JP6004415B2 - Peptide and self-assembly method thereof, aggregate thereof, cell culture substrate using these, and method for producing cell sheet - Google Patents

Description

  The present invention relates to a peptide, a self-assembly method thereof, an assembly thereof, a cell culture substrate using these, and a method for producing a cell sheet.

  Elastin is known as a protein constituting animal tissues including the human body. This elastin is a fibrous protein that functions extracellularly like collagen, and is involved in imparting flexibility to tissues because it has the property of stretching (elasticity) like rubber. . For this reason, it is widely distributed in tissues and organs (for example, the dermis of skin, ligaments, tendons, lungs, blood vessel walls, etc.) that require stretchability.

  In vivo, this elastin is first biosynthesized in vascular smooth muscle cells and fibroblasts as a precursor protein, tropoelastin (molecular weight of about 70,000). Tropoelastin then self-assembles around and between the glycoproteins called microfibrils, and is then appropriately crosslinked between the molecules to become insoluble elastin. The regular self-assembly of tropoelastin, which is the first step, is important for the formation of normal elastin in vivo, and this self-assembly phenomenon is called “coacervation”. In other words, coacervation is deeply involved in normal elastin formation.

  The elastin coacervation properties can be observed in vitro (FIG. 1). That is, an aqueous solution of tropoelastin or an elastin-derived peptide is a transparent and uniform solution at a low temperature (25 ° C. or lower). However, when the temperature is raised above the body temperature (37 ° C.), the molecules self-assemble and as a result the solution becomes cloudy. When the cloudy solution is cooled again, it returns to a transparent solution, but if left uncooled, the solution system separates into two layers. Specifically, it is separated into two layers, an equilibrium solution (upper layer) containing only a few elastin molecules and a viscous coacervate layer (lower layer) formed by concentrating the molecules. This layer separation process is also reversible. When the temperature is lowered to 25 ° C. or lower, the original transparent uniform solution is obtained again. This reversible self-assembly / dissociation characteristic is “coacervation”, which is an important characteristic for normal elastin fibril formation and the development of elastic function.

The primary structural feature of tropoelastin, the precursor of elastin, is that a hydrophobic region containing a large amount of hydrophobic amino acids and a cross-linked region related to cross-linking between molecules are alternately repeated. Various hydrophobic amino acid repeating sequences exist in the hydrophobic region, and one of the repeating sequences, Val-Pro-Gly-Val-Gly (hereinafter referred to as “VPGVG”); The pentapeptide sequence consisting of the amino acid sequence written from left to right (hereinafter the same) from the N-terminal side to the C-terminal side is almost all animal species that have been reported so far. Exists. In addition, since the synthetic polypeptide (VPGVG) _n (n ≧ 40) having this repeating sequence exhibits coacervation characteristics, it is suggested that the VPGVG repeating sequence is a sequence responsible for the elastic function of elastin.

  Thus, a polypeptide having a repetitive amino acid sequence of VPGVG is highly useful as a base material such as a biomaterial or a drug delivery system (DDS) material because of its self-assembly ability. However, in order to industrially use the polypeptide, it is necessary to synthesize a high molecular weight peptide, which takes time and cost. For this reason, there has been little industrial use so far. In addition, as a report from overseas, the use of a genetically synthesized polymer having a repetitive sequence of Ile-Pro-Gly-Val-Gly (hereinafter sometimes abbreviated as “IPGVG”) as a biomaterial is Dan. Proposed by Professor W. Urry (see Non-Patent Document 1). However, there is no report so far that peptides having other repetitive amino acid sequences are industrially advantageous.

  In addition, as a novel cell culture substrate used for culturing various cells, it is widely used to use a polymer material such as poly N-isopropylacrylamide (PNIPAAm). However, the Ministry of Health, Labor and Welfare has pointed out that trace amounts of unpolymerized acrylamide monomer may remain in the polymer during the production process of the culture substrate, and the safety of the material when applied to a living body is regarded as a problem. ing. On the other hand, it has not been conventionally known to use the above-mentioned tropoelastin or elastin-derived peptide alone as a cell culture substrate.

J. Phys. Chem. B, 101, 11007-11028 (1997)

According to the study of the present inventor, conventionally reported synthetic polypeptides (polypentapeptide; (VPGVG) _n and (IPGVG) _n ) are reduced in molecular weight while ensuring coacervation (self-assembly) ability. It was confirmed that it was necessary to increase the molecular weight to some extent in order to exhibit coacervation (self-assembly) ability for these polypeptides.

Specifically, in the synthetic polypeptide (VPGVG) _n , a polymer having a polymerization degree n of 40 or more shows coacervation (self-assembly) ability, but a monomer (VPGVG) having a polymerization degree n of 1 is coacervation. In addition, a slight increase in turbidity was first observed at n = 10 ((VPGVG) ₁₀ ). That is, it was shown that (GVGPV) _n having a naturally occurring amino acid sequence requires n ≧ 10 in order to exhibit coacervation characteristics. However, this (VPGVG) _n oligopeptide had a low turbidity strength compared to a polymer of n ≧ 40, and did not show sufficient coacervation ability.

On the other hand, in the synthetic polypeptide (IPGVG) _n in which valine, which is the terminal amino acid of the repetitive sequence, is substituted with isoleucine (Ile) having higher hydrophobicity, the coacervation (self-assembly) ability is shown when n ≧ 7 Was also confirmed.

  Thus, in a conventionally known synthetic polypeptide, the increase in the turbidity of the solution or coacervation is not achieved until the repeat number (polymerization degree) of 5 amino acid repeats is as large as 10 or 7 or more. Only the expression of (self-assembly) ability is confirmed. In other words, the present inventors have found that there is still room for improvement from the viewpoint of achieving both low molecular weight and self-assembly ability.

  Therefore, an object of the present invention is to provide means capable of expressing coacervation (self-assembly) ability in peptides even at a lower molecular weight.

In view of the current state of the prior art as described above, the present inventors have intensively studied. In the process, surprisingly, in the conventionally reported synthetic polypeptides (polypentapeptides; (VPGVG) _n and (IPGVG) _n ), the N-terminal amino acid (valine, isoleucine) is replaced with phenylalanine, whereby The present inventors have found that the problem can be solved and have completed the present invention.

That is, according to one aspect of the present invention, the following peptide (a) or (b) is provided:
(A) The following chemical formula 1:

In the formula, n is an integer of 4 to 300.
(Hereinafter also referred to as “(FPGVG) _n ”);
(B) A peptide obtained by deleting, substituting, and / or adding one or several amino acids in the peptide of (a), and exhibiting temperature-dependent self-assembly.

  Moreover, in the preferable form of the said form, n is an integer of 4-10, In a more preferable form, a peptide is represented by the amino acid sequence in any one of sequence number 2-sequence number 6.

  Furthermore, according to another aspect of the present invention, there is provided a method for self-assembling a peptide, comprising a step of heating an aqueous solution in which the peptide is dissolved.

  According to still another aspect of the present invention, there is provided a peptide aggregate formed by self-assembling the above peptides.

  Furthermore, according to the further another form of this invention, the manufacturing method of the cell culture | cultivation base material containing the said peptide or the said peptide assembly, and a cell sheet using the same is also provided.

  According to the present invention, it is possible to provide a means that allows a peptide to exhibit coacervation (self-assembly) ability even at a lower molecular weight.

It is explanatory drawing for demonstrating the coacervation characteristic of elastin. It is explanatory drawing for demonstrating the mode of the chemical synthesis of the peptide by a solid-phase method in an Example. It is explanatory drawing for demonstrating the mode of the chemical synthesis of the peptide by a solid-phase method in an Example. It is a graph which shows the result of having evaluated the coacervation characteristic of 2 types of peptides synthesize | combined in the Example. 4A shows the results for (FPGVG) ₄ and FIG. 4B shows the results for (FPGVG) ₅ . In an Example, it is a figure which shows the experimental flow at the time of examining preparation of the cell sheet using (FPGVG) ₅ , and the observation result after cell collection | recovery.

  Hereinafter, specific embodiments for carrying out the present invention will be described in detail, but the technical scope of the present invention is not limited to the following specific embodiments.

One embodiment of the present invention is the following peptide (a) or (b):
(A) The following chemical formula 1:

In the formula, n is an integer of 4 to 300.
A peptide represented by:
(B) A peptide obtained by deleting, substituting, and / or adding one or several amino acids in the peptide of (a), and exhibiting temperature-dependent self-assembly.

  In the peptide of (a) (peptide represented by Chemical Formula 1), n is the number of repetitions (degree of polymerization) of a repeating sequence consisting of 5 amino acids (SEQ ID NO: 1: Phe-Pro-Gly-Val-Gly (FPGVG)). Represents. And this n must be an integer of 4 or more in the present invention. This is because the peptide expresses coacervation (self-assembly) ability only when n ≧ 4. On the other hand, regarding the upper limit, n is an integer of 300 or less as a molecular size that can be handled chemically. Here, about a minimum, n becomes like this. Preferably it is an integer greater than or equal to 5, More preferably, it is an integer greater than or equal to 6. On the other hand, about an upper limit, n becomes like this. Preferably it is an integer of 100 or less, More preferably, it is an integer of 10 or less.

An example of a preferred embodiment of the peptide (a) (peptide represented by Chemical Formula 1) is a peptide represented by any one of the amino acid sequences of SEQ ID NO: 2 to SEQ ID NO: 6. The peptide represented by the amino acid sequence of SEQ ID NO: 2 is a peptide in which n is 4 in Chemical Formula 1 (that is, (FPGVG) ₄ ). In addition, the peptide represented by the amino acid sequence of SEQ ID NO: 3 is a peptide in which n is 5 in Formula 1 (that is, (FPGVG) ₅ ). Furthermore, the peptide represented by the amino acid sequence of SEQ ID NO: 4 is a peptide in which n is 6 in Formula 1 (that is, (FPGVG) ₆ ). The peptide represented by the amino acid sequence of SEQ ID NO: 5 is a peptide in which n is 8 in Formula 1 (that is, (FPGVG) ₈ ). Furthermore, the peptide represented by the amino acid sequence of SEQ ID NO: 6 is a peptide in which n is 10 in Formula 1 (that is, (FPGVG) ₁₀ ). Among these, the peptide represented by the amino acid sequence of SEQ ID NO: 3 ((FPGVG) ₅ ) is particularly preferable in that it has a better self-assembly ability.

The C-terminus of the peptide provided by the present invention may be any of a carboxyl group (—COOH), a carboxylate (—COO ⁻ ), an amide (—CONH ₂ ), or an ester (—COOR). Here, as R in the case where the C-terminal is an ester (—COOR), for example, C _1-6 alkyl group such as methyl, ethyl, n-propyl, isopropyl, n-butyl, C ₃ such as cyclopentyl, cyclohexyl, etc. _-8 cycloalkyl groups, C _6-12 aryl groups such as phenyl and α-naphthyl, phenyl-C _1-2 alkyl groups such as benzyl and phenethyl or α-naphthyl-C _1-2 alkyl groups such as α-naphthylmethyl C _7-14 aralkyl group such as, pivaloyloxymethyl group and the like.

In addition, the N-terminal amino group of the peptide provided by the present invention may be protected with a protecting group (for example, a C _1-6 acyl group such as C _1-6 alkanoyl such as formyl group, acetyl group, etc.). Good.

  Furthermore, the peptide provided by the present invention may be a free form or a salt. Such salts include salts with physiologically acceptable acids (eg, inorganic acids, organic acids) and bases (eg, alkali metal salts), and particularly physiologically acceptable acid addition salts. Is preferred. Examples of such salts include salts with inorganic acids (eg, hydrochloric acid, phosphoric acid, hydrobromic acid, sulfuric acid), or organic acids (eg, acetic acid, formic acid, propionic acid, fumaric acid, maleic acid, succinic acid). Salts with acid, tartaric acid, citric acid, malic acid, oxalic acid, benzoic acid, methanesulfonic acid, benzenesulfonic acid, trifluoroacetic acid), and the like.

  According to the present invention, in addition to the peptide represented by Chemical Formula 1 shown in (a) above, a peptide comprising an amino acid sequence obtained by slightly modifying the amino acid sequence of the peptide also has a temperature-dependent self-assembly ability. As long as it has, it can be included in the technical scope of the present invention. Specifically, as described in the above (b), 1 or several (preferably 1 to 8, more preferably 1 to 4, more preferably 1 to 2) of the peptide of (a). Peptides in which amino acids are deleted, substituted, and / or added can also be included in the technical scope of the present invention as long as they have temperature-dependent self-assembly ability. Note that whether or not a specific peptide exhibits a temperature-dependent self-assembly ability can be determined by the method described in the examples described later.

  The peptide provided by the present invention is designed based on the amino acid sequence of elastin. For this reason, the amino acid sequence of “Pro-Gly” located at the center of the repetitive sequence (Phe-Pro-Gly-Val-Gly) is preferably conserved in all repetitive sequences. In other words, it is preferable that all of the amino acid sequences of “Pro-Gly” derived from the original repetitive sequence are conserved even by the deletion, substitution and / or addition of one or several amino acids described above. It is. According to such a form, it becomes possible to reliably preserve the temperature-dependent self-assembly ability.

  Furthermore, D-type and L-type optical isomers exist in amino acids. In the examples described later, examples using L-type amino acids are described. However, peptides in which at least a part is replaced with D-type amino acids are also considered to exhibit similar self-assembly properties. It can be included in the technical scope of the invention. That is, the amino acids constituting the peptide of the present invention represented by Chemical Formula 1 may be L-type or D-type at all sites, but preferably all amino acids are L-type.

  There is no restriction | limiting in particular about the method of manufacturing the peptide provided by this invention, The conventionally well-known knowledge regarding acquisition of a peptide can be referred suitably. For example, the peptide of the present invention can be produced according to a known peptide synthesis method.

As a peptide synthesis method, for example, either a solid phase synthesis method or a liquid phase synthesis method may be used. That is, the target peptide can be produced by condensing the amino acid constituting the peptide of the present invention and the remaining part and removing the protecting group when the product has a protecting group. Examples of known condensation methods and protecting group removal include the methods described in the following (i) to (v).
(I) M.M. Bodanszky and M.M. A. Ondetti, Peptide Synthesis, Interscience Publishers, New York (1966)
(Ii) Schroeder and Luebke, The Peptide, Academic Press, New York (1965)
(Iii) Nobuo Izumiya et al., Basics and Experiments of Peptide Synthesis, Maruzen Co., Ltd. (1975)
(Iv) Haruaki Yajima and Shunpei Sugawara, Biochemistry Experiment Course 1, Protein Chemistry IV, 205, (1977)
(V) Supervised by Haruaki Yajima, Development of follow-up pharmaceuticals, Volume 14, Peptide synthesis, Hirokawa Shoten The peptides thus obtained can be purified and isolated by known purification methods. Here, examples of the purification method include solvent extraction, distillation, column chromatography, liquid chromatography, and recrystallization.

  When the peptide obtained by the above method is a free form, it can be converted to an appropriate salt by a known method or a method similar thereto, and conversely, when the peptide is obtained as a salt, the known method or It can be converted to a free form or other salt by a similar method.

  For the synthesis of the peptide of the present invention, a commercially available resin for protein synthesis can be used. Examples of such resins include chloromethyl resin, hydroxymethyl resin, benzhydrylamine resin, aminomethyl resin, 4-benzyloxybenzyl alcohol resin, 4-methylbenzhydrylamine resin, PAM resin, 4-hydroxymethylmethyl. Examples include phenylacetamidomethyl resin, polyacrylamide resin, 4- (2 ′, 4′-dimethoxyphenyl-hydroxymethyl) phenoxy resin, 4- (2 ′, 4′-dimethoxyphenyl-Fmocaminoethyl) phenoxy resin, and the like. Using such a resin, an amino acid having an α-amino group and a side chain functional group appropriately protected is condensed on the resin according to various known condensation methods according to the sequence of the target peptide. At the end of the reaction, the peptide is excised from the resin, and at the same time, various protecting groups are removed to obtain the desired peptide.

  For the above-mentioned condensation of protected amino acids, various activating reagents that can be used for peptide synthesis can be used, and carbodiimides are particularly preferable. Examples of carbodiimides include DCC, N, N′-diisopropylcarbodiimide, N-ethyl-N ′-(3-dimethylaminoprolyl) carbodiimide, and the like. It is also possible to use a condensing agent such as TBTU, HBTU, HATU, BOP, PyBOP. For activation by these, a protected amino acid is added directly to the resin together with a racemization inhibitor (eg, HOBt, HOBt, HOAt), or the protected amino acid is activated in advance as a symmetric acid anhydride, HOBt ester or HOBt ester. After being done, it can be added to the resin.

  The solvent used for the activation of the protected amino acid and the condensation with the resin can be appropriately selected from solvents known to be used for the peptide condensation reaction. For example, acid amides such as N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, halogenated hydrocarbons such as methylene chloride and chloroform, alcohols such as trifluoroethanol, dimethyl sulfoxide, etc. Examples include sulfoxides, ethers such as pyridine, dioxane and tetrahydrofuran, nitriles such as acetonitrile and propionitrile, esters such as methyl acetate and ethyl acetate, and appropriate mixtures thereof. The reaction temperature is appropriately selected from a range known to be used for peptide bond formation reaction, and is usually in the range of about -20 to about 50 ° C. The activated amino acid derivative is usually used in an excess of 1.1 to 10 times. As a result of the test using the ninhydrin reaction, when the condensation is insufficient, sufficient condensation can be performed by repeating the condensation reaction without removing the protecting group. When sufficient condensation is not obtained even if the reaction is repeated, acetylation of the unreacted amino acid with acetic anhydride or acetylimidazole can prevent the subsequent reaction from being affected.

  The protection of the functional group that should not be involved in the reaction of the raw material, the protecting group, the elimination of the protecting group, the activation of the functional group involved in the reaction, etc. can be appropriately selected from known groups or known means.

  Examples of the protective group for the amino group of the raw material include Z, Boc, t-pentyloxycarbonyl, isobornyloxycarbonyl, 4-methoxybenzyloxycarbonyl, Cl-Z, Br-Z, adamantyloxycarbonyl, and trifluoroacetyl. , Phthaloyl, formyl, 2-nitrophenylsulfenyl, diphenylphosphinothioyl, Fmoc and the like.

  The carboxyl group is, for example, alkyl esterified (eg, linear, branched or cyclic alkyl ester such as methyl, ethyl, propyl, butyl, t-butyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, 2-adamantyl, etc. ), Aralkyl esterification (eg, benzyl ester, 4-nitrobenzyl ester, 4-methoxybenzyl ester, 4-chlorobenzyl ester, benzhydryl esterification), phenacyl esterification, benzyloxycarbonylhydrazide, t-butoxy It can be protected by carbonyl hydrazation, trityl hydrazation or the like.

  Examples of the method for removing (eliminating) the protecting group include catalytic reduction in a hydrogen stream in the presence of a catalyst such as Pd-black or Pd-carbon, anhydrous hydrogen fluoride, methanesulfonic acid, trifluoro. Acid treatment with romethanesulfonic acid, trifluoroacetic acid or a mixture thereof, base treatment with diisopropylethylamine, triethylamine, piperidine, piperazine, etc., reduction with sodium in liquid ammonia, and the like are also used. The elimination reaction by the acid treatment is generally performed at a temperature of about −20 ° C. to about 40 ° C. In the acid treatment, for example, anisole, phenol, thioanisole, metacresol, paracresol, dimethyl sulfide, 1, Addition of a cation scavenger such as 4-butanedithiol and 1,2-ethanedithiol is effective. The 2,4-dinitrophenyl group used as the imidazole protecting group of histidine is removed by thiophenol treatment, and the formyl group used as the indole protecting group of tryptophan is the above 1,2-ethanedithiol, 1,4-butane. In addition to deprotection by acid treatment in the presence of dithiol or the like, it can also be removed by alkali treatment with dilute sodium hydroxide solution, dilute ammonia or the like.

  Examples of the activated carboxyl group of the raw material include a corresponding acid anhydride, azide, active ester [alcohol (eg, pentachlorophenol, 2,4,5-trichlorophenol, 2,4-dinitrophenol, And esters thereof with cyanomethyl alcohol, paranitrophenol, HONB, N-hydroxysuccinimide, N-hydroxyphthalimide, HOBt) and the like. As the activated amino group of the raw material, for example, a corresponding phosphoric acid amide is used.

  As another method for obtaining the peptide, for example, first, the α-carboxyl group of the carboxy terminal amino acid is amidated and protected, then the peptide chain is extended to the amino group side to the desired chain length, and then the peptide chain N A peptide from which only the terminal α-amino group protecting group is removed and a peptide from which only the C-terminal carboxyl group protecting group has been removed are produced, and these peptides are condensed in a mixed solvent as described above. The details of the condensation reaction are the same as described above. After purifying the protected peptide obtained by the condensation, all the protecting groups are removed by the above method to obtain the desired crude peptide. This crude peptide can be purified using various known purification means, and the main fraction can be lyophilized to obtain an amide of the desired peptide.

  To obtain an ester of a peptide, for example, after condensing the α-carboxyl group of the carboxy terminal amino acid with a desired alcohol to form an amino acid ester, an ester of the desired peptide is obtained in the same manner as the peptide amide. be able to.

  Furthermore, the peptide provided by the present invention can be produced by culturing a transformant containing a polynucleotide encoding the peptide, and separating and purifying the peptide from the resulting culture. The polynucleotide encoding the peptide of the present invention may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The polynucleotide may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid. In the case of a single strand, it may be a sense strand (ie, a coding strand) or an antisense strand (ie, a non-coding strand).

  Examples of the polynucleotide encoding the peptide of the present invention include genomic DNA, genomic DNA library, mammal (eg, human, cow, monkey, horse, pig, sheep, goat, dog, cat, guinea pig, rat, mouse, rabbit). , Hamsters, etc.) [eg hepatocytes, spleen cells, neurons, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells , Fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (eg, macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), Megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or precursor cells, stem cells or Or any tissue in which those cells exist [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, thalamus, hypothalamus, cerebral cortex, medulla, cerebellum), spinal cord , Pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral blood , Prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), skeletal muscle, etc.]. The genomic DNA and cDNA encoding the protein or its partial peptide used in the present invention are the polymerase chain reaction (Polymerase Chain) using the genomic DNA fraction and total RNA or mRNA fraction prepared from the cells / tissues described above as templates, respectively. Direct amplification can also be performed by RT-PCR using Reaction (PCR method), (Loop-Mediated Isothermal Amplification) LAMP method, and Reverse Transcriptase (Reverse Transcriptase).

  The peptide provided by the present invention has a property of self-assembly in a temperature-dependent manner (temperature-dependent self-assembly). That is, when the temperature of the solution in which it is dissolved rises, it self-assembles reversibly at a certain point to form a condensate, and with cooling, the self-assembly is canceled and returns to the original dissolved state. Utilizing this property, according to another aspect of the present invention, there is provided a method for self-assembling a peptide, comprising the step of heating an aqueous solution in which the peptide of the present invention is dissolved. According to still another aspect of the present invention, there is provided a peptide aggregate formed by self-assembling the above-described peptides of the present invention.

  The self-assembly of the peptide of the present invention can be performed by heating a solution in which the peptide of the present invention is dissolved.

  Although there is no restriction | limiting in particular in the density | concentration of the peptide in a peptide solution, Preferably it is about 1-100 mg / ml. Also, the heating means is not particularly limited, and heating means such as a water bath, a block heater, and an incubator that are generally used in the technical field can be used as well. In addition, there is no restriction | limiting in particular also about heating temperature, What is necessary is just the temperature which the coacervation (self-assembly) of a peptide produces. The lower the temperature at which peptide coacervation (self-assembly) occurs, the better. As an example, when the turbidity is measured by the method described in the examples described later, the temperature of the solution having a turbidity of 1.0 is preferably 60 ° C. or lower, more preferably 50 ° C. or lower, Preferably it is 40 degrees C or less, Most preferably, it is 30 degrees C or less, Most preferably, it is 25 degrees C or less.

  The peptides provided by the present invention are expected to be used in a wide range of applications as raw materials for tissue engineering biomaterials, drug delivery systems (DDS), cosmetic base materials, immobilization materials such as microorganisms and enzymes, etc. .

  In addition, the peptides and aggregates provided by the present invention are also useful as cell culture substrates (see Examples described later). That is, according to still another aspect of the present invention, a cell culture substrate containing the peptide or peptide aggregate according to the present invention is also provided.

  In this case, the peptides and aggregates provided by the present invention may be used alone as a cell culture substrate as they are, or may be used in combination with other conventionally known cell culture substrates. Examples of other conventionally known cell culture substrates that can be used in combination with the peptides and aggregates provided by the present invention include, for example, collagen, gelatin, chitin-chitosan, hyaluronic acid, fibronectin, laminin, proteoglycan, tenascin, Agarose, alginic acid, cellulose, hydroxyapatite, polylactic acid (PLA), polylactic acid glycol (PLGA), polyacrylic acid (PAAc), poly (2-hydroxyethyl methacrylate) (PHEMA), polycaprolactone, fluororesin (polytetrafluoro) Ethylene) and the like.

  Furthermore, according to the further another form of this invention, the manufacturing method of a cell sheet is also provided. Specifically, the method for producing a cell sheet provided by the present invention includes a step of culturing cells using the cell culture substrate containing the peptide or peptide aggregate according to the present invention described above (culture step), And a step (collecting step) of collecting a cell sheet containing the treated cells. Thereby, the cell sheet containing the cultured cell is manufactured.

  There is no restriction | limiting in particular about the specific method of implementing the manufacturing method mentioned above, A conventionally well-known knowledge can be referred suitably. As an example of the culture method, for example, a method of culturing cells on a culture dish on which the cell culture substrate according to the present invention is spread is exemplified. According to this method, a two-dimensional tissue (cell sheet) is formed, which can be easily separated and collected. At this time, in order to form a two-dimensional tissue body (cell sheet) and collect it, the cell culture substrate according to the present invention is spread on a culture dish, and normal cell culture is performed thereon. Then, after confirming that the cells have sufficiently proliferated, the cell sheet can be peeled from the culture dish by adding a large amount of culture solution and shaking the culture dish.

  Since the cell culture substrate provided by the present invention is composed of the peptide according to the present invention or an aggregate thereof, it exhibits coacervation ability (temperature-dependent self-assembly). Therefore, when cells are cultured on the cell culture substrate according to the present invention and then the culture is cooled, the cell culture substrate is liquefied. Thus, even if the cells cultured after liquefying the cell culture substrate are recovered, the recovered cells still retain the sheet-like shape by experiments described in the examples described later. It was done.

  That is, in a preferred embodiment of the method for producing a cell sheet described above, the collecting step includes a step of collecting the cell sheet after cooling the culture and liquefying the cell culture substrate. The temperature for cooling the culture is not particularly limited as long as the cell culture substrate can be liquefied, but as an example, it is preferably 25 ° C. or lower, more preferably 15 ° C. or lower. It becomes possible to sufficiently liquefy this by utilizing the coacervation ability of the cell culture substrate. On the other hand, the lower limit of the temperature at which the culture is cooled is not particularly limited, but is preferably 5 ° C. or higher from the viewpoint of allowing cells to survive in a normal state.

  Hereinafter, preferred embodiments of the present invention will be described in more detail using examples. However, the technical scope of the present invention should not be construed as being limited to the following examples.

≪Chemical synthesis of peptides≫
By chemical synthesis (solid phase method), (FPGVG) ₄ (peptide represented by the amino acid sequence of SEQ ID NO: 2), (FPGVG) ₅ (peptide represented by the amino acid sequence of SEQ ID NO: 3), (FPGVG) ₆ (Peptide represented by the amino acid sequence of SEQ ID NO: 4), (FPGVG) ₈ (peptide represented by the amino acid sequence of SEQ ID NO: 5), and (FPGVG) ₁₀ (peptide represented by the amino acid sequence of SEQ ID NO: 6) Was synthesized. The Fmoc method was used as the chemical synthesis method (solid phase method). In addition, as the resin for peptide synthesis and the Fmoc amino acid, products manufactured by Watanabe Chemical Co., Ltd. were all used. As synthesis reagents, products manufactured by Watanabe Chemical Co., Ltd. and Wako Pure Chemical Industries, Ltd. were used.

  Specifically, the target peptide was synthesized by sequentially binding Fmoc amino acids to a synthetic resin (Fmoc-NH-SAL-Resin) (see FIGS. 2 and 3). At this time, 0.45M HBTU-HOBt and 2M DIEA were used for amino acid condensation, 95% TFA was added to the resin after the introduction of the peptide, and the mixture was stirred at 25 ° C. for 1 hour, and then TFA was removed under a nitrogen stream. The obtained residue was dissolved in 3% acetic acid (3 ml), washed with diethyl ether and lyophilized. The obtained peptide was purified using high performance liquid chromatography (HPLC, manufactured by Tosoh Corporation) and subjected to mass spectrometry (equipment: Voyager-DE, manufactured by Applied Biosystems) by MALDI TOF-MS measurement. It was confirmed that the peptide was obtained with high purity.

≪Measurement of coacervation (self-assembly) characteristics≫
For each of (FPGVG) ₄ , (FPGVG) ₅ , (FPGVG) ₆ , (FPGVG) ₈ , and (FPGVG) ₁₀ obtained above, a peptide aqueous solution (50 mg / ml) was prepared, and a Peltier temperature controller Turbidity was measured under a nitrogen stream using a spectrophotometer equipped with a hood (JASCO Ubest V-560, manufactured by JASCO Corporation). The measurement conditions were a measurement wavelength of 400 nm, a temperature range of 5 to 50 ° C., and a temperature change rate of 0.5 ° C./min. Some results are shown in FIG. As shown in FIG. 4 (b), (FPGVG) ₅ consisting of 25 amino acids showed sufficient coacervation (self-assembly) ability equivalent to a conventionally known high molecular weight polypeptide. In addition, as shown in FIG. 4 (a), a tendency to exhibit coacervation (self-assembly) was also observed for (FPGVG) ₄ having a small number of repetitions of 1 and 20 amino acids. Although not shown, it was confirmed that (FPGVG) ₆ , (FPGVG) ₈ , and (FPGVG) ₁₀ also show coacervation (self-assembly) as shown in FIG.

From the above, it can be seen that according to the present invention, a peptide having a coacervation (self-assembly) ability can be provided even with a low molecular weight. Furthermore, the peptide provided by the present invention exhibits a self-assembly start temperature near body temperature (37 ° C.) and has a reversible assembly / desorption, similar to a high molecular weight polypeptide, despite its small molecular weight size. It was also confirmed to show the nature of the set. Although not shown, n = 3 (FPGVG) ₃ does not exhibit self-assembling properties at a concentration of 50 mg / ml, and finally appears cloudy at a high concentration (100 mg / ml). It was. However, no temperature-dependent turbidity increase was observed. From this, it is considered that the cloudiness of the solution in the high concentration (FPGVG) ₃ is not due to the temperature-dependent self-assembly ability but to aggregation at a high concentration generally found in peptides and proteins. Therefore, it was also shown that when examining the presence or absence of coacervation ability peculiar to elastin, it is important to make a distinction from the peptide aggregation action.

  The peptide provided by the present invention can be easily synthesized by the chemical synthesis method as described above (of course, it may be genetically synthesized). Moreover, since the length of the amino acid sequence is shorter than that of conventionally known polypentapeptide, there is an advantage that the labor required for purification after synthesis can be remarkably reduced.

  In addition, in the conventional solid phase synthesis of peptides, when each amino acid is bound to a resin as a solid phase, the introduction rate is not perfect at 95-99%. The content of the target product is about 95 to 97%, and it is general that amino acids missing one residue, four residues, etc. are mixed as impurities. On the other hand, since the peptide provided by the present invention has a simple sequence structure, an amino acid that does not require a side chain protecting group can be used in its synthesis. Therefore, if a single peptide can be synthesized completely by introducing each amino acid into the synthesis resin at a yield of 100%, the peptide cleaved from the resin can be obtained without purification by HPLC. It can also be used as it is. As described above, the peptide of the present invention having advantages from the viewpoint of synthesis is expected as an industrially very useful material.

  Specifically, the peptides provided by the present invention are water-soluble and can be used as various medical materials, such as the ability to easily synthesize efficient DDS carriers that can contain drugs inside. It is expected to be possible.

_{«(FPGVG) 5} study of cell sheets prepared using»
Using (FPGVG) ₅ obtained above as a cell culture substrate, cell culture was attempted by the following method.

First, a solution in which (FPGVG) ₅ obtained above was dissolved in a DMEM (−) medium to a concentration of 20 mg / mL was prepared and used as a peptide sample.

  The peptide sample prepared above was sterilized with a 0.22 μm sterilizing filter and added to each well of a 24-well plate. At this time, the amount of peptide sample added to each well was 200 μL. Subsequently, the 24-well plate to which the peptide sample was added was allowed to stand in an incubator at 37 ° C. for 1 to 2 hours to form a sheet composed of peptide aggregates as a cell culture substrate.

Subsequently, NHDF (p14) cells were prepared as cells to be cultured, seeded at a concentration of 4 × 10 ⁴ cells / well in each well of the 24-well plate obtained above, and incubator (37 ° C., CO ₂ 5%). Two days after the start of the culture, the medium was removed for the purpose of observing cell adhesion and proliferation, and the cells were washed with physiological saline. Then, after adding 400 μL of physiological saline to each well, the culture was cooled to room temperature (25 ° C.) and allowed to stand for 90 minutes to liquefy the cell culture substrate. (Cell sheet) was observed. For this reason, the cells were collected and stained with trypan blue for observation. The flow of this experiment including the observation results is shown in FIG. As shown in FIG. 5, it was confirmed that the cell-to-cell binding remained retained after being recovered from the plate. From this, it is possible to produce a cell sheet in which cells are bound in a sheet form by culturing cells using the peptide according to the present invention or an aggregate of the peptides as a cell culture substrate. It was shown that. A similar experiment was conducted using water-soluble elastin isolated from animal living tissue as a cell culture substrate. However, the cells could not be recovered in a sheet form, and the above results were confirmed. There wasn't.

  As described above, since the peptide according to the present invention and the aggregate of the peptide have a characteristic of being composed only of amino acids derived from living organisms, poly N-isopropyl currently used as a cell culture substrate. It is expected to be a highly promising and highly safe material applicable to a wide range of applications that can be substituted for a polymer material such as acrylamide (PNIPAAm).

[SEQ ID NO: 1]
It is the repeating amino acid sequence (FPGVG) which the peptide of (a) in this invention has.
[SEQ ID NO: 2]
It is the amino acid sequence of the peptide [(FPGVG) ₄ ] synthesized by repeating the repeated amino acid sequence of SEQ ID NO: 1 four times.
[SEQ ID NO: 3]
It is an amino acid sequence of a peptide [(FPGVG) ₅ ] synthesized by repeating the amino acid sequence of SEQ ID NO: 1 and repeated 5 times.
[SEQ ID NO: 4]
It is an amino acid sequence of a peptide [(FPGVG) ₆ ] synthesized by repeating the repeated amino acid sequence of SEQ ID NO: 1 6 times in the Examples.
[SEQ ID NO: 5]
It is an amino acid sequence of a peptide [(FPGVG) ₈ ] synthesized in the examples and having the repeated amino acid sequence of SEQ ID NO: 1 repeated 8 times.
[SEQ ID NO: 6]
It is the amino acid sequence of the peptide [(FPGVG) ₁₀ ] synthesized by repeating the amino acid sequence of SEQ ID NO: 1 and repeated 10 times.

Claims (8)

The following peptide (a) or (b):
(A) The following chemical formula 1:
In the formula, n is an integer of 4 to 300.
A peptide represented by:
(B) A peptide in which 1 to 2 amino acids are deleted, substituted, and / or added in the peptide of (a), and exhibits temperature-dependent self-assembly.   The peptide according to claim 1, wherein n is an integer of 4 to 10.   The peptide according to claim 2, represented by the amino acid sequence of any one of SEQ ID NO: 2 to SEQ ID NO: 6.   A method for self-assembling a peptide, comprising a step of heating an aqueous solution in which the peptide according to any one of claims 1 to 3 is dissolved.   The peptide assembly formed by self-assembling the peptide of any one of Claims 1-3.   A cell culture substrate comprising the peptide according to any one of claims 1 to 3 or the peptide aggregate according to claim 5. A culture step of culturing cells using the cell culture substrate according to claim 6;
A recovery step of recovering a cell sheet containing cultured cells;
A method for producing a cell sheet, comprising:   The manufacturing method according to claim 7, wherein the recovery step includes a step of recovering the cell sheet after cooling the culture and liquefying the cell culture substrate.