WO2015167003A1 - Fragment de collagène ou atélocollagène, et procédé de fabrication ainsi qu'application de celui-ci - Google Patents

Fragment de collagène ou atélocollagène, et procédé de fabrication ainsi qu'application de celui-ci Download PDF

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WO2015167003A1
WO2015167003A1 PCT/JP2015/063044 JP2015063044W WO2015167003A1 WO 2015167003 A1 WO2015167003 A1 WO 2015167003A1 JP 2015063044 W JP2015063044 W JP 2015063044W WO 2015167003 A1 WO2015167003 A1 WO 2015167003A1
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collagen
atelocollagen
degradation product
chemical bond
amino acid
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PCT/JP2015/063044
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English (en)
Japanese (ja)
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康一 森本
沙織 國井
衛 山本
久保木　芳徳
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学校法人近畿大学
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Priority to JP2016516418A priority Critical patent/JP6521461B2/ja
Publication of WO2015167003A1 publication Critical patent/WO2015167003A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/04—Animal proteins
    • A23J3/06—Gelatine
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23J—PROTEIN COMPOSITIONS FOR FOODSTUFFS; WORKING-UP PROTEINS FOR FOODSTUFFS; PHOSPHATIDE COMPOSITIONS FOR FOODSTUFFS
    • A23J3/00—Working-up of proteins for foodstuffs
    • A23J3/30—Working-up of proteins for foodstuffs by hydrolysis
    • A23J3/32—Working-up of proteins for foodstuffs by hydrolysis using chemical agents
    • A23J3/34—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

Definitions

  • the present invention relates to a degradation product of collagen or atelocollagen, a method for producing the degradation product, and use of the degradation product.
  • Collagen is one of the proteins constituting the dermis, ligaments, tendons, bones and cartilage, and is the main component of the extracellular matrix of multicellular organisms. As research progresses, it has become clear that collagen has various physiological functions, and research for finding new physiological functions of collagen molecules and research for finding new uses of collagen molecules are still ongoing. Is underway.
  • one collagen molecule is composed of three polypeptide chains, and that these three polypeptide chains form a helical structure to form one collagen molecule. It has become.
  • a region in each polypeptide chain for forming a helical structure is called a triple helical domain, and the triple helical domain has a characteristic amino acid sequence.
  • the triple helical domain has a characteristic amino acid sequence in which the amino acid sequence represented by “Gly-XY” appears repeatedly and continuously.
  • amino acids other than glycine that is, X and Y can be various amino acids.
  • the telopeptide which is the main antigenic site of collagen, is present at the amino terminus and / or carboxyl terminus of the collagen molecule (in other words, the amino terminus and carboxyl terminus of each polypeptide chain constituting the collagen molecule).
  • the telopeptide is present on the amino terminal side and / or the carboxyl terminal side of the above-described triple helical domain in each polypeptide chain constituting the collagen molecule.
  • telocollagen A collagen molecule from which such a telopeptide has been partially excised is called atelocollagen.
  • Patent Document 1 discloses a technique of using a degradation product obtained by treating collagen or atelocollagen with a protease (for example, pepsin and actinidine) as a medical material for hemostasis. More specifically, in Patent Document 1, first, the skin of yellowfin tuna is subjected to pepsin treatment to obtain an aqueous solution containing atelocollagen, and further, sodium chloride is added to the aqueous solution to obtain atelocollagen. Precipitation and recovery. In addition, when recovering atelocollagen as a precipitate, sodium chloride is removed together with the supernatant.
  • a protease for example, pepsin and actinidine
  • Patent Document 2 discloses a technique in which a degradation product obtained by treating collagen or atelocollagen with a protease is used as a composition for preventing or treating arteriosclerosis and diseases caused by arteriosclerosis. More specifically, Patent Document 2 discloses a composition for preventing or treating arteriosclerosis and diseases caused by arteriosclerosis, which is obtained by degrading collagen after removing minerals with protease. Techniques used as objects are disclosed.
  • the present invention has been made in view of the above-described conventional problems, and its purpose is to provide a degradation product of collagen or atelocollagen having a novel physiological function, a method for producing the degradation product, and use of the degradation product. It is to provide.
  • A) to C) have completed the present invention. That means A) Under conditions where the salt concentration is high, the cysteine protease cleaves collagen or atelocollagen at a location that has not been known so far (specifically, a specific location within the triple helical domain); B) Collagen or a degradation product of atelocollagen cleaved at a previously unknown location has a spheroid-inducing ability; C) Collagen or atelocollagen degradation product cleaved at a previously unknown location has a higher ability to induce spheroids than collagen or atelocollagen degradation product known at a previously known location. .
  • Degradation product of collagen or atelocollagen of the present invention in order to solve the above problems, the amino acid sequence represented by the following (1) or (2) in the triple helical domain of the collagen or atelocollagen, X 1 and X 2 chemical bond, chemical bond between X 2 and G, the chemical bond between G and X 3, the chemical bond between X 4 and G, chemical bond between X 6 and G between the A degradation product of collagen or atelocollagen in which the chemical bond between G and X 7 or the chemical bond between X 14 and G is cleaved: (1) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -G- (2) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -GX 7 -X 8 -GX 9 -X 10 -GX 11 -X 12 -GX 13 -X 14 -G- (However, G is glycine, and
  • the present invention has an effect that spheroids can be formed in various cells.
  • the present invention has an effect that a larger spheroid can be formed as compared with the conventional degradation product of collagen or atelocollagen.
  • the present invention has the effect of being able to form spheroids in a shorter time than conventional collagen or atelocollagen degradation products.
  • the present invention is capable of realizing a collagen or atelocollagen degradation product that can be in a liquid state even at a temperature close to human body temperature, in other words, a collagen or atelocollagen degradation product that is easily adapted to human skin and the like. Play.
  • (A) is a photomicrograph of a spheroid of normal human dermal fibroblast NHDF induced by the degradation product of the example of the present invention, and (b) is a normal human dermal fibroblast induced by atelocollagen. It is a microscope picture of NHDF.
  • (A) is a photomicrograph of spheroids of normal human umbilical vein endothelial cells HUVEC induced by degradation products of the examples of the present invention, and (b) is normal human umbilical vein endothelial cells induced by atelocollagen. It is a photomicrograph of HUVEC.
  • (A) is a photomicrograph of a spheroid of the mouse osteoblast progenitor cell line MC3T3-E1 subclone 4 induced by the degradation product of the example of the present invention, (b) is induced by atelocollagen, 2 is a photomicrograph of mouse osteoblast precursor cell line MC3T3-E1 subclone 4.
  • (A) is a photomicrograph of a spheroid of the mouse adipocyte progenitor cell line MC3T3-G2 / PA6 induced by the degradation product of the example of the present invention, and (b) is a mouse fat induced by atelocollagen.
  • FIG. 2 is a photomicrograph of cell precursor cell line MC3T3-G2 / PA6.
  • A is a micrograph of a spheroid of human bone marrow mesenchymal stem cells MSC induced by the degradation product of the example of the present invention
  • (b) is a human bone marrow mesenchymal stem cell induced by atelocollagen. It is a microscope picture of MSC.
  • A) is a photomicrograph of a spheroid of rat bone marrow mesenchymal stem cell MSC induced by the degradation product of the example of the present invention, and (b) is a rat bone marrow mesenchymal stem cell induced by atelocollagen.
  • FIG. 2 shows a photomicrograph of a mouse fibroblast NIH / 3T3 spheroid induced by a degradation product of an example of the present invention.
  • (A)-(j) are photomicrographs of various cell spheroids induced by the degradation products of the examples of the present invention.
  • (A) And (b) is the photograph which left still for 11 days, after making a bone fragment and each enzyme contact in the Example of this invention.
  • derived with the bone fragment decomposition product of the Example of this invention is shown.
  • the collagen or atelocollagen degradation product of the present embodiment is a chemical bond between X 1 and X 2 of the amino acid sequence shown in the following (1) in the triple helical domain of collagen or atelocollagen, X 2 and G chemical bond, chemical bond between G and X 3, the chemical bond between X 4 and G, or a chemical bond between X 6 and G is disconnected, collagen or atelocollagen between Is a decomposition product of:
  • the collagen or atelocollagen degradation product of the present embodiment is a chemical bond between X 1 and X 2 of the amino acid sequence represented by the following (1) in the triple helical domain of collagen or atelocollagen, X 2 Any one selected from a chemical bond between G and X, a chemical bond between G and X 3 , a chemical bond between X 4 and G, and a chemical bond between X 6 and G It may be a degradation product of collagen or atelocollagen in which one chemical bond
  • the collagen or degradation product of atelocollagen of the present embodiment is a chemical bond between X 1 and X 2 of the amino acid sequence shown in the following (2) in the triple helical domain of collagen or atelocollagen.
  • a chemical bond between X 2 and G, a chemical bond between G and X 3 , a chemical bond between X 4 and G, a chemical bond between X 6 and G, and a G and X 7 chemical bonding between or chemical bonding between X 14 and G is disconnected
  • a degradation product of collagen or atelocollagen The collagen or atelocollagen degradation product of the present embodiment is a chemical bond between X 1 and X 2 of the amino acid sequence represented by the following (2) in the triple helical domain of collagen or atelocollagen, X 2 Chemical bond between G and X, chemical bond between G and X 3 , chemical bond between X 4 and G, chemical bond between X 6 and G, between G and X 7 It may be a degradation product of collagen or
  • the degradation product of the present embodiment is a degradation product of collagen or atelocollagen.
  • the collagen and atelocollagen used as the material of the degradation product are not particularly limited, and may be any known collagen and atelocollagen.
  • Collagen that becomes the material of the degradation product includes mammals (eg, cow, pig, rabbit, human, rat or mouse), birds (eg, chicken), or fish (eg, shark, carp, eel, tuna ( For example, yellowfin tuna), tilapia, Thailand, salmon, etc.) can be used.
  • mammals eg, cow, pig, rabbit, human, rat or mouse
  • birds eg, chicken
  • fish eg, shark, carp, eel, tuna ( For example, yellowfin tuna), tilapia, Thailand, salmon, etc.) can be used.
  • collagen derived from the dermis, tendon, bone or fascia of mammals or birds, or collagen derived from the skin or scales of fish, etc. is used as the collagen used as the degradation product. Can do.
  • telopeptide As the atelocollagen that becomes the material of the degradation product, telopeptide is partially obtained from the amino terminus and / or carboxyl terminus of the collagen molecule obtained by treating the above-mentioned mammalian, avian or fish collagen with a protease (for example, pepsin). The removed atelocollagen can be used.
  • a protease for example, pepsin
  • chicken, pig, human or rat collagen or atelocollagen can be preferably used as the degradation material, and porcine or human collagen or atelocollagen can be more preferably used as the degradation material.
  • the material can be obtained simply, safely, and in large quantities, and to realize a collagen or atelocollagen degradation product that is safer for humans. Can do.
  • fish collagen or atelocollagen as a material of the degradation product, it is preferable to use shark, carp, eel, tuna (eg yellowfin tuna), tilapia, Thai or salmon collagen or atelocollagen, tuna, tilapia, More preferably, tie or salmon collagen or atelocollagen is used.
  • tuna eg yellowfin tuna
  • tilapia tilapia
  • Thai or salmon collagen or atelocollagen tuna, tilapia
  • tie or salmon collagen or atelocollagen is used.
  • telocollagen When using atelocollagen as the material of the decomposition product, it is preferable to use atelocollagen having a heat denaturation temperature of preferably 15 ° C. or higher, more preferably 20 ° C. or higher.
  • telo eg, yellowfin tuna
  • carp and other atelocollagens have a heat denaturation temperature of 25 ° C. or higher, and therefore it is preferable to use these atelocollagens.
  • the denaturation temperature of the collagen or atelocollagen degradation product of the present embodiment can be adjusted to preferably 15 ° C. or higher, more preferably 20 ° C. or higher.
  • the degradation product of collagen or atelocollagen excellent in the stability at the time of storage and the stability at the time of utilization is realizable.
  • Collagen and atelocollagen that are the materials of the degradation product can be obtained by a known method.
  • collagen can be eluted by putting a tissue rich in collagen of mammals, birds or fish into an acidic solution of about pH 2-4.
  • a protease such as pepsin is added to the eluate to partially remove the amino terminal and / or carboxyl terminal telopeptide of the collagen molecule.
  • atelocollagen can be precipitated by adding a salt such as sodium chloride to the eluate.
  • the degradation product of collagen or atelocollagen of the present embodiment is a chemical bond between X 1 and X 2 of the amino acid sequence represented by the following (1) in the above-described triple helical domain of collagen or atelocollagen, X 2 Collagen in which the chemical bond between G and G, the chemical bond between G and X 3 , the chemical bond between X 4 and G, or the chemical bond between X 6 and G is broken Or a degradation product of atelocollagen: (1) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -G- (However, G is glycine, and X 1 to X 6 are arbitrary amino acids).
  • the decomposition of collagen or atelocollagen of this embodiment the amino acid sequence represented by the following (2) in the triple helical domain of collagen or atelocollagen as described above, between the X 1 and X 2 chemical bond, chemical bond between X 2 and G, the chemical bond between G and X 3, the chemical bond between X 4 and G, chemical bond between X 6 and G, G and X
  • the “triple helical domain” refers to an amino acid sequence represented by “Gly-XY” (X and Y are arbitrary amino acids), at least 3 or more, more preferably at least 80 or more, Preferably, it is a domain comprising at least 300 or more consecutive amino acid sequences, which contributes to the formation of a helical structure.
  • the polypeptide chain in which the chemical bond is broken in the triple helical domain may be any polypeptide chain among a plurality of types of polypeptide chains constituting collagen or atelocollagen.
  • polypeptide chain in which the chemical bond is broken in the triple helical domain may be any of ⁇ 1 chain, ⁇ 2 chain, and ⁇ 3 chain.
  • the polypeptide chain in which the chemical bond is broken in the triple helical domain is preferably at least one of the ⁇ 1 chain and the ⁇ 2 chain among the polypeptide chains described above.
  • the polypeptide chain in which the chemical bond is broken in the triple helical domain is more preferably an ⁇ 1 chain among the above-described polypeptide chains.
  • the degradation product of collagen or atelocollagen according to the present embodiment is prepared by enzymatic treatment, it can be easily cleaved only with a specific polypeptide chain.
  • the collagen or atelocollagen degradation product of the present embodiment may be one in which three polypeptide chains form a helical structure.
  • the collagen or atelocollagen degradation product of the present embodiment is one in which three polypeptide chains do not form a helical structure, or in which three polypeptide chains do not partially form a helical structure. There may be. Whether or not the three polypeptide chains form a helical structure can be confirmed by a known method (for example, circular dichroism spectrum).
  • the degradation product of collagen or atelocollagen of the present embodiment basically includes three polypeptide chains, but is chemically bonded only in the triple helical domain of one of the three polypeptide chains. Cleavage may occur, chemical bond breakage may occur only in the triple helical domain of two polypeptide chains of three polypeptide chains, or triple helical domain of three polypeptide chains Chemical bonds may be broken in all of the above.
  • a network-like association may be formed by a plurality of helical structures, or a fibrous association may be formed.
  • the network means a structure in which molecules are linked by hydrogen bonds, electrostatic interaction, van der Waals bonds, etc. to form a three-dimensional network, and a gap is formed between the networks.
  • the term “fibrous” means a substantially linear structure in which molecules are connected by hydrogen bonding, electrostatic interaction, van der Waals bonding, or the like.
  • an aggregate is intended to mean a single structural unit in which two or more molecules interact with each other and are linked by the same kind of molecules without being based on a covalent bond. Whether or not a network-like or fibrous aggregate is formed can be confirmed by observing with an electron microscope.
  • the collagen or atelocollagen degradation product of the present embodiment may have a crosslinked structure.
  • the polypeptide chain and the polypeptide chain may be cross-linked by a cross-linking agent between the helical structure and the helical structure, or the polypeptide chain and the helical structure.
  • the cross-linked structure can be formed by a well-known cross-linking method. Examples thereof include a chemical crosslinking method, a crosslinking method by heat treatment, and a crosslinking method by irradiation with radiation such as ultraviolet rays.
  • crosslinking agent used for chemical crosslinking examples include water-soluble carbodiimide compounds such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, diepoxy compounds such as epichlorohydrin and bisepoxydiethylene glycol, NaBH 4 and the like. Is mentioned.
  • the concentration of the crosslinking agent with respect to degradation of the present embodiment is preferably 10 -3 to 10 wt%.
  • a crosslinked structure can be formed by bringing the decomposition product of this embodiment into contact with the crosslinking agent at 5 to 40 ° C. for 3 to 48 hours.
  • a crosslinked structure can be formed by irradiating the decomposed product of the present embodiment with ultraviolet rays for 3 to 48 hours, for example, at room temperature using an ultraviolet lamp or the like.
  • the collagen or atelocollagen degradation product of the present embodiment having a cross-linked structure has the advantage of improved collagenase resistance and strength.
  • the collagen or atelocollagen degradation product of the present embodiment may be subjected to desired chemical modification as necessary.
  • the chemical modification include acylation, myristylation, and polyethylene glycol modification.
  • a decomposition product subjected to succinylation which is a kind of acylation
  • succinic anhydride in a neutral pH solvent such as a phosphate buffer. it can.
  • succinylation the solubility of the decomposition product in a solvent having a neutral pH can be improved.
  • the degradation product subjected to polyethylene glycol modification can be obtained by reacting polyethylene glycol activated with cyanuric chloride with the degradation product of the present embodiment.
  • the position in the triple helical domain of the amino acid sequence shown in (1) or (2) above is not particularly limited.
  • the amino acid sequence represented by (1) or (2) above may be present inside the triple helical domain, but is preferably present at the amino terminus of the triple helical domain (in other words, In the amino acid sequence represented by the above (1) or (2), “G” arranged at the most amino terminal side in the amino acid sequence is “G” arranged at the most amino terminal side in the triple helical domain. Is preferred).
  • the specific position of the amino acid sequence represented by (1) or (2) is not particularly limited. . 1 or more, 5 or more, 10 or more, 50 or more, 100 or more, 150 or more, 200 or more, 250 on the amino terminal side of the amino acid sequence represented by (1) or (2)
  • amino acid sequence represented by (1) or (2) There may be an amino acid sequence in which 250 or more or 300 or more “Gly-XY” (X and Y are arbitrary amino acids) are continuous.
  • Each of the above X 1 to X 6 can be any amino acid, and the type of amino acid is not particularly limited. Each of X 1 to X 6 may be at least partly the same type of amino acid, or all may be different types of amino acid.
  • each of X 1 to X 6 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tyrosine, Any of tryptophan, hydroxyproline, and hydroxylysine may be used.
  • X 1 to X 6 may be the same amino acid, and the other may be different amino acids.
  • At least one selected from the group consisting of X 1 , X 3 and X 5 among X 1 to X 6 is proline, and the other may be any amino acid.
  • X 1 may be proline and X 2 to X 6 may be any amino acid.
  • X 1 and X 3 may be proline, and X 2 and X 4 to X 6 may be any amino acid.
  • X 1 , X 3 and X 5 may be proline, and X 2 , X 4 and X 6 may be any amino acid.
  • X 1 , X 3 and X 5 are proline
  • X 2 is an amino acid containing a sulfur atom in the side chain (eg cysteine or methionine) or an amino acid containing a hydroxyl group in the side chain (eg hydroxyproline).
  • X 4 and X 6 may be any amino acid.
  • X 1 , X 3 and X 5 are proline
  • X 2 is an amino acid containing a sulfur atom in the side chain (eg, cysteine or methionine)
  • X 4 has an aliphatic side chain.
  • An amino acid for example, glycine, alanine, valine, leucine or isoleucine
  • an amino acid having a hydroxyl group in the side chain for example, hydroxyproline, hydroxylysine or serine
  • X 6 may be any amino acid.
  • X 1 , X 3 and X 5 are proline
  • X 2 is an amino acid containing a sulfur atom in the side chain (eg, cysteine or methionine)
  • X 4 has an aliphatic side chain.
  • An amino acid eg, glycine, alanine, valine, leucine or isoleucine
  • an amino acid containing a hydroxyl group in the side chain eg, hydroxyproline, hydroxylysine or serine
  • X 6 is an amino acid containing a base in the side chain (eg, arginine) Lysine or histidine).
  • Each of X 7 to X 14 may be any amino acid, and the type of amino acid is not particularly limited. Each of X 7 to X 14 may be at least partly the same type of amino acid, or all may be different types of amino acid.
  • each of X 7 to X 14 is glycine, alanine, valine, leucine, isoleucine, serine, threonine, tyrosine, cysteine, methionine, aspartic acid, asparagine, glutamic acid, glutamine, arginine, lysine, histidine, phenylalanine, tyrosine, Any of tryptophan, hydroxyproline, and hydroxylysine may be used.
  • X 7 to X 14 , X 8 , X 9 , X 10 , X 12 and X 13 may be the same amino acid, and the other may be different amino acids.
  • At least one selected from the group consisting of X 8 , X 9 , X 10 , X 12 and X 13 is proline or hydroxyproline, and the other is any amino acid It may be.
  • X 8 may be proline or hydroxyproline, and the other may be any amino acid.
  • X 7 to X 14 may be proline or hydroxyproline, and the other may be any amino acid.
  • X 7 to X 14 , X 8 , X 9 and X 10 may be proline or hydroxyproline, and the other may be any amino acid.
  • X 7 to X 14 , X 8 , X 9 , X 10 and X 12 may be proline or hydroxyproline, and the other may be any amino acid.
  • X 7 to X 14 , X 8 , X 9 , X 10 , X 12 and X 13 may be proline or hydroxyproline, and the other may be any amino acid.
  • an amino acid X 7 has a side chain aliphatic (e.g., Glycine, alanine, valine, leucine or isoleucine), and the other may be any amino acid.
  • a side chain aliphatic e.g., Glycine, alanine, valine, leucine or isoleucine
  • X 7 to X 14 , X 8 , X 9 , X 10 , X 12 and X 13 are proline or hydroxyproline
  • X 7 and X 11 are amino acids having an aliphatic side chain.
  • glycine, alanine, valine, leucine or isoleucine may be any amino acid.
  • an amino acid X 7 and X 11 has a side chain aliphatic (e.g., glycine, alanine, valine, leucine or isoleucine), and amino acid X 14 has is and undissociated side chains are hydrophilic (serine, threonine, asparagine or glutamine) may be used.
  • X 7 to X 14 , X 8 , X 9 , X 10 , X 12 and X 13 are proline or hydroxyproline
  • X 7 is leucine
  • X 11 is alanine
  • X 14 may be a glutamine.
  • the collagen or degradation product of atelocollagen of the present embodiment can be in a liquid state even at a temperature close to human body temperature. Therefore, if the degradation product of collagen or atelocollagen according to the present embodiment is applied to human skin or the like, the degradation product and the skin are easily adapted.
  • the collagen or atelocollagen degradation product of the present embodiment has a higher concentration at which gelation starts than the conventional collagen or atelocollagen degradation product. Therefore, the collagen or atelocollagen degradation product of the present embodiment having the same concentration as that at which the conventional collagen or atelocollagen degradation product gels can be stably stored at room temperature.
  • the chemical bond between X 1 and X 2 in the amino acid sequence represented by (1) or (2) in the triple helical domain of collagen or atelocollagen X Chemical bond between 2 and G, Chemical bond between G and X 3 , Chemical bond between X 4 and G, Chemical bond between X 6 and G, Between G and X 7 The chemical bond between X14 and G is broken.
  • the above cutting can be appropriately performed by a desired method.
  • collagen or atelocollagen that has already been cut can be produced by a chemical synthesis method.
  • a chemical synthesis method a general well-known chemical synthesis method can be used.
  • DNA encoding collagen or atelocollagen that has already been cut is inserted into a known protein expression vector. Then, after introducing the protein expression vector into a desired host (for example, E. coli, yeast, insect cells, animal cells, etc.), expression of collagen or atelocollagen that has already been cleaved is induced in the host. In this way, it is also possible to produce collagen or atelocollagen that has already been cut.
  • a desired host for example, E. coli, yeast, insect cells, animal cells, etc.
  • the method for producing a collagen or atelocollagen degradation product according to the present embodiment includes a chemical bond between X 1 and X 2 of the amino acid sequence represented by the following (1) in the triple helical domain of collagen or atelocollagen, X A cleavage step that cleaves a chemical bond between 2 and G, a chemical bond between G and X 3 , a chemical bond between X 4 and G, or a chemical bond between X 6 and G A manufacturing method comprising:
  • the method for producing a collagen or atelocollagen degradation product according to the present embodiment includes a chemical bond between X 1 and X 2 in the amino acid sequence represented by the following (1) in the triple helical domain of collagen or atelocollagen.
  • a chemical bond between X 2 and G the chemical bond between G and X 3, the chemical bond between X 4 and G, and is selected from a chemical bond between X 6 and G
  • It may be a production method including a cutting step for cutting any one chemical bond: (1) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -G- (However, G is glycine, and X 1 to X 6 are arbitrary amino acids).
  • a production method including a cleavage step of cleaving any one chemical bond selected from a chemical bond between X 14 and a chemical bond between X 14 and G: (2) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -GX 7 -X 8 -GX 9 -X 10 -GX 11 -X 12 -GX 13 -X 14 -G- (However, G is glycine, and X 1 to X 14 are arbitrary amino acids).
  • the above-mentioned cleavage step may be a step for cleaving a chemical bond at a specific position of the amino acid sequence represented by (1) or (2), and the specific configuration is not particularly limited.
  • the cutting step may be a step of actually cleaving a chemical bond in the triple helical domain to produce a degradation product of collagen or atelocollagen (for example, an enzymatic method).
  • a step of producing a degradation product of collagen or atelocollagen in which chemical bonds in the triple helical domain have already been cleaved may be included in the concept of “cleavage step” in the present application. it can.
  • the cleavage step can be configured as follows.
  • the above-mentioned cutting step can be performed by degrading collagen or atelocollagen with an enzyme (for example, protease (for example, cysteine protease)).
  • an enzyme for example, protease (for example, cysteine protease)
  • the enzyme is not particularly limited, but for example, cysteine protease is preferably used.
  • cysteine protease it is preferable to use a cysteine protease having a larger amount of acidic amino acids than a basic amino acid amount, or a cysteine protease active at a hydrogen ion concentration in an acidic region.
  • cysteine proteases include cathepsin B [EC 3.4.22.1], papain [EC 3.4.22.2], ficin [EC 3.4.22.3], actinidin [EC 3. 4.2.14], cathepsin L [EC 3.4.22.15], cathepsin H [EC 3.4.222.16], cathepsin S [EC 3.4.22.27], bromelain [EC 3 4.2.32], cathepsin K [EC 3.4.22.38], alloline, calcium-dependent protease, and the like.
  • papain, ficin, actinidine, bromelain, cathepsin K, or alloline are preferably used, and papain, ficin, actinidine, and cathepsin K are more preferably used.
  • the enzyme described above can be obtained by a known method.
  • it can be obtained by preparation of an enzyme by chemical synthesis; extraction of an enzyme from cells or tissues of bacteria, fungi, various animals and plants; preparation of an enzyme by genetic engineering means;
  • commercially available enzymes can also be used.
  • the cleavage step can be performed according to the following method (i) or (ii).
  • an enzyme for example, protease
  • the cleavage step can be performed according to the following method (i) or (ii).
  • the following methods (i) and (ii) are merely examples of the cutting step, and the present invention is not limited to these methods (i) and (ii).
  • Specific examples of the method (i) described above include a method of bringing collagen or atelocollagen into contact with an enzyme in an aqueous solution containing a high concentration of salt.
  • Specific examples of the method (ii) described above include, for example, a method in which an aqueous solution containing a high concentration salt and an enzyme are contacted in advance, and then the enzyme is contacted with collagen or atelocollagen.
  • aqueous solution is not particularly limited, for example, water can be used.
  • the specific structure of the salt is not particularly limited, but chloride is preferably used.
  • the chloride is not particularly limited, but for example, it is possible to use NaCl, KCl, and LiCl or MgCl 2.
  • the concentration of the salt in the aqueous solution is not particularly limited, but it can be said that a higher concentration is preferable.
  • the concentration is preferably 200 mM or higher, more preferably 500 mM or higher, more preferably 1000 mM or higher, more preferably 1500 mM or higher, and most preferably 2000 mM or higher.
  • the upper limit value of the salt concentration in the aqueous solution is not particularly limited, but may be, for example, 2500 mM.
  • the salt concentration is higher than 2500 mM, most of the protein is salted out, and as a result, the degradation efficiency of collagen or atelocollagen by the enzyme tends to be lowered.
  • the salt concentration is 2500 mM or less, the degradation efficiency of collagen or atelocollagen by the enzyme can be increased.
  • the salt concentration in the aqueous solution is preferably 200 mM or more and 2500 mM or less, more preferably 500 mM or more and 2500 mM or less, more preferably 1000 mM or more and 2500 mM or less, and more preferably 1500 mM or more and 2500 mM or less. More preferably, it is 2000 mM or more and 2500 mM or less.
  • the upper limit value of the salt concentration in the aqueous solution is preferably 500 mM or 800 mM.
  • the higher the salt concentration in the aqueous solution the higher the specificity of the enzyme or atelocollagen cleavage site.
  • the collagen or atelocollagen degradation product of the present embodiment can be made more uniform and highly bioactive.
  • the amount of collagen or atelocollagen dissolved in the aqueous solution is not particularly limited.
  • the amount of the enzyme to be added to the aqueous solution is not particularly limited. For example, it is preferable to add 10 to 20 parts by weight of the enzyme with respect to 100 parts by weight of collagen or atelocollagen.
  • conditions for contacting collagen or atelocollagen with an enzyme in an aqueous solution are not particularly limited, and can be set as appropriate, but within the following ranges. Is preferred.
  • the pH of the aqueous solution is preferably pH 2.0 to 7.0, and more preferably pH 2.5 to 6.5.
  • a known buffer can be added to the aqueous solution. If it is the said pH, collagen or atelocollagen can be melt
  • the temperature is not particularly limited, and the temperature may be selected according to the enzyme used.
  • the temperature is preferably 15 ° C. to 40 ° C., and more preferably 20 ° C. to 35 ° C.
  • the contact time is not particularly limited, and the contact time may be selected according to the amount of enzyme and / or the amount of collagen or atelocollagen.
  • the time is preferably 1 hour to 60 days, more preferably 1 day to 7 days, and even more preferably 3 days to 7 days.
  • the step of removing the impurities can be performed by a general method for separating substances.
  • the step of removing the impurities can be performed, for example, by dialysis, salting out, gel filtration chromatography, isoelectric precipitation, ion exchange chromatography, hydrophobic interaction chromatography, or the like.
  • the cutting step can be performed by degrading collagen or atelocollagen with an enzyme.
  • the collagen or atelocollagen to be decomposed may be contained in the living tissue. That is, the cutting step can be performed by bringing a living tissue and an enzyme into contact with each other.
  • the biological tissue is not particularly limited, and examples thereof include mammalian or avian dermis, tendon, bone or fascia, or fish skin or scales.
  • the acidic condition is preferably pH 2.5 to 6.5, more preferably pH 2.5 to 5.0, more preferably pH 2.5 to 4.0, and most preferably pH 2.5 to 3.5. It is.
  • the collagen contained in the bone is brought into contact with the cysteine protease by bringing the cysteine protease into contact with the bone. It is preferable to make it.
  • bone and cysteine protease are contacted in the presence of a salt having a concentration of 200 mM or more.
  • the cutting step can be configured as follows.
  • polypeptide chain constituting collagen or atelocollagen
  • the polypeptide chain may be appropriately selected according to the type of collagen or atelocollagen, and may be one type of polypeptide chain or multiple types of polypeptide chains.
  • polypeptide chain containing the chemical bond to be cleaved and the position of the chemical bond to be cleaved are determined from the above polypeptides, and the desired polypeptide is assumed when the chemical bond is cleaved. Determine the amino acid sequence of the chain.
  • a desired polypeptide chain is synthesized by a well-known chemical synthesis method according to the determined amino acid sequence.
  • the cutting process can be performed as described above.
  • the method for producing a collagen or atelocollagen degradation product according to the present embodiment may include steps other than the cutting step described above.
  • the method for producing a degradation product of collagen or atelocollagen according to the present embodiment may include a step of purifying a synthesized polypeptide chain after a desired polypeptide chain is synthesized by a well-known chemical synthesis method. Good. Note that the purification may be appropriately performed using a well-known column.
  • the method for producing a degradation product of collagen or atelocollagen according to the present embodiment may include a step of mixing a desired polypeptide chain and another polypeptide chain.
  • strand it does not specifically limit as another polypeptide chain
  • the cleavage step can be configured as follows.
  • polypeptide chain constituting collagen or atelocollagen
  • the polypeptide chain may be appropriately selected according to the type of collagen or atelocollagen, and may be one type of polypeptide chain or multiple types of polypeptide chains.
  • polypeptide chain containing the chemical bond to be cleaved and the position of the chemical bond to be cleaved are determined from the above polypeptides, and the desired polypeptide is assumed when the chemical bond is cleaved.
  • the amino acid sequence and DNA sequence of the chain are determined.
  • DNA encoding the desired polypeptide chain is inserted into a known protein expression vector. Then, after the protein expression vector is introduced into a desired host (for example, E. coli, yeast, insect cell, animal cell, etc.), the polypeptide chain after the chemical bond is cleaved is expressed in the host.
  • a desired host for example, E. coli, yeast, insect cell, animal cell, etc.
  • the cutting process can be performed as described above.
  • the method for producing a collagen or atelocollagen degradation product according to the present embodiment may include steps other than the cutting step described above.
  • the method for producing a collagen or atelocollagen degradation product of the present embodiment may include a step of purifying the expressed polypeptide chain after expressing the desired polypeptide chain in the host.
  • the purification may be appropriately performed using a well-known column.
  • the method for producing a degradation product of collagen or atelocollagen according to the present embodiment may include a step of mixing a desired polypeptide chain and another polypeptide chain.
  • strand it does not specifically limit as another polypeptide chain
  • Examples of uses of the collagen or atelocollagen degradation product of the present invention include spheroid-forming compositions.
  • the spheroid-forming composition of the present embodiment contains the collagen or atelocollagen degradation product of the present invention.
  • the amount of collagen or atelocollagen degradation product contained in the spheroid-forming composition of the present embodiment is not particularly limited.
  • the spheroid-forming composition of the present embodiment may contain 0.1% to 100% by weight of collagen or atelocollagen degradation product, or 50% to 100% by weight. It may be contained in an amount of 90% by weight to 100% by weight.
  • the present invention is not limited to the configuration described above.
  • composition for forming spheroids of the present embodiment may contain a composition other than collagen or atelocollagen degradation products. These configurations are not particularly limited, and a desired configuration can be appropriately added.
  • the present invention can also be configured as follows.
  • ⁇ 1> degradation product of collagen or atelocollagen of the present invention the amino acid sequence represented by the following (1) or (2) in the triple helical domain of the collagen or atelocollagen, chemical between X 1 and X 2 bonding, chemical bonding between X 2 and G, the chemical bond between G and X 3, the chemical bond between X 4 and G, chemical bond between X 6 and G, G and X 7 chemical bonding, between or chemical bonding between X 14 and G is disconnected, and characterized in that it is a degradation product of collagen or atelocollagen: (1) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -G- (2) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -GX 7 -X 8 -GX 9 -X 10 -GX 11 -X 12 -GX 13 -X 14 -G- (However, G is glycine, and
  • the amino acid sequence represented by (1) or (2) is preferably the amino acid sequence of the amino terminal of the triple helical domain.
  • the cleavage is performed in at least one of the ⁇ 1 chain and the ⁇ 2 chain of the collagen or atelocollagen.
  • the cleavage may be performed by contacting the collagen or atelocollagen with a cysteine protease in the presence of a salt having a concentration of 200 mM or more. preferable.
  • the cleavage may be performed by bringing the cysteine protease after contact with a salt having a concentration of 200 mM or more into contact with the collagen or atelocollagen. preferable.
  • the spheroid-forming composition of the present invention is characterized by containing the degradation product of collagen or atelocollagen of the present invention.
  • Method for producing a ⁇ 7> collagen or degradation product of the atelocollagen of the present invention the amino acid sequence represented by the following in a triple helical domain of collagen or atelocollagen (1) or (2), between X 1 and X 2 Chemical bond between X 2 and G, chemical bond between G and X 3 , chemical bond between X 4 and G, chemical bond between X 6 and G, G and It is characterized by comprising a cleavage step that cleaves the chemical bond between X 7 or the chemical bond between X 14 and G: (1) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -G- (2) -GX 1 -X 2 -GX 3 -X 4 -GX 5 -X 6 -GX 7 -X 8 -GX 9 -X 10 -GX 11 -X 12 -GX 13 -X 14 -G- (However, G is glycine, and X 1 to
  • the amino acid sequence represented by (1) or (2) is preferably the amino terminal amino acid sequence of the triple helical domain.
  • the cleavage is performed in at least one of the ⁇ 1 chain and ⁇ 2 chain of the collagen or atelocollagen.
  • the collagen or atelocollagen is contacted with cysteine protease in the presence of a salt having a concentration of 200 mM or more.
  • the cysteine protease after contact with a salt having a concentration of 200 mM or more is brought into contact with the collagen or atelocollagen.
  • the method for producing a degradation product of collagen or atelocollagen according to the present invention comprises contacting the collagen contained in the bone with the cysteine protease by bringing the cysteine protease into contact with the bone in the cutting step. Is preferred.
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • actinidine what was refine
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 1 shows the amino terminus of the ⁇ 1 chain degradation product and the amino acid sequence in the vicinity thereof when the salt concentration is 0 mM, 200 mM, 1000 mM, 1500 mM, or 2000 mM.
  • the cutting site when the salt concentration was high was a new cutting site found by the present inventors.
  • the amount of the degradation product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 2 and the degradation product having the amino terminus of the amino acid sequence shown in SEQ ID NO: 3 contained in the degradation product The ratio of the amount of was different.
  • NaCl became insoluble when the salt concentration exceeded 2000 mM.
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • type I collagen derived from rat tail or type I collagen derived from chicken skin was dissolved in 50 mM citrate buffer (pH 3.0) containing salt.
  • An aqueous solution containing actinidine and type I collagen derived from rat tail or type I collagen derived from chicken skin were contacted at 20 ° C. for 10 days or longer to prepare a degradation product of type I collagen.
  • actinidine the same thing as what was used in the Example of ⁇ 1> mentioned above was used.
  • type I collagen derived from rat tail and type I collagen derived from chicken skin were purified based on a well-known method (see, for example, Non-Patent Document 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 2 shows the amino terminus of the rat ⁇ 1-chain degradation product at a salt concentration of 2000 mM and the amino acid sequence in the vicinity thereof, and the partial structure of the rat ⁇ 1-chain that has not been degraded (the column for salt concentration is “ Refer to data that is “-”).
  • Table 3 shows amino acid sequences at and near the amino terminus of the chicken ⁇ 1-chain degradation product when the salt concentration is 2000 mM, and the partial structure of the undegraded chicken-derived ⁇ 1 chain (the salt concentration column is “ Refer to data that is “-”).
  • the cutting site when the salt concentration was high was a new cutting site found by the present inventors.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 4 shows amino acid sequences at and near the amino terminus of the ⁇ 1-chain degradation product derived from pigs when an aqueous solution with a concentration of MgCl 2 of 500 mM and an aqueous solution with a concentration of KCl of 200 mM are used. , And the partial structure of the undegraded ⁇ 1 chain derived from swine (see data in which the salt concentration column is “ ⁇ ”).
  • the cutting site was a new cutting site found by the present inventors.
  • cathepsin K which is a kind of cysteine protease, was used to study the ⁇ 1 chain cleavage site under high salt conditions. The test method and test results will be described below.
  • a 50 mM citrate buffer solution (pH 3.0) having a sodium chloride concentration of 2000 mM was prepared. Note that water was used as the solvent of the aqueous solution.
  • chicken-derived type I collagen or porcine-derived type I collagen was dissolved in 50 mM citrate buffer (pH 3.0) containing salt.
  • An aqueous solution containing cathepsin K and the solution containing chicken-derived type I collagen or porcine-derived type I collagen were contacted at 20 ° C. for 10 days or longer to prepare a degradation product of type I collagen.
  • chicken type I collagen and porcine type I collagen were purified based on a well-known method (see, for example, Non-Patent Document 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 5 shows amino acid sequence at and near the amino terminus of the degradation product of swine-derived ⁇ 1 chain, and the partial structure of undegraded porcine-derived ⁇ 1 chain (salt concentration column is “ ⁇ ”) ).
  • cathepsin K which is a kind of cysteine protease, is cleaved inside the triple helical domain when the salt concentration is high.
  • Actinidine was put into a dialysis tube, and the actinidine was dialyzed against an external dialysis solution having a sodium chloride concentration of 2000 mM. Thereafter, the external dialyzate was changed to distilled water, and dialysis was continued to obtain actinidine.
  • actinidine what was refine
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • porcine type I collagen was dissolved in 50 mM citrate buffer (pH 3.0) containing salt. An aqueous solution containing actinidine was contacted with porcine-derived type I collagen at 20 ° C. for 3 days or longer to prepare a degradation product of type I collagen. Further, porcine type I collagen was purified based on a well-known method (for example, see Non-Patent Document 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 6 shows the amino terminal sequence of the ⁇ 1-chain degradation product derived from swine when the dialysis salt concentration is 2000 mM and the amino acid sequence in the vicinity thereof, and the partial structure of undegraded porcine-derived ⁇ 1 chain (in the column for salt concentration). Refer to data with “-”).
  • the cutting site when the salt concentration was high was a new cutting site found by the present inventors.
  • Actinidine was put into a dialysis tube, and the actinidine was dialyzed against an external dialysis solution having a sodium chloride concentration of 2000 mM. Thereafter, the external dialyzate was changed to distilled water, and dialysis was continued to obtain actinidine.
  • actinidine what was refine
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • human-derived type I collagen was dissolved in 50 mM citrate buffer (pH 3.5) containing salt. An aqueous solution containing actinidine was contacted with human-derived type I collagen at 20 ° C. for 10 days or longer to prepare a degradation product of type I collagen. Moreover, human-derived type I collagen was purified based on a well-known method (for example, refer nonpatent literature 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the ⁇ 1 chain degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • the cutting site when the salt concentration was high was a new cutting site found by the present inventors.
  • the cells after a predetermined time elapsed from the start of the culture were observed with a microscope.
  • 1 to 7 show micrographs of each cell.
  • FIGS. 1 (a) and (b) show normal human dermal fibroblast NHDF (C-12302, PromoCell), and FIGS. 2 (a) and (b) show normal human umbilical vein endothelial cells HUVEC ( C-12203, PromoCell),
  • FIGS. 3 (a) and 3 (b) are mouse osteoblast precursor cell line MC3T3-E1 subclone 4 (ATCC® CRL-2593, Sumisho Pharma International Co., Ltd.)
  • FIG. 4 (a) and (b) are mouse adipocyte progenitor cell lines MC3T3-G2 / PA6 (RBRC-RCB1127, RIKEN BioResource Center), FIGS.
  • FIGS. 5 (a) and (b) are human bone marrow Mesenchymal stem cells MSC (PT-034, Lonza Japan)
  • FIGS. 6 (a) and (b) are rat bone marrow mesenchymal stem cells MS (BMC01, Ltd. primary cell)
  • FIG. 7 (a) and (b) shows a photomicrograph of primary mouse embryonic fibroblasts MEF (R-PMEF-CFL, DS Pharma Biomedical Co., Ltd.).
  • PHY indicates the result of a culture plate in contact with commercially available pepsin-treated type I collagen
  • LASCol indicates culture in contact with a collagen degradation product of this example. The result of the plate is shown.
  • Cells (mouse fibroblast NIH / 3T3 (RBRC-RCB2767, RIKEN BioResource Center)) were seeded on each culture plate, and cultured under conditions of 37 ° C. and 5% CO 2 .
  • the collagen degradation product of this example can not only form spheroids earlier, but also can form large spheroids as compared with the conventional collagen degradation products.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • Primary human mesenchymal stem cells (LONZA) are suspended in a growth medium (MSCBM + MSCGM, LONZA) dedicated to the cells, and then seeded on the culture plate described above, under conditions of 37 ° C. and 5% CO 2 .
  • the primary human mesenchymal stem cells were allowed to adhere onto the culture plate. Thereafter, differentiation was induced for 5 days according to a known method, and primary human mesenchymal stem cells were differentiated into human osteoblasts.
  • FIG. 9B shows the state of osteoblasts after differentiation induction. As is apparent from FIG. 9B, the collagen degradation product of this example significantly promoted the formation of spheroids in osteoblasts.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • Mouse MC3T3-G2 / PA6 cells (RIKEN BRC, adipocytes) were suspended in a growth medium (10% FBS DMEM) and then seeded on the above-described culture plate, and conditions of 37 ° C. and 5% CO 2 were obtained. Under culture for 1 day, mouse MC3T3-G2 / PA6 cells were allowed to adhere onto the culture plate. The state of mouse MC3T3-G2 / PA6 cells (in other words, the fat cells) after adhesion is shown in FIG. 9 (c). As is clear from FIG. 9 (c), the collagen degradation product of this example significantly promoted the formation of spheroids in the preadipocytes.
  • FIG. 9 (d) shows the state of the human breast cancer cells MCF-7 (in other words, breast cancer cells) after adhesion.
  • the collagen degradation product of this example significantly promoted spheroid formation of breast cancer cells.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • Human lung cancer cells A549 (RIKEN BRC, RCB0098) are suspended in a growth medium (10% FBS DMEM), seeded on the above-described culture plate, and cultured for 7 days under conditions of 37 ° C. and 5% CO 2. Then, human lung cancer cells A549 were adhered on the culture plate.
  • the state of human lung cancer cells A549 after adhesion is shown in FIG.
  • FIG. 9 (e) the collagen degradation product of this example significantly promoted spheroid formation of lung cancer cells.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • Primary cells collected from the rat ovary are suspended in a growth medium (10% FBS DMEM), seeded on the above-described culture plate, and cultured for 1 day under conditions of 37 ° C. and 5% CO 2 .
  • the ovary-derived primary cells were allowed to adhere on the culture plate.
  • the state of primary cells derived from ovaries after adhesion is shown in FIG.
  • FIG. 9 (f) the collagen degradation product of this example significantly promoted spheroid formation of ovary-derived primary cells.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • Mouse embryo-derived fibroblasts (MEF) are suspended in a growth medium (10% FBS DMEM), seeded on the above-described culture plate, and cultured for one day under conditions of 37 ° C. and 5% CO 2. Then, the fetal fibroblasts were adhered on the culture plate. The state of the fibroblast after adhesion is shown in FIG. 9 (g). As is apparent from FIG. 9 (g), the collagen degradation product of this example significantly promoted spheroid formation of fibroblasts.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • growth medium (10% FBS DMEM)
  • the mouse ES cells were allowed to adhere on a culture plate.
  • the state of the ES cells after adhesion is shown in FIG. 9 (h).
  • the collagen degradation product of this example significantly promoted spheroid formation of ES cells.
  • a commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine collagen at a salt concentration of 200 mM) was used for the test.
  • a cell line derived from mouse embryocarcinoma (P19.CL6) was suspended in a growth medium (10% FBS MEM ⁇ ), seeded on the above-mentioned culture plate, and 1 under 37 ° C. and 5% CO 2 conditions.
  • the cell line derived from the embryonic carcinoma was allowed to adhere on the culture plate.
  • the state of the cell line derived from embryonic carcinoma after adhesion is shown in FIG. 9 (i).
  • the collagen degradation product of this example markedly promoted spheroid formation of cell lines derived from embryonic carcinoma.
  • a commercial culture plate contacted with yellowfin tuna collagen degradation product (collagen degradation product derived from yellowfin tuna at a salt concentration of 200 mM) was used for the test. After suspending the mouse NIH / 3T3 fibroblasts in growth medium (10% CS DMEM), seeded on culture plates as described above, 37 ° C., and cultured for 1 day under the condition of 5% CO 2, The fibroblasts were allowed to adhere on the culture plate. The appearance of the fibroblast after adhesion is shown in FIG. As is clear from FIG. 9 (j), the collagen degradation product of this example significantly promoted the formation of spheroids in fibroblasts.
  • Actinidine was put into a dialysis tube, and the actinidine was dialyzed against an external dialysis solution having a sodium chloride concentration of 2000 mM. Thereafter, the external dialyzate was changed to distilled water, and dialysis was continued to obtain actinidine.
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • fish type I collagen (specifically yellowfin tuna) was dissolved in 50 mM citrate buffer (pH 3.0) containing salt.
  • An aqueous solution containing actinidine and fish-derived type I collagen were contacted at 20 ° C. for 3 days or longer to prepare a degradation product of type I collagen.
  • actinidine the same thing as what was used in the Example of ⁇ 1> mentioned above was used.
  • fish-derived type I collagen was purified based on a well-known method (for example, see Non-Patent Document 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the degradation product of ⁇ 1 chain (fish-derived type I collagen) transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 8 shows the amino terminus and the amino acid sequence in the vicinity of the ⁇ 1-chain degradation product derived from fish when the salt concentration of the dialysis external solution is 2000 mM. As shown in Table 8, two types of degradation products of ⁇ 1 chain (fish-derived type I collagen) were detected, and amino acid sequences at the amino terminal of these degradation products are shown in SEQ ID NOs: 17 and 18, respectively. The amino acid sequence was successfully identified.
  • cathepsin K was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 45 minutes.
  • human-derived type I collagen was dissolved in 50 mM phosphate buffer (pH 6.0) containing salt.
  • An aqueous solution containing cathepsin K was contacted with human-derived type I collagen at 20 ° C. for 10 days or longer to prepare a degradation product of type I collagen.
  • cathepsin K the same thing as what was used in the Example of ⁇ 1> mentioned above was used.
  • human-derived type I collagen was purified based on a well-known method (for example, refer nonpatent literature 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the degradation product of ⁇ 2 chain (human-derived type I collagen) transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 9 shows the amino terminus and the amino acid sequence in the vicinity of the ⁇ 2-chain degradation product derived from human when the salt concentration of the reaction solution is 200 mM.
  • Actinidine was put into a dialysis tube, and the actinidine was dialyzed against an external dialysis solution having a sodium chloride concentration of 2000 mM. Thereafter, the external dialyzate was changed to distilled water, and dialysis was continued to obtain actinidine.
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • chicken-derived type I collagen was dissolved in 50 mM citrate buffer (pH 3.0) containing salt.
  • An aqueous solution containing actinidine was contacted with chicken-derived type I collagen at 20 ° C. for 7 days or longer to prepare a degradation product of type I collagen.
  • actinidine the same thing as what was used in the Example of ⁇ 1> mentioned above was used.
  • chicken type I collagen was purified based on a well-known method (for example, see Non-Patent Document 2).
  • the degradation product described above was subjected to polyacrylamide gel electrophoresis to separate the degradation product of type I collagen.
  • the degradation product of type I collagen was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the degradation product of ⁇ 2 chain (chicken-derived type I collagen) transferred to the PVDF membrane was determined by the Edman degradation method.
  • Table 10 shows the amino terminus of the chicken ⁇ 2-chain degradation product and the amino acid sequence in the vicinity thereof when the salt concentration of the dialysis external solution is 2000 mM.
  • Actinidine was placed in a dialysis tube, and the actinidine was dialyzed against an external dialysis solution having a sodium chloride concentration of 2000 mM. Thereafter, the external dialyzate was changed to distilled water, and dialysis was continued to obtain actinidine.
  • actinidine what was refine
  • actinidine was dissolved in 50 mM phosphate buffer (pH 6.5) containing 10 mM dithiothreitol and allowed to stand at 25 ° C. for 90 minutes.
  • the buffer solution and the bone were mixed in the following combinations (i) to (iii). That is, (i) 50 mM citrate buffer (pH 3.0) containing salt and chicken ulna (45 mg (wet weight)) were mixed, (ii) 50 mM citrate buffer (pH 2.5, 3. 0) and porcine tibia (20 mg (dry weight)), or (iii) 50 mM oxalate buffer (pH 4.5, 5.0, 5.5) containing salt and porcine tibia (20 mg (dry weight)) ).
  • the aqueous solution containing actinidine and the solutions (i) to (iii) containing bone were brought into contact at 20 ° C. for 10 days or longer to prepare a degradation product of collagen.
  • the buffer solution and the bone were mixed in the following combinations (iv) to (v). That is, (iv) 50 mM citrate buffer solution (pH 3.0) containing salt dissolved in porcine pepsin and chicken ulna (45 mg (wet weight)), (v) salt dissolved in porcine pepsin 50 mM citrate buffer solution (pH 3.0) and porcine tibia (20 mg (dry weight)) were mixed.
  • aqueous solution containing porcine pepsin and the solutions (iv) to (v) containing bone were contacted at 20 ° C. for 7 days or longer to prepare a degradation product of collagen.
  • the difference in the recovery rate is obvious, and the bone-derived solubilized collagen obtained by the method of this example can be used for the same purpose as collagen derived from the dermis.
  • Bone and dentin are classified as hard tissues, and unlike soft tissues such as dermis and tendons, they have been considered to be unfavorable raw materials for recovering collagen or collagen degradation products. .
  • a method for extracting collagen having a triple-stranded helical structure from bone has not been reported so far, and it has been limited to extracting gelatin from bone by heat-denaturing the bone.
  • the solid content of the bone was completely dissolved, and a large amount of collagen degradation product and bone matrix protein were successfully recovered.
  • FIG. 10 (a) and FIG. 10 (b) show photographs of standing for 11 days after contacting the bone fragment and each enzyme.
  • actinidine was used, bone fragments remaining without digestion were not confirmed.
  • pepsin was used, a large bone fragment remaining without being digested was confirmed.
  • the bone matrix protein contained in the degradation product was identified by a peptide mass fingerprint method using a mass spectrometer. It can be confirmed that useful bone matrix proteins (for example, osteocalcin, etc.) unique to bones other than collagen can be efficiently recovered by solubilizing the bone tissue. Degradation products containing matrix proteins can be made. Bone submerged in a buffer solution to which no enzyme was added was not solubilized at all, and the shape of the bone fragment did not change over time.
  • the collagen degradation product was transferred to a PVDF (Polyvinylidene Difluoride) membrane by a conventional method.
  • the amino terminal amino acid sequence of the degradation product transferred to the PVDF membrane was determined by the Edman degradation method.
  • the obtained degradation product contained a degradation product in which the amino terminus and the amino acid sequence in the vicinity thereof correspond to the amino acid sequence represented by SEQ ID NO: 14.
  • a commercially available culture plate contacted with a collagen degradation product prepared from bone tissue (a collagen degradation product derived from porcine tibia) was used for the test.
  • a collagen degradation product prepared from bone tissue a collagen degradation product derived from porcine tibia
  • MSCGM human bone marrow mesenchymal stem cells
  • the cells are seeded on the above-mentioned culture plate and cultured for 1 day under conditions of 37 ° C. and 5% CO 2.
  • the appearance of the fibroblast after adhesion is shown in FIG.
  • the bone-derived collagen degradation product of this example significantly promoted spheroid formation of fibroblasts.
  • the present invention relates to a composition for forming a spheroid, a food additive, a medical material, a cosmetic material, a culture material for culturing cells or embryos (eg, a coating material of a culture apparatus (eg, a culture dish), a medium component) It can be used for

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Abstract

L'invention fournit un fragment de collagène ou atélocollagène possédant de nouvelles fonctions physiologiques, et un procédé de fabrication ainsi qu'une application de celui-ci. Dans cet objectif, l'invention met en œuvre le fragment de collagène ou atélocollagène dans lequel une liaison chimique de position spécifique à l'intérieur d'un collagène ou d'un domaine hélicoïdal triple d'atélocollagène, est coupée.
PCT/JP2015/063044 2014-04-30 2015-04-30 Fragment de collagène ou atélocollagène, et procédé de fabrication ainsi qu'application de celui-ci WO2015167003A1 (fr)

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JP6991570B2 (ja) 2015-10-30 2022-02-03 学校法人近畿大学 分化誘導剤、分化誘導方法、および、これらに用いられる骨組織分解物の製造方法
JPWO2017073785A1 (ja) * 2015-10-30 2018-08-16 学校法人近畿大学 分化誘導剤、分化誘導方法、および、これらに用いられる骨組織分解物の製造方法
WO2017073785A1 (fr) * 2015-10-30 2017-05-04 学校法人近畿大学 Inducteur de différentiation et procédé d'induction de la différentiation, et procédé de production d'un tissu osseux décomposé utilisé à cet effet
WO2018186185A1 (fr) * 2017-04-06 2018-10-11 日機装株式会社 Procédé de culture de cellules, procédé de fabrication d'un composite de support de cellules, cellules cultivées et composite de support de cellules
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JPWO2018186185A1 (ja) * 2017-04-06 2019-12-19 日機装株式会社 細胞の培養方法、細胞支持複合体の製造方法、培養細胞及び細胞支持複合体
JP6995112B2 (ja) 2017-04-06 2022-02-04 日機装株式会社 細胞の培養方法及び細胞支持複合体の製造方法
WO2019151450A1 (fr) * 2018-01-31 2019-08-08 学校法人近畿大学 Substance de culture de cellules nerveuses et agent thérapeutique pour lésion nerveuse
JPWO2019151444A1 (ja) * 2018-01-31 2021-01-07 国立大学法人神戸大学 椎間板変性の治療剤および椎間板細胞培養材
JPWO2019151450A1 (ja) * 2018-01-31 2021-01-28 学校法人近畿大学 神経細胞培養材および神経損傷治療剤
JP7012970B2 (ja) 2018-01-31 2022-01-31 学校法人近畿大学 神経損傷治療剤
CN111868226A (zh) * 2018-01-31 2020-10-30 学校法人近畿大学 神经细胞培养材料及神经损伤治疗剂
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CN111867645B (zh) * 2018-01-31 2022-05-27 国立大学法人神户大学 椎间盘退变的治疗剂及椎间盘细胞培养材料
US11951231B2 (en) 2018-01-31 2024-04-09 Kinki University Therapeutic agent for intervertebral disc degeneration and material for culturing inter vertebral disc cells
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