JP3936704B2 - Method for detecting human β-defensin 2 - Google Patents

Description

  The present invention relates to a factor that induces and / or enhances the production of an antimicrobial peptide contained in a food material, and a method for detecting human β-defensin 2.

Living organisms that do not have an immune mechanism create antibacterial substances as a defense mechanism against the body and protect themselves from infection by microorganisms such as bacteria, but vertebrates that have an immune mechanism are also called antimicrobial (antimicrobial activity) peptides. It is known to make substances. Although the size and structure of these antimicrobial peptides shows considerable diversity, in general they are membrane active amphiphilic molecules with a net positive charge at neutral pH.
These cationic peptides include two broadly divided families: linear peptides (eg, cecropins) and cysteine-rich peptides. The latter include mammalian defensins, tracheal antimicrobial peptides and bovine bactenines (bovine
bactenecines) and the like. There is a substance called defensin as a human antibacterial peptide, and it is constantly or transiently expressed in various tissues in the body in addition to neutrophils and Paneth cells of the small intestinal mucosa. It is known.
Defensins are the main constituents of neutrophil azurophilic granules. So far, four types of defensins called HNP-1, -2, -3 and -4 have been isolated and their structures have been elucidated. Such a defensin is a basic peptide consisting of 29 to 34 amino acids, and a gene family (hBD-1) in which 6 cysteines and 1 glycine are conserved in common
Is configured.
The existence of a defensin family gene expressed in human Paneth cells was also proved. These digestive defensins are called human defensins 5 and 6, and their structures were also determined (Japanese Patent Publication No. 7-507213).
From the above, it is suggested that human defensin contributes to maintenance of normal cell flora and protection from foreign bacteria.

Recently, new antimicrobial peptides have been discovered in human epidermal keratinocytes. This antimicrobial peptide was named human β-defensin 2 (hBD-2) because it has a common sequence found in the defensin gene family and has high homology with them. Furthermore, it was found that the level of transcription of hBD-2 is changed by bacterial infection. This hBD-2 was also found to be expressed in the lungs and trachea (J. Harder et al., Nature)
387, 861 (1997). However, its regulatory mechanism is still unclear.
Special table flat 7-507213 Jay Harder et al., Nature, 387, 861, 1997

  Therefore, if a factor capable of inducing and / or enhancing the production of such an antibacterial peptide can be found, for example, in food ingredients, the biological defense mechanism can be strengthened by adding such a factor. It becomes possible to develop a drug effective for treating a disorder such as a food or digestive system.

  In order to solve the above-mentioned problems, the present inventor uses human keratinocytes and human nasal mucosal epithelial cells and adds dead bacteria or food material extracts to detect changes in the expression level of antibacterial peptides by RT-PCR method. The present inventors have succeeded in constructing an experimental system that detects factors that induce and / or enhance the production of antibacterial peptides, and found components that can induce and / or enhance the production of antibacterial peptides. Completed.

That is, the present invention relates to a food extract containing a factor that induces expression of a human antibacterial peptide and a food extract containing a factor that enhances the expression of a human antibacterial peptide.
As human antimicrobial peptides, various conventionally known substances can be targeted. A suitable example is human β-defensin 2 (hBD-2).
The food extract of the present invention can be prepared by subjecting various food materials to any extraction operation known to those skilled in the art. For example, extraction with water or hot water containing inorganic acid, organic acid, salt, saccharide, etc., extraction with alcohol, extraction with liquefied carbon dioxide gas, etc. can be mentioned. The food composition can take any form such as solid, liquid, sol, gel, powder, granule and the like.

  As used herein, the term “antimicrobial” or “antimicrobial activity” refers to the ability of a compound to inhibit or irreversibly block microbial growth. This type of inhibition or prevention may be via microbicidal action or microbial stasis inhibition. As used herein, the term “microbicidal action” refers to the ability of a compound to kill or irreversibly damage a target microorganism. The term “inhibition of microbial stasis” does not lead to the death of microorganisms, but means to suppress their growth and growth.

Therefore, the present invention also relates to a food composition characterized by blending the above-mentioned factor or a food extract containing the factor.
Ingestion of the food composition of the present invention is expected to induce or enhance the expression of antimicrobial peptides such as human defensin in the digestive system and prevent or eliminate potential microbial pathogens. The food composition can take any form such as solid, liquid, sol, gel, powder, and granule. The manufacturing method can be any method known in the art.
The content of the above-mentioned factor or food extract in the food composition of the present invention can be appropriately selected by those skilled in the art according to the blending purpose and the type / form of the food composition.

Furthermore, the present invention also relates to a pharmaceutical composition comprising the above factor or a food extract containing the factor. The pharmaceutical composition of the present invention can be used for the prevention and treatment of microbial infections or digestive system inflammation.
The pharmaceutical composition of the present invention may contain an appropriate pharmaceutically acceptable carrier known to those skilled in the art, in addition to the above-mentioned factors effective for the prevention and treatment or a food extract containing the factors. it can.
The pharmaceutical composition of the present invention is effective for infections caused by a wide variety of microorganisms such as fungi and bacteria (both gram positive and negative) or inflammation of the digestive system.
The amount of the above-mentioned factor or food extract incorporated in the pharmaceutical composition is appropriately selected by those skilled in the art according to the properties of other ingredients, the purpose of use, the age and weight of the patient, the degree of required effect, etc. be able to.

The pharmaceutical composition of the present invention can take any form known in the art, for example, liquid formulations such as solutions, suspensions, emulsions and the like buffered with various salts and buffers. can do. The salts can be mostly alkali and alkaline earth halides, phosphates and sulfates such as sodium chloride, potassium chloride or sodium sulfate. Various buffers such as citrate, phosphate, HEPES, Tris, etc. can be used to the extent that is physiologically acceptable to the subject to which this type of buffer is treated.
In addition, when the pharmaceutical composition of the present invention is a tablet, granule, powder, gel, sol or the like, various excipients or other additives known to those skilled in the art can be used. . Furthermore, the above-mentioned factor or food extract and the above-mentioned various additives can be made into a pharmaceutical form encapsulated with pharmaceutically acceptable liposomes.
The pharmaceutical composition of the present invention can be administered in various ways depending on the form of the pharmaceutical composition and the properties of the administration target. For example, it can be administered orally, intravenously, subcutaneously, intramuscularly, intraperitoneally, nasopharynx and the like.

The present invention further relates to a method for detecting human antimicrobial peptide and / or enhancing factor. In such a detection method, various cultured cells capable of expressing a human antimicrobial peptide, for example, human epidermal keratinocytes and human nasal mucosal epithelial cells are used. Such cells are cultured in the presence of samples such as extracts from various food materials that are believed to contain human antimicrobial peptide induction and / or enhancement factors.
As a detection method, various methods known to those skilled in the art, for example, quantitative RT-PCR method, reporter gene assay, antibacterial test, immunoassay method and the like can be used. Measurement conditions and the like in each detection method can be experimentally selected as appropriate by those skilled in the art.

That is, the present invention uses an hBD-2 f primer (5′-CCAGCCATCAGCCATGAGGGT-3 ′) and an hBD-2 r primer (5′-GGAGCCCTTTCTGAATCCGCA-3 ′), which is a human by RT-PCR method. The method for detecting β-defensin 2 mRNA is applied.
Furthermore, the present invention uses qRThBD2f primer (5'-cttccaggtgtttttggtggta-3 ') and qRThBD2r primer (5'-ggagccatatgtcatccagtc-3'), and quantifies human β-defensin 2 mRNA by real-time RT-PCR. In particular, this method uses hBD2 probe (5′-aggcgatcctgttacctgccttaagag-3 ′) as a TaqMan / TaqMan® probe.
The present invention also provides a method for detecting the effect of inducing and / or enhancing the expression of human β-defensin 2 in a food material extract by measuring the expression level of human β-defensin 2 mRNA using the detection method described above. Also related.

According to the present invention, an antibacterial peptide induction and / or enhancement factor, or a food material (food extract) containing the factor can be detected, and a food composition or a pharmaceutical composition containing the factor or food extract The product is effective in strengthening the defense of the body and treating various diseases.
Further, according to the present invention, substances that have been conventionally treated as industrial waste and caused environmental pollution such as shochu moromi can be reused as useful materials.

  EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.

Example 1: Determination of minimum induction concentration of human β-defensin 2 A human epidermis keratinocyte (Nissui Pharmaceutical Co., Ltd.) cultured to a confluence in a 10φ dish was subjected to autoclave-sterilized PBS suspension of E. coli. The final concentrations were 0, 0.2, 1, 5, and 25 ng / ml, and the cells were cultured at 37 ° C. in a 5% CO ₂ atmosphere. After about 16 hours, the medium was removed and washed with PBS, and the cells were collected with a scraper. Total RNA was extracted from the collected cells using RNeasy (Qiagen). The concentration of each total RNA solution was adjusted to 1 μg / μl. The target mRNA was amplified by RT-PCR using each extracted RNA as a template.

The RT-PCR conditions were as follows. First, based on the cDNA sequence of human β-defensin 2 (Accession No. Z71389), hBD-2 f primer (5′-CCAGCCATCAGCCATGAGGGT-3 ′) and hBD-2
r Primer (5'-GGAGCCCTTTCTGAATCCGCA-3 ') was prepared. GAPDH (glyceraldehyde-3-phosphate dehydrogenase)
GAPDH f primer (5'-ACCACAGTCCATGCCATCAC-3 ') and GAPDH with reference to the cDNA sequence (Accession No. M33197)
r Primer (5'-TCCACCACCCTGTTGCTGTA-3 ') was prepared and used as an internal standard.
The RT-PCR reaction solution was prepared using a OneStep RNA PCR Kit (Takara Shuzo Co., Ltd.) and a master mix was prepared so as to have the composition shown in Table 1 below per tube. The mixture was mixed so as to have the composition shown in 2, and the reaction was carried out in a system of 25 μl.

RT-PCR was performed for 25 cycles under conditions of reverse transcription reaction at 50 ° C. for 30 minutes, 94 ° C. for 2 minutes, and PCR reaction at 94 ° C. for 30 seconds and 68 ° C. for 30 seconds.
After the reaction, 10 μl of the reaction solution was analyzed by 2% agarose gel electrophoresis. As shown in Table 3, it was found that β defensin 2 mRNA can be detected in a concentration-dependent manner by adding the E. coli solution.
When the final concentration of E. coli was 1 ng / ml or more, β defensin 2 mRNA was observed (Table 3). From this, the defensin minimum induction concentration was determined to be 1 ng / ml. As the limit detection sensitivity, the amount of total RNA was 1 μg, and the number of amplification cycles was 25.

Example 2: Preparation of food material extract 1 g of each food material was suspended in purified water to 10 ml, and then autoclaved. These were centrifuged, and the supernatant was used as a food material extract (food extract).

Example 3: Induction of human β-defensin 2 Using the experimental system of Example 1, each food material extract prepared in Example 2 was added to a final concentration of 0.1% instead of the E. coli solution. As a result, the same effect of inducing β-defensin 2 mRNA as in the case of E. coli was observed when the miso miso concentrate and shochu mash were added.

Example 4: Enhancement of human β-defensin 2 The food material extract that did not show the induction effect in Example 3 was further added to the experimental system of Example 1, and the expression of β-defensin 2 mRNA was examined in the presence of an E. coli solution. As a result, enhanced expression was observed for roast maranth, roasted wheat germ, concentrated yogurt extract for haichu, and amazake compared to the case of E. coli solution alone.
Table 4 shows the results obtained in Examples 3 and 4 above.

As is clear from the above results, it was found that factors having an antibacterial peptide induction and / or enhancement effect exist in various food materials.
These inducers have the function of regulating the expression of antimicrobial peptides by themselves, whereas the enhancer has the function of making the signal stronger after the antimicrobial peptide expression is switched on by infection with E. coli. Conceivable.
The effect of inducing and / or enhancing the antibacterial peptide of the factors in various food materials thus confirmed was further quantitatively measured in the following examples.

Example 5: Setting of expression induction conditions for human β-defensin 2 Human epidermal keratinocytes (manufactured by Nissui Pharmaceutical Co., Ltd.) cultured to confluence on a 6-well multiplate (manufactured by Falcon) were used for autoclaved E. coli. The PBS suspension was added so that the final concentration of E. coli was 0, 0.07, 0.7, 7, 70, 700 ug / ml, and cultured at 37 ° C. in a 5% CO ₂ atmosphere. After about 16 hours, the medium was removed and washed with PBS. Total RNA was extracted from the collected cells using RNeasy (Qiagen). Using each extracted RNA as a template, the target mRNA was amplified by quantitative RT-PCR.
The conditions for quantitative RT-PCR are as follows. First, based on the cDNA sequence of human β-defensin 2 (Accession No. Z71389), qRThBD2f primer (5'-cttccaggtgtttttggtggta-3 '), qRThBD2r primer (5'-ggagccatatgtcatccagtc-3' ) And hBD2 as ABI PRISM TaqMan ^TM probe
A probe (5'-aggcgatcctgttacctgccttaagag-3 ') was produced.
GAPDH (glyceraldehyde-3-phosphate
dehydrogenase) mRNA was measured simultaneously using TaqMan ^™ GAPDH Control Regents (manufactured by PerkinElmer Japan Co., Ltd.), and used as an internal standard for the amount of mRNA in the template.
A RT-PCR reaction solution was prepared using a TaqMan EZ RT-PCR Kit (manufactured by PerkinElmer Japan Co., Ltd.), and a master mix was prepared so as to have the composition shown in Table 5 below per tube. Furthermore, each total RNA was mixed so as to have the composition shown in Table 6, and an amplification reaction was performed in a system of 25 μl × 3 per sample.
Real-time detection was performed using PRISM 7700 sequence detector (manufactured by PerkinElmer Japan Co., Ltd.).
RT-PCR performs reverse transcription at 60 ° C
The PCR reaction was performed for 40 to 45 cycles at 94 ° C. for 20 sec and 60 ° C. for 1 min at 30 min, 95 ° C. for 5 min. In addition, the included analysis software (Sequence
The threshold was set on Detector v1.6.3) and the expression level was calculated. The relative expression level was determined with the sample to which only PBS was added as 100%.
As shown in FIG. 1, it was found that β defensin 2 mRNA can be detected in a concentration-dependent manner by adding an E. coli solution.
When the final E. coli concentration was 7 ug / ml or more, an increase in defensin mRNA expression was observed. (FIG. 1) Therefore, the defensin induction concentration was set to 7 ug / ml.

Example 6: Induction and Enhancement of Human β-Defensin 2 by Food Using the experimental system of Example 5, each food material extract prepared in Example 2 was added to a final concentration of 0.1%. A comparison was made between the case of adding only the food and the case of adding the E. coli solution at the same time. As a result, β-defensin 2 is the same as in E. coli when shochu moromi and amazake are added.
An inducing effect of mRNA was observed. In the state of coexisting with the Escherichia coli solution, the expression-enhancing effect of shochu moromi, roast maranth, and amazake was observed more than that of the Escherichia coli solution alone or the food extract alone. The obtained results are shown in FIG.

The concentration-dependent expression of β-defensin 2 mRNA by addition of an E. coli solution is shown. The inducing effect of β-defensin 2 mRNA expression and the induction / enhancement effect by adding each food material extract are shown.

Claims (2)

qRThBD2f primer (5'-cttccaggtgtttttggtggta-3 '), qRThBD2r primer (5'-ggagccatatgtcatccagtc-3'), and hBD2 probe (5'-aggcgatcctgttacctgccttaagag-3 ') used as Tackman / TaqMan (registered trademark) probe A method for quantitative detection of human β-defensin 2 mRNA by real-time RT-PCR. A method for detecting an effect of inducing and / or enhancing expression of human β-defensin 2 in a food material extract by measuring the expression level of human β-defensin 2 mRNA using the detection method according to claim 1.

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