm the effect of Corynebacterium glutamicum-derived vesicles (Corynebacterium glutamicum EV) on the secretion of inflammatory mediators (IL-6, TNF-α) in inflammatory cells, Raw 264.7 cells, a mouse macrophage strain After treating Corynebacterium glutamicum-derived vesicles at various concentrations (0.1, 1, 10 μg/ml), cell killing and ELISA were performed.
より具体的に、４８－ｗｅｌｌ細胞培養プレート中に５×１０４個ずつ分注したＲａｗ２６４．７細胞にＤＭＥＭ（Ｄｕｌｂｅｃｃｏ’ｓ Ｍｉｎｉｍｕｍ Ｅｓｓｅｎｔｉａｌ Ｍｅｄｉｕｍ）無血清培地で希釈した多様な濃度のコリネバクテリウム・グルタミクム由来の小胞を処理して１２時間の間培養した。その後、細胞培養液を１．５ｍｌチューブに集めて３０００×ｇで５分間遠心分離し、上澄液を集めて－８０℃で保管した後、ＥＬＩＳＡを進めた。 More specifically, 5×10 4 cells were dispensed into Raw 264.7 cells in a 48-well cell culture plate, and various concentrations of Corynebacterium were diluted with DMEM (Dulbecco's Minimum Essential Medium) serum-free medium. Glutamicum-derived vesicles were treated and cultured for 12 hours. Then, the cell culture was collected in a 1.5 ml tube, centrifuged at 3000×g for 5 minutes, and the supernatant was collected and stored at −80° C. before proceeding with ELISA.
ＥＬＩＳＡを行うために、捕捉抗体をＰＢＳに希釈させて９６ ｗｅｌｌのポリスチレンプレートに作用濃度に合わせて５０μｌずつ分注した後、４℃で一晩反応させた。その後、ＰＢＳＴ（０．０５％ ｔｗｅｅｎ－２０が入っているＰＢＳ）溶液１００μｌで３回ずつ洗った後、ＲＤ（１％ ＢＳＡが入っているＰＢＳ）溶液１００μｌを分注して常温で１時間の間ブロッキングした。サンプル及びスタンダードを濃度に合わせて５０μｌずつ分注して常温で２時間の間反応させた後、ＰＢＳＴ １００μｌで３回洗った後、検出抗体をＲＤに希釈させて作用濃度に合わせて５０μｌずつ分注して常温で２時間の間反応させた。ＰＢＳＴ １００μｌで３回洗った後、Ｓｔｒｅｐｔａｖｉｄｉｎ－ＨＲＰ（Ｒ＆Ｄ ｓｙｓｔｅｍ、ＵＳＡ）をＲＤに１／４０で希釈させて５０μｌずつ分注して常温で２０分間反応させた。最後に、ＰＢＳＴ １００μｌで３回洗った後、ＴＭＢ（３，３’，５，５’－Ｔｅｔｒａｍｅｔｈｙｌｂｅｎｚｉｄｉｎｅ）基質（ＳｕｒＭｏｄｉｃｓ、ＵＳＡ）５０μｌを分注して５分～２０分後に発色が進行されたとき、１Ｍ硫酸溶液を５０μｌずつ分注して反応を止め、ＳｐｅｃｔｒａＭａｘ Ｍ３ ｍｉｃｒｏｐｌａｔｅ ｒｅａｄｅｒ（Ｍｏｌｅｃｕｌａｒ Ｄｅｖｉｃｅｓ、ＵＳＡ）を用いて４５０ｎｍで吸光度を測定した。
For ELISA, the capture antibody was diluted in PBS, dispensed into a 96-well polystyrene plate at 50 μl each according to the working concentration, and allowed to react at 4° C. overnight. Then, after washing three times with 100 μl of PBST (PBS containing 0.05% tween-20) solution, 100 μl of RD (PBS containing 1% BSA) solution was dispensed and incubated at room temperature for 1 hour. blocked for a while. Samples and standards were dispensed in 50 μl aliquots according to concentration, reacted at room temperature for 2 hours, washed 3 times with
その結果、図１２に示したように、コリネバクテリウム・グルタミクム由来の小胞（ＣＧＴ１０１）の処理による細胞死滅は観察されなかった（図１２参照）。また、図１３ａ及び図１３ｂに示したように、炎症細胞での炎症メディエーターの分泌様相を評価した結果、陽性対照群である大腸菌由来の小胞（Ｅ．ｃｏｌｉ ＥＶ １μｇ／ｍｌ）の処理時に比べてコリネバクテリウム・グルタミクム由来の小胞（ＣＧＴ１０１）の処理時、ＩＬ－６（図１３ａ）及びＴＮＦ－α（図１３ｂ）の分泌がより一層減少していることを確認した。
As a result, as shown in FIG. 12, no cell death was observed due to treatment with Corynebacterium glutamicum-derived vesicles (CGT101) (see FIG. 12). In addition, as shown in FIGS. 13a and 13b, the secretion of inflammatory mediators in inflammatory cells was evaluated, and the results showed that the positive control group, E. coli-derived vesicles (
［実施例１５．コリネバクテリウム・グルタミクム由来の小胞の抗炎症効果］
前記実施例１４の結果を土台に、コリネバクテリウム・グルタミクム由来の小胞の抗炎症効果を評価するために、多様な濃度（０．１、１、１０μｇ／ｍｌ）のコリネバクテリウム・グルタミクム由来の小胞（ＣＧＴ１０１）をマウスマクロファージ株に１２時間前処理した後、病原性原因因子である大腸菌由来の小胞１μｇ／ｍｌを処理し、１２時間後に炎症性サイトカインの分泌をＥＬＩＳＡで測定した。
[Example 15. Anti-inflammatory effect of vesicles derived from Corynebacterium glutamicum]
Based on the results of Example 14, in order to evaluate the anti-inflammatory effect of Corynebacterium glutamicum-derived vesicles, various concentrations (0.1, 1, 10 µg/ml) of Corynebacterium glutamicum-derived vesicles (CGT101) were pretreated in a mouse macrophage strain for 12 hours, followed by treatment with 1 μg/ml of vesicles derived from Escherichia coli, a virulence causative factor, and 12 hours later, inflammatory cytokine secretion was measured by ELISA.
その結果、コリネバクテリウム・グルタミクム由来の小胞を前処理するとき、大腸菌由来の小胞の刺激による炎症細胞に分泌されるＴＮＦ－αの量が顕著に抑制されることを確認した（図１４参照）。これは、病原性小胞である大腸菌由来の小胞のような炎症誘発因子により誘導される炎症反応をコリネバクテリウム・グルタミクム由来の小胞が効率的に抑制できることを意味する。 As a result, it was confirmed that pretreatment of Corynebacterium glutamicum-derived vesicles markedly suppressed the amount of TNF-α secreted by inflammatory cells stimulated by E. coli-derived vesicles (FIG. 14). reference). This means that Corynebacterium glutamicum-derived vesicles can efficiently suppress inflammatory reactions induced by proinflammatory factors such as pathogenic vesicles, Escherichia coli-derived vesicles.
上述した本発明の説明は例示のためのもので、本発明が属する技術分野において通常の知識を有した者は、本発明の技術的思想や必須的な特徴を変更しなくても他の具体的な形態に容易に変形が可能であることが理解できる。したがって、以上で記述した実施例は、全ての面で例示的なものであり、限定的ではないことで理解すべきである。 The above description of the present invention is for illustrative purposes only, and those skilled in the art to which the present invention pertains will be able to implement other specific embodiments without changing the technical spirit or essential features of the present invention. It can be understood that it can be easily transformed into a typical form. Accordingly, the embodiments set forth above are to be considered in all respects as illustrative and not restrictive.
本発明によるコリネバクテリウム属細菌由来の小胞は、粘膜の防御膜を通過して粘膜の上皮細胞に吸収されて全身的に分布し、腎臓、肝臓、肺を通じて体外に排泄されることが確認され、肝硬変、脳卒中、糖尿病、喘息、アトピー性皮膚炎、鬱病、乳癌、認知症及び鼻ポリープ患者の血液又は鼻粘膜で有意に減少していることが確認され、病原性小胞によるＴＮＦ－αなどの炎症メディエーターの分泌を有意に抑制できるところ、炎症性疾患の予防又は治療用組成物に有用に用いられ得るという点で、産業的利用価値が大きいと予想される。 It was confirmed that the Corynebacterium-derived vesicles according to the present invention pass through the protective membrane of the mucosa, are absorbed by the epithelial cells of the mucosa, are distributed systemically, and are excreted to the outside of the body through the kidney, liver, and lungs. was confirmed to be significantly decreased in the blood or nasal mucosa of patients with cirrhosis, stroke, diabetes, asthma, atopic dermatitis, depression, breast cancer, dementia and nasal polyps, and TNF-α by pathogenic vesicles Since the secretion of inflammatory mediators such as inflammatory mediators can be significantly suppressed, it is expected to have great industrial utility value in that it can be usefully used in compositions for the prevention or treatment of inflammatory diseases.
Claims (9)
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| KR20180158622 | 2018-12-10 | ||
| KR10-2018-0158622 | 2018-12-10 | ||
| KR10-2019-0132137 | 2019-10-23 | ||
| KR1020190132137A KR102223406B1 (en) | 2018-12-10 | 2019-10-23 | Nanovesicles derived from Corynebacterium bacteria and Use thereof |
| PCT/KR2019/015901 WO2020122449A1 (en) | 2018-12-10 | 2019-11-20 | Nano-vesicle derived from corynebacterium sp. bacteria and use thereof |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| JP2022512469A JP2022512469A (en) | 2022-02-04 |
| JP2022512469A5 JP2022512469A5 (en) | 2022-04-20 |
| JP7186470B2 true JP7186470B2 (en) | 2022-12-09 |
Family
ID=71075621
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2021532852A Active JP7186470B2 (en) | 2018-12-10 | 2019-11-20 | Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof |
Country Status (2)
| Country | Link |
|---|---|
| JP (1) | JP7186470B2 (en) |
| WO (1) | WO2020122449A1 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013503858A (en) | 2009-09-04 | 2013-02-04 | イオン メディックス インコーポレイテッド | Gram-positive bacteria-derived extracellular vesicles and uses thereof |
| JP2013507354A (en) | 2009-10-08 | 2013-03-04 | イオン メディックス インコーポレイテッド | Composition comprising indoor air-derived extracellular vesicle and use thereof |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011027971A2 (en) * | 2009-09-01 | 2011-03-10 | 주식회사이언메딕스 | Gut flora-derived extracellular vesicles, and method for searching for a disease model, vaccine, and candidate drug and for diagnosis using same |
| KR101629525B1 (en) * | 2010-01-11 | 2016-06-13 | 이화여자대학교 산학협력단 | Composition comprising extracellular vesicles derived from fermented food, and use thereof |
| KR101940426B1 (en) * | 2016-12-28 | 2019-01-18 | 주식회사 엠디헬스케어 | Method for diagnosis of colon tumor using analysis of bacteria metagenome |
| KR101944665B1 (en) * | 2017-02-24 | 2019-02-01 | 주식회사 엠디헬스케어 | Method for diagnosis of chronic obstructive airway disease using analysis of bacteria metagenome |
| KR102597602B1 (en) | 2018-05-18 | 2023-11-02 | 삼성전자주식회사 | thin film formation apparatus and thin film formation apparatus using the same |
-
2019
- 2019-11-20 JP JP2021532852A patent/JP7186470B2/en active Active
- 2019-11-20 WO PCT/KR2019/015901 patent/WO2020122449A1/en not_active Ceased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013503858A (en) | 2009-09-04 | 2013-02-04 | イオン メディックス インコーポレイテッド | Gram-positive bacteria-derived extracellular vesicles and uses thereof |
| JP2013507354A (en) | 2009-10-08 | 2013-03-04 | イオン メディックス インコーポレイテッド | Composition comprising indoor air-derived extracellular vesicle and use thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2022512469A (en) | 2022-02-04 |
| WO2020122449A1 (en) | 2020-06-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP7300762B2 (en) | Nanovesicles derived from bacteria belonging to the genus Lactobacillus and uses thereof | |
| JP7132661B2 (en) | Nanovesicles derived from bacteria of the genus Sphingomonas and uses thereof | |
| JP7433665B2 (en) | Nanovesicles derived from Micrococcus bacteria and their uses | |
| JP2019528688A (en) | Nanovesicles derived from Bacillus bacteria and uses thereof | |
| JP7186470B2 (en) | Nanovesicles derived from bacteria of the genus Corynebacterium and uses thereof | |
| JP7291424B2 (en) | Deinococcus-derived nanovesicles and uses thereof | |
| JP7240031B2 (en) | Nanovesicles derived from Weissella bacteria and uses thereof | |
| KR102223406B1 (en) | Nanovesicles derived from Corynebacterium bacteria and Use thereof | |
| EP3910074B1 (en) | Nanovesicles derived from bacteria of genus deinococcus, and use thereof | |
| KR102124794B1 (en) | Nanovesicles derived from Geobacillus bacteria and Use thereof | |
| CN113286897A (en) | Nanocysomes derived from Rhodococcus bacteria and uses thereof | |
| HK40042844A (en) | Nanovesicles derived from bacteria of genus sphingomonas and uses of same |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20210708 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210708 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20220412 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220712 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20221011 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20221108 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20221121 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7186470 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |