WO2015167004A1 - 分化誘導用組成物 - Google Patents
分化誘導用組成物 Download PDFInfo
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- WO2015167004A1 WO2015167004A1 PCT/JP2015/063045 JP2015063045W WO2015167004A1 WO 2015167004 A1 WO2015167004 A1 WO 2015167004A1 JP 2015063045 W JP2015063045 W JP 2015063045W WO 2015167004 A1 WO2015167004 A1 WO 2015167004A1
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- Prior art keywords
- collagen
- atelocollagen
- degradation product
- amino acid
- chemical bond
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Images
Classifications
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- 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
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
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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
- A23J3/30—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
- A23J3/341—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins
- A23J3/342—Working-up of proteins for foodstuffs by hydrolysis using chemical agents using enzymes of animal proteins of collagen; of gelatin
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- 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
-
- 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
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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
- A23J3/30—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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2513/00—3D culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
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- 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
Definitions
- the present invention relates to a composition for inducing differentiation.
- 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-mentioned conventional problems, and an object of the present invention is to provide a composition for inducing differentiation containing a degradation product of collagen or atelocollagen.
- the composition for inducing differentiation of the present invention is a composition for inducing differentiation for inducing cell differentiation, including a degradation product of collagen or atelocollagen, It is characterized by containing at least part of the triple helical domain of collagen or atelocollagen.
- the present invention has the effect of being able to induce cell differentiation.
- the present invention has an effect that differentiation of many cells can be induced.
- the present invention has an effect that cell differentiation can be induced quickly.
- the present invention has an effect that cell differentiation can be maintained.
- FIG. 2 shows a photomicrograph of a mouse fibroblast NIH / 3T3 spheroid induced by a degradation product of an example of the present invention.
- cultivation plate contacted with the decomposition product of the Example of this invention is shown.
- cultivation contacted with the commercially available pepsin-treated type I collagen is shown.
- cultivation which is not made to contact the degradation product of collagen is shown.
- staining at the time of using commercially available Nano Culture Dish is shown.
- staining at the time of using various types of culture plates is shown. It is a graph which shows the result of the elemental analysis of the cell at the time of using the commercially available culture
- the composition for inducing differentiation of the present embodiment is a composition for inducing cell differentiation, and contains a degradation product of collagen or atelocollagen as a main component, and the degradation product is a triple helical of the collagen or atelocollagen. It contains at least part of the domain. That is, the degradation product may include the entire triple helical domain of collagen or atelocollagen, or may include a part of the triple helical domain.
- the differentiation-inducing composition of the present embodiment is a differentiation-inducing composition containing a degradation product of collagen or atelocollagen, and may be the following:
- composition for inducing differentiation of the present embodiment contains collagen or a degradation product of atelocollagen as a main component.
- 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 composition for differentiation induction of this Embodiment can be adjusted to 15 degreeC or more preferably, More preferably, 20 degreeC or more.
- the composition for differentiation induction 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 “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 may be any of ⁇ 1 chain, ⁇ 2 chain, and ⁇ 3 chain.
- the polypeptide chain in which the chemical bond is broken is at least one of ⁇ 1 chain and ⁇ 2 chain among the above-mentioned polypeptide chains.
- the polypeptide chain in which the chemical bond is broken is an ⁇ 1 chain among the polypeptide chains described above.
- a degradation product of collagen or atelocollagen is prepared by enzymatic treatment, it can be easily cleaved only with a specific polypeptide chain.
- the degradation product of collagen or atelocollagen may be one in which three polypeptide chains form a helical structure.
- the degradation product of collagen or atelocollagen may be one in which three polypeptide chains do not form a helical structure, or three polypeptide chains do not partially form a helical structure. Whether or not the three polypeptide chains form a helical structure can be confirmed by a known method (for example, circular dichroism spectrum).
- Collagen or atelocollagen degradation products basically contain three polypeptide chains, but chemical bond breakage may occur in one of the three polypeptide chains. Chemical bond breakage may occur in two polypeptide chains of one polypeptide chain, or chemical bond breakage may occur in all three polypeptide chains.
- 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 degradation product of collagen or atelocollagen 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 cross-linking agent is preferably 10 ⁇ 3 to 10% by mass with respect to the degradation product of collagen or atelocollagen.
- a crosslinked structure can be formed by bringing a degradation product of collagen or atelocollagen into contact with a crosslinking agent at 5 to 40 ° C. for 3 to 48 hours.
- a crosslinked structure can be formed by irradiating collagen or atelocollagen degradation products with ultraviolet rays for 3 to 48 hours, for example, at room temperature using an ultraviolet lamp or the like.
- a crosslinked structure can be formed by heating a degradation product of collagen or atelocollagen under reduced pressure, preferably at a temperature of about 110 to 160 ° C. for about 3 to 48 hours.
- a collagen or atelocollagen degradation product having a cross-linked structure has the advantage of improved collagenase resistance and strength.
- the collagen or atelocollagen degradation product may be subjected to a desired chemical modification, if necessary.
- the chemical modification include acylation, myristylation, and polyethylene glycol modification.
- a degradation product subjected to succinylation which is a kind of acylation can be obtained by reacting a degradation product of collagen or atelocollagen with succinic anhydride in a neutral pH solvent such as a phosphate buffer.
- a neutral pH solvent such as a phosphate buffer.
- the degradation product subjected to polyethylene glycol modification can be obtained by reacting polyethylene glycol activated with cyanuric chloride with a degradation product of collagen or atelocollagen.
- the chemical bond between X 1 and X 2 and the chemistry between X 2 and G of the amino acid sequence represented by the following (1) in the above-described triple helical domain The bond, 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; (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 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).
- X 1 , X 3 and X 5 may be proline
- X 2 may be methionine
- X 4 may be alanine or serine
- X 6 may be arginine
- each of X 1 ⁇ X 6 may be the same configuration as the X 1 ⁇ X 6 described above.
- a specific configuration of X 7 to X 14 will be described below.
- 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 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.
- X 7 to X 14 , X 8 , X 9 , X 10 , X 12 and X 13 are proline or hydroxyproline
- X 7 is an amino acid having an aliphatic side chain (for example, Glycine, alanine, valine, leucine or isoleucine), and the other may be any amino acid.
- 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.
- 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.
- amino acid X 14 has is and undissociated side chains are hydrophilic (serine, threonine, asparagine or glutamine) may be used.
- X 7 ⁇ X 14, X 8 , X 9, X 10, X 12 and X 13 is proline or hydroxyproline
- X 7 is leucine
- X 11 is alanine
- X 14 may be glutamine.
- the amino acid sequence shown in (3) above is located at the amino terminus of the triple helical domain. That is, G located between Y 3 and Y 4 indicates glycine located on the most amino terminal side in the triple helical domain.
- Y 1 , Y 2 and Y 3 represent amino acids located on the amino terminal side of the triple helical domain in a plurality of types of polypeptide chains constituting collagen or atelocollagen.
- each of Y 1 ⁇ Y 9 are 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.
- Y 3 may be proline and Y 1 and Y 2 may be any amino acid.
- Y 3 is proline and Y 1 and Y 2 are amino acids having an aliphatic side chain (for example, glycine, alanine, valine, leucine or isoleucine) or amino acids having a hydroxyl group in the side chain (hydroxy Proline, hydroxylysine or serine).
- amino acids having an aliphatic side chain for example, glycine, alanine, valine, leucine or isoleucine
- amino acids having a hydroxyl group in the side chain hydroxy Proline, hydroxylysine or serine
- Y 3 may be proline
- Y 1 may be alanine or serine
- Y 2 may be valine
- Y 4 to Y 9 is not particularly limited, but Y 4 and X 1 are the same amino acid, Y 5 and X 2 are the same amino acid, and Y 6 and X 3 May be the same amino acid, Y 7 and X 4 may be the same amino acid, Y 8 and X 5 may be the same amino acid, and Y 9 and X 6 may be the same amino acid.
- the degradation product of collagen or atelocollagen used in the present invention can be liquid even at a temperature close to human body temperature. Therefore, if the composition for inducing differentiation of the present embodiment is used for human tissues (for example, bone, fat, cartilage, nerve), etc., the composition for inducing differentiation and the tissues can be easily adapted.
- the collagen or atelocollagen degradation product used in the present invention has a higher concentration at which gelation starts than the conventional collagen or atelocollagen degradation product. Therefore, the collagen or atelocollagen degradation product used in the present invention at the same concentration as the gelation of the conventional collagen or atelocollagen degradation product can be stably stored at room temperature. Therefore, the composition for inducing differentiation of the present embodiment can be stably stored at room temperature.
- a chemical bond between X 1 and X 2 of the amino acid sequence represented by (1) in the triple helical domain of collagen or atelocollagen, X 2 and G The chemical bond between G, X 3 , the chemical bond between X 4 and G, or the chemical bond between X 6 and G is broken.
- a chemical bond between X 1 and X 2 of the amino acid sequence represented by (2) in the triple helical domain of collagen or atelocollagen, X 2 and G A chemical bond between G, X 3 , a chemical bond between X 4 and G, a chemical bond between X 6 and G, a chemical bond between G and X 7 , or , The chemical bond between X14 and G is broken.
- the chemical bond between Y 1 and Y 2 in the amino acid sequence represented by (3) at the amino terminal of the triple helical domain of collagen or atelocollagen is cleaved. ing.
- 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.
- a method for producing a composition for inducing differentiation, comprising a degradation product of collagen or atelocollagen of the present embodiment A) Chemical bond between X 1 and X 2 , chemical bond between X 2 and G, G and X 3 in the amino acid sequence shown in the following (1) in the triple helical domain of collagen or atelocollagen chemical bonding, between the chemical binding, X 4 and G between or to cut the chemical bond between X 6 and G, the cutting process, or B) Chemical bond between X 1 and X 2 , chemical bond between X 2 and G, G and X 3 in the amino acid sequence shown in the following (2) in the triple helical domain of collagen or atelocollagen chemical bond between the chemical bond between X 4 and G, chemical bond between X 6 and G, chemical bond between G and X 7, or, between X 14 and G Breaking process, breaking chemical bonds, or C) A production method comprising a
- the manufacturing method of the composition for differentiation induction containing the degradation product of collagen or atelocollagen of this Embodiment may be a manufacturing method including the following cutting processes. That means D) Chemical bond between X 1 and X 2 , chemical bond between X 2 and G, G and X 3 in the amino acid sequence shown in the following (1) in the triple helical domain of collagen or atelocollagen chemical bond, chemical bond between X 4 and G, and disconnects any one chemical bond selected from a chemical bond between X 6 and G between the cutting step or, The amino acid sequence represented by the following (2) in the triple helical domain of E) collagen or atelocollagen, a chemical bond between X 1 and X 2, the chemical bond between X 2 and G, G and X 3 chemical bond between the chemical bond between X 4 and G, chemical bond between X 6 and G, chemical bond between G and X 7, and, between X 14 and G A cleavage step of cleaving any one chemical bond selected from chemical bonds, or F) A
- the cutting step is not particularly limited as long as it is a step of cutting a chemical bond at a specific position of the amino acid sequence represented by (1) to (3).
- 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).
- the cleavage step can be configured as follows.
- 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, cathepsin K, alloline or bromelain 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 methods (i) to (iii).
- an enzyme eg, protease
- the cleavage step can be performed according to the following methods (i) to (iii).
- the following methods (i) to (iii) are merely examples of the cutting step, and the present invention is not limited to these methods (i) to (iii).
- the following methods (i) and (ii) are examples of methods used for cleaving a chemical bond at a specific position of the amino acid sequence represented by (1) or (2).
- the method (iii) is an example of a method used for breaking a chemical bond at a specific position of the amino acid sequence shown in (3).
- 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.
- Specific examples of the method (iii) described above include a method of bringing collagen or atelocollagen into contact with an enzyme in an aqueous solution containing a low concentration salt.
- 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 containing the high concentration salt 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 of the salt concentration in the aqueous solution containing the high-concentration salt 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 concentration of the salt in the aqueous solution containing the high-concentration salt 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. It is more preferably 2500 mM or less, and most preferably 2000 mM or more and 2500 mM or less.
- the specificity of the cleavage site of collagen or atelocollagen by the enzyme can be increased as the salt concentration in the aqueous solution containing the high-concentration salt is higher.
- the degradation product of collagen or atelocollagen used in the present invention can be made more uniform and highly bioactive.
- the concentration of the salt in the aqueous solution containing the low-concentration salt is not particularly limited.
- the concentration is preferably lower than 200 mM, more preferably 150 mM or less, more preferably 100 mM or less, more preferably 50 mM or less, and most preferably about 0 mM.
- 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.
- bone and cysteine protease are contacted in the presence of a salt having a concentration lower than 200 mM.
- the cutting process 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 the composition for inducing differentiation of the present embodiment can include steps other than the cutting step described above.
- the method for producing a composition for inducing differentiation of the present embodiment may include a step of purifying a synthesized polypeptide chain after synthesizing a desired polypeptide chain by a well-known chemical synthesis method. . Note that the purification may be appropriately performed using a well-known column.
- the method for producing the composition for inducing differentiation of the present embodiment may include a step of mixing a desired polypeptide chain with 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 the composition for inducing differentiation of the present embodiment can include steps other than the cutting step described above.
- the method for producing the composition for inducing differentiation 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 the composition for inducing differentiation of the present embodiment may include a step of mixing a desired polypeptide chain with another polypeptide chain.
- strand it does not specifically limit as another polypeptide chain
- composition for inducing differentiation of the present embodiment induces cell differentiation.
- composition for inducing differentiation of the present embodiment induces differentiation of cells into bone (specifically, osteoblasts), fat, cartilage, nerves and the like.
- collagen or a degradation product of atelocollagen is included as a main component having differentiation inducing activity.
- the amount of collagen or atelocollagen degradation product contained in the differentiation-inducing composition of the present embodiment is not particularly limited, but the larger the amount of collagen or atelocollagen degradation product, the higher the differentiation-inducing effect, which is preferable.
- the composition for inducing differentiation of the present embodiment may contain 0.1 to 100% by weight of collagen or atelocollagen degradation product, of which 50 to 100% by weight is contained. More preferably, it is more preferably 90 to 100% by weight, and most preferably 100% by weight.
- composition for inducing differentiation of the present embodiment may be added with 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.
- the differentiation-inducing composition of the present invention is a differentiation-inducing composition for inducing cell differentiation, including a degradation product of collagen or atelocollagen, in order to solve the above problems, Includes at least a part of the triple helical domain of the collagen or atelocollagen.
- the composition for inducing differentiation of the present invention comprises 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 the collagen or atelocollagen, X 2 and A chemical bond between 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, Degradation product of atelocollagen, or In the triple helical domain of the collagen or atelocollagen, the chemical bond between X 1 and X 2 , the chemical bond between X 2 and G, and the G and X 3 in the amino acid sequence shown in the following (2) chemical bonding between the chemical bond between X 4 and G, chemical bond between X 6 and G, chemical bond between G and X 7, or a chemical between X 14 and G Collagen or atelocollagen degradation products that have broken bonds, or A degradation product of collagen or atelocollagen in which the chemical bond between Y 1 and Y 2
- the amino acid sequence represented by (1) or (2) is preferably the amino acid sequence at the amino terminal of the triple helical domain.
- the cleavage in the amino acid sequence represented by any one of (1) to (3) is at least one of the ⁇ 1 chain and ⁇ 2 chain of the collagen or atelocollagen. Preferably it is done.
- 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
- porcine type I collagen was dissolved in 50 mM citrate buffer (pH 3.0) containing salt. An aqueous solution containing actinidine and the solution containing porcine type I collagen were contacted at 20 ° C. for 10 days or longer to prepare a degradation product of type I collagen.
- porcine 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.
- 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.
- 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.
- phosphate buffer pH 6.5
- the commercially available thing was utilized as cathepsin K.
- 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.
- the chicken-derived ⁇ 1 chain degradation product includes the chicken-derived ⁇ 1 chain degradation product corresponding to SEQ ID NOS: 11 and 12 below and the amino terminal in SEQ ID NO: 10 below.
- the ⁇ 1-chain degradation product derived from the chicken corresponding to the broken chemical bond between the 10th “S” and the 11th “G” was confirmed.
- 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.
- 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 amino acid sequences of these degradation products are shown in SEQ ID NOs: 18 and 19, 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.
- 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. 1A and 1B show micrographs of a mouse osteoblast precursor cell line MC3T3-E1 subclone 4 (ATCC (registered trademark) CRL-2593, Sumisho Pharma International Co., Ltd.).
- PHE indicates the result of the culture plate contacted with commercially available pepsin-treated collagen type I
- LASCol indicates the culture plate contacted with the collagen degradation product of this example. Shows the results.
- the time described in each drawing indicates the culture time.
- Test for Spheroid Inducibility of Collagen Degradation Products-2> A commercially available culture plate contacted with the above-described collagen degradation product (degradation product of porcine ⁇ 1 chain at a salt concentration of 200 mM) was used for the test.
- FIG. 2 A photomicrograph is shown in FIG. In FIG. 2, “LASCol” indicates the result of the culture plate brought into contact with the collagen degradation product of this Example, and “CPCol” is brought into contact with the conventional collagen degradation product (see WO2004 / 020470). The results of the culture plates are shown.
- the collagen degradation product of this example can form not only spheroids but also large spheroids faster than conventional collagen degradation products.
- Test for differentiation-inducing ability-1> A commercially available culture plate (micro dish 35 mm ( ⁇ -Dish 35 mm) uncoated, Cat. #: Ib811511, Nippon Genetics Co., Ltd.), which was brought into contact with the collagen degradation product described in ⁇ 1> above, Used for testing.
- a culture plate ibidi ⁇ -Dish 35 mm uncoated, Cat. #: Ib81151
- commercially available pepsin-treated type I collagen Cell Matrix Type-IC, Nitta Gelatin Co., Ltd.
- culture plates not contacted with collagen degradation products ibidi ⁇ -Dish 35 mm surface treatment ibiTreat, Cat. #: Ib81156, Nippon Genetics Co., Ltd.
- Nano Culture Three types of culture plates with a registered trademark MH pattern (Cat. #: NCD-LH35-5, SCIVAX) were used.
- mice osteoblast progenitor cell line MC3T3-E1 subclone 4 (ATCC (registered trademark) CRL) suspended in the basic medium ( ⁇ -MEM, 10% FBS) was added to each of the four types of culture plates described above. -2593, Sumisho Pharma International Co., Ltd.). Then, the culture plate was cultured for 24 hours under the conditions of 37 ° C. and 5% CO 2 .
- osteoblast differentiation induction medium ⁇ -MEM, 10% FBS, 50 ⁇ g-ascorbic acid / mL, 3.5 mM ⁇ -glycerophosphate. did.
- the culture was continued every 3 days while replacing the osteoblast differentiation medium in the culture plate with a fresh osteoblast differentiation medium.
- mouse osteoblasts were obtained with Alizarin Red Staining Kit (Part No. ARD-A1, PG Co., Ltd.) 10 days, 14 days, and 21 days later.
- the progenitor cell line MC3T3-E1 subclone 4 was stained to confirm the mineralization of the mouse osteoblast progenitor cell line MC3T3-E1 subclone 4.
- Alizarin red staining was performed according to a known method.
- FIG. 3 shows a photograph of the staining result of alizarin red staining when a commercially available culture plate in contact with the collagen degradation product described in ⁇ 1> above is used.
- Fig. 5 shows a photograph of the staining result of alizarin red staining when a culture plate contacted with commercially available pepsin-treated type I collagen is used, and Fig. 5 shows a culture plate not contacted with a degradation product of collagen.
- FIG. 6 shows a photograph of the staining result of alizarin red staining when using commercially available Nano Culture Dish (SCIVAX).
- SCIVAX Nano Culture Dish
- the basic medium in the culture plate is used as the osteoblast differentiation induction medium.
- the vicinity of the center of the spheroid was stained red, and calcification of the cells was confirmed.
- Calcification amount increased with differentiation induction days.
- calcification was not confirmed in cells other than the spheroids of the present application. That is, it was shown that the collagen degradation product described in ⁇ 1> described above promotes calcification of cells.
- Test for differentiation-inducing ability-2> A commercially available culture plate contacted with the collagen degradation product described in ⁇ 1> above was used for the test.
- rat bone marrow mesenchymal stem cells BMC01: Primary Cell Co., Ltd
- BMCM Primary Cell Co., Ltd
- the culture plate was cultured for 24 hours under the conditions of 37 ° C. and 5% CO 2 .
- the culture was continued every 3 days while replacing the bone formation medium in the culture plate with fresh bone formation medium.
- the bone marrow cell culture medium in the culture plate was replaced with an osteogenic medium, and over time, rat bone marrow mesenchymal stem cells were stained with alizarin red staining to confirm calcification of rat bone marrow mesenchymal stem cells. .
- Alizarin red staining was performed according to a known method.
- FIG. 7 shows the results of alizarin red staining.
- “LASCol” in FIG. 7 shows a photograph of the staining results of alizarin red staining when using a commercially available culture plate in contact with the collagen degradation product described in ⁇ 1> above.
- “PHCol” shows a photograph of the staining result of alizarin red staining when using a culture plate contacted with commercially available pepsin-treated type I collagen.
- “PHCol (IWAKI)” in FIG. FIG. 7 shows a photograph of the staining result of alizarin red staining when a commercially available culture plate (manufactured by IWAKI Cytec) is used, and “NonCoat” in FIG. 7 uses a culture plate that is not in contact with a collagen degradation product. The photograph of the dyeing result of the alizarin red dyeing
- staining of the case is shown.
- LASCol calcification was remarkably observed 9 days after the medium was changed. At this time, when observed with a microscope, the number of cells present on the culture plate was significantly smaller in “LASCol” than in “PHCol (IWAKI)” and “NonCoat”. This indicates that “LASCol” has a remarkably high calcification capacity per cell number as compared with “PHCol (IWAKI)” and “NonCoat”.
- Test for differentiation induction ability-3> A commercially available culture plate (ibidi ⁇ -Dish (uncoated), Nippon Genetics) contacted with the collagen degradation product described in ⁇ 1> above was used for the test.
- a culture plate ibidi ⁇ -Dish (uncoated), Nippon Genetics Co., Ltd.
- commercially available pepsin-treated type I collagen Cell Matrix Type-IC, Nitta Gelatin Co., Ltd.
- two types of culture plates i.e., culture plates not contacted with collagen degradation products (ibidi ⁇ -Dish (uncoated), Nippon Genetics Co., Ltd.) were used.
- Each culture plate was seeded with the same number of rat bone marrow mesenchymal stem cells.
- the medium in each culture plate was replaced with an osteoblast differentiation medium (manufactured by Primary Cell) every few days.
- the cells in each culture plate were immobilized using a glutaraldehyde solution.
- each culture plate was observed with a scanning electron microscope (SEM) (manufactured by Hitachi High-Tech Co., Ltd., SU3500), and elemental analysis was performed.
- SEM scanning electron microscope
- FIG. 8 shows the results of elemental analysis when using a commercially available culture plate brought into contact with the collagen degradation product described in ⁇ 1> above
- FIG. 9 shows a commercially available pepsin treatment
- FIG. 10 shows the results of elemental analysis when using a culture plate in contact with the type I collagen
- FIG. 10 shows the results of elemental analysis when using a culture plate that is not in contact with collagen degradation products. Show.
- Test for differentiation induction ability-4> In this example, the bone formation ability of the collagen degradation product described in ⁇ 1> above was tested in vivo. The test method and test results will be described below.
- Pentobarbital (Nembutal) was injected into the abdominal cavity of a rat (male, SPF, Kwl: SD, 12 weeks old), and the rat was anesthetized.
- the rat body weight was measured.
- the weight of the rat before implantation was approximately 400 g.
- the above-mentioned rat's thigh was incised to expose the bone. Specifically, the skin and flesh of the thigh were separated using a scalpel and scissors. Next, after removing the periosteum using tweezers and a scalpel, the bone was exposed.
- a hole with a diameter of 2.5 mm was opened in the vicinity of the attachment part of the medial collateral ligament of the tibia. The hole was deep enough to reach the bone marrow cavity without penetrating the bone.
- freeze-dried collagen degradation product approximately 2-3 mg
- freeze-dried collagen degradation product (2-3 mg)
- rat bone marrow mesenchymal stem cells cultured in osteogenic medium for 1 day
- Rat bone marrow mesenchymal stem cells washed with PBS ( ⁇ )) were placed.
- rats were prepared in which holes were opened but nothing was placed in the holes.
- collagen degradation product a) the collagen degradation product described in ⁇ 1> above or b) a commercially available pepsin-treated type I collagen was used.
- the muscle tissue under the skin was sutured about 3 to 5 places surgically.
- the rats were bred in isolation.
- the breeding environment was a room temperature of 23 ⁇ 2 ° C. and a humidity of 50% with an air conditioning system, feed was constantly fed, and drinking water was freely consumed.
- the tissue near the hole was stained with hematoxylin and eosin (HE staining) according to a well-known method.
- Fig. 13 shows a stained image when a mixture of the bone marrow and rat bone marrow mesenchymal stem cells is placed in the hole, and Fig. 13 shows a stained image when only commercially available pepsin type I collagen is placed in the hole.
- FIG. 14 shows a stained image when a mixture of commercially available pepsin-treated collagen type I and rat bone marrow mesenchymal stem cells is placed in the hole.
- Table 11 shows the body weight (A) of the rat before implanting the collagen degradation product, the body weight (B) of the rat 15 days after implantation of the collagen degradation product (immediately before euthanasia), and the increase in body weight. Amount (C) and weight gain (D) are shown.
- LASCol indicates the result when only the collagen degradation product described in ⁇ 1> described above is placed in the hole
- LASCol + rMSC is described in ⁇ 1> above.
- PHY shows the case where only commercially available pepsin type I collagen is placed in the hole.
- PPHCol + rMSC shows the results when a mixture of commercially available pepsin-treated collagen type I collagen and rat bone marrow mesenchymal stem cells is placed in the hole, and “Control” indicates that the hole is opened. Although the hole is formed, the result when nothing is arranged in the hole is shown.
- the differentiation-inducing composition of the present example can maintain the soundness of the activity and food intake of the rat and is very suitable for the induction of cell differentiation.
- the average increase in the body weight of the normally bred rats that had not undergone any surgery was about 100 g during the test period, whereas in the case of “LASCol” and “LASCol + rMSC”, the body weight was as shown in Table 11. The average increase amount was 90 g. Therefore, this comparison also shows that the composition for inducing differentiation of this example can maintain the soundness of the activity amount and the intake amount of rats, and the bone healing condition is extremely good.
- LASCol indicates the result of the culture plate contacted with the collagen degradation product of this example
- NonCoat (Plastic) indicates the result of a commercially available plastic plate
- Telocollagen The result of a commercially available atelocollagen coated plate is shown.
- the cell nuclei cultured with “LASCol” were fluorescently stained with Hoechst 33342 (Dojin Corporation) to confirm the survival of the cells. From the above results, it was revealed that the collagen degradation product of this example is suitable for long-term culture of 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 human mesenchymal stem cells (LONZA) are suspended in a growth medium (MSCBM + MSCGM, LONZA) dedicated to the cells, and then seeded on the above-described culture plate, at 37 ° C. and 5% CO 2 .
- the cells were cultured for 1 day, and the cells were allowed to adhere on the culture plate.
- the entire amount of the medium was replaced with an osteoblast induction medium (MSCGM, LONZA). Thereafter, the cells were cultured under conditions of 37 ° C. and 5% CO 2 for 15 days while changing the osteoblast induction medium every 3 days. After 15 days, the degree of differentiation from primary human mesenchymal stem cells to osteoblasts was examined by alkaline phosphatase (ALP) staining (Takara Bio).
- ALP alkaline phosphatase
- FIG. 16 The test results are shown in FIG. In FIG. 16, “LASCol” indicates the result of the culture plate contacted with the collagen degradation product of this example, “Atelocollagen” indicates the result of the commercially available atelocollagen-coated plate, and “NonCoat (Plastic)” indicates The results for a commercially available plastic plate are shown. As is clear from FIG. 16, it was revealed that the collagen degradation product of this example significantly promotes differentiation into 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.
- Primary human mesenchymal stem cells (LONZA) are suspended in a growth medium (MSCBM + MSCGM, LONZA) dedicated to the cells, and then seeded on the above-described culture plate, at 37 ° C. and 5% CO 2 .
- the cells were cultured for 1 day, and the cells were allowed to adhere on the culture plate.
- chondrocyte induction medium GEBCO
- the cells were cultured for 15 days under conditions of 37 ° C. and 5% CO 2 while changing the chondrocyte induction medium every 3 days.
- the cartilage matrix produced by the cells was examined by Alcian blue staining (pH 2.5) (Nacalai Tesque, 37154-44).
- FIG. 17 The test results are shown in FIG. In FIG. 17, “LASCol” indicates the result of the culture plate brought into contact with the collagen degradation product of this example, and “NonCoat (Plastic)” indicates the result of the commercially available plastic plate.
- the collagen degradation product of this example induces spontaneous formation of spheroids effective for chondrocyte differentiation by primary human mesenchymal stem cells, and further differentiates into chondrocytes. It was revealed that the production of the shown cartilage matrix positive for Alcian blue staining was promoted.
- 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 (RIKEN BRC) was 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. Then, the cells were allowed to adhere on the culture plate.
- the entire amount of medium was replaced with fresh medium (10% FBS DMEM). Thereafter, the cells were cultured for 6 days under conditions of 37 ° C. and 5% CO 2 while changing the medium every 3 days.
- LASCol indicates the result of the culture plate brought into contact with the collagen degradation product of this example
- Atelocollagen indicates the result of pepsin-treated collagen
- acid extracted collagen is shown
- NonCoat (Plastic) shows the results for a commercially available plastic plate.
- 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.
- Rat C6 cells (RIKEN BRC) are suspended in a growth medium (10% FBS DMEM), seeded on the above-mentioned culture plate, cultured at 37 ° C. under 5% CO 2 , and the cells are cultured. Adhered onto the culture plate.
- 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. After suspending primary human mesenchymal stem cells (PromoCell) in a growth medium dedicated to the cells (Mesenchymal Stem Cell Growth Medium (C-28010), PromoCell), the cells are seeded on the culture plate described above, The cells were cultured at 37 ° C. and 5% CO 2 for 2 days.
- the entire amount of the medium was replaced with a neural cell induction medium (Ready-to-use) (C-28015), PromoCell Co., Ltd. (Mechanical Stem Cell Neurogenic Differentiation Medium). Thereafter, the cells were cultured for 5 days under conditions of 37 ° C. and 5% CO 2 while changing the nerve cell induction medium every two days. Five days later, the degree of differentiation from primary human mesenchymal stem cells to neurons was examined by observing the intercellular network by neurites using a phase contrast microscope.
- a neural cell induction medium Ready-to-use
- PromoCell Co., Ltd. Mechanism of Stem Cell Neurogenic Differentiation Medium
- FIG. 20 The test results are shown in FIG. In FIG. 20, “LASCol” indicates the result of the culture plate contacted with the collagen degradation product of this example, and “Fibronectin” indicates the result of the plate obtained by applying fibronectin to a commercially available plate according to the manual. “NonCoat (Plastic)” shows the results for a commercially available plastic plate. As is clear from FIG. 20, it was revealed that the collagen degradation product of this example significantly promotes differentiation into nerve cells constructing a neurite network.
- actinidine Purification of collagen from bone> 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. 21 (a) and FIG. 21 (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 state 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 differentiation-inducing composition, a food additive, a medical material, a cosmetic material, a culture material for culturing cells or embryos (eg, a coating material for a culture apparatus (eg, a culture dish), a culture medium component) It can be used for
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Abstract
Description
本実施の形態の分化誘導用組成物は、細胞の分化を誘導するための組成物であって、コラーゲンまたはアテロコラーゲンの分解物を主成分として含み、上記分解物は、上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインの少なくとも一部分を含んでいるものである。つまり、上記分解物は、コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインの全体を含んでいてもよいし、トリプルヘリカルドメインの一部分を含んでいてもよい。
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(1)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、または、X6とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、または、X14とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、を含む分化誘導用組成物である。
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(1)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、および、X6とGとの間の化学結合から選択される何れか1つの化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、および、X14とGとの間の化学結合から選択される何れか1つの化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、を含む分化誘導用組成物である。
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-(配列番号14):
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-(配列番号13):
(但し、Gは、グリシンであり、X1~X14およびY1~Y9は、任意のアミノ酸である)。
上述したコラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、または、X14とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
上述したコラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、を含んでいる:
(1)-G-X1-X2-G-X3-X4-G-X5-X6-G-:
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-:
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-:
(但し、Gは、グリシンであり、X1~X14およびY1~Y9は、任意のアミノ酸である)。
(1)-G-X1-X2-G-X3-X4-G-X5-X6-G-:
(但し、Gは、グリシンであり、X1~X6は、任意のアミノ酸である)。
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-:
(但し、Gは、グリシンであり、X1~X14は、任意のアミノ酸である)。
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-
(但し、Gは、グリシンであり、Y1~Y9は、任意のアミノ酸である)。
本実施の形態のコラーゲンまたはアテロコラーゲンの分解物を含む分化誘導用組成物の製造方法は、
A)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(1)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、または、X6とGとの間の化学結合を切断する、切断工程、または、
B)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、または、X14とGとの間の化学結合を切断する、切断工程、または、
C)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合を切断する、切断工程、を含む製造方法である。
D)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(1)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、および、X6とGとの間の化学結合から選択される何れか1つの化学結合を切断する、切断工程、または、
E)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、および、X14とGとの間の化学結合から選択される何れか1つの化学結合を切断する、切断工程、または、
F)コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合を切断する、切断工程。
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-:
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-:
(但し、Gは、グリシンであり、X1~X14およびY1~Y9は、任意のアミノ酸である)。
酵素法に基づく切断工程を採用する場合には、例えば、以下のように切断工程を構成することができる。
化学合成法に基づく切断工程を採用する場合には、例えば、以下のように切断工程を構成することができる。
組み換えタンパク質の発現に基づく切断工程を採用する場合には、例えば、以下のように切断工程を構成することができる。
本実施の形態の分化誘導用組成物は、細胞が分化することを誘導するものである。
上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、または、X14とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、を含む、ことが好ましい。つまり、
(1)-G-X1-X2-G-X3-X4-G-X5-X6-G-;
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-;
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-;
(但し、Gは、グリシンであり、X1~X14およびY1~Y9は、任意のアミノ酸である)。
塩化ナトリウムの濃度が0mM、200mM、1000mM、1500mMまたは2000mMである50mM クエン酸緩衝液(pH3.0)を準備した。なお、当該水溶液の溶媒としては、水を用いた。
塩化ナトリウムの濃度が2000mMである50mM クエン酸緩衝液(pH3.0)を準備した。なお、当該水溶液の溶媒としては、水を用いた。
本実施例では、システインプロテアーゼの一種であるカテプシンKを用いて、高塩濃度条件下におけるα1鎖の切断箇所を検討した。以下に、試験方法および試験結果を説明する。
透析チューブにアクチニダインを入れ、当該アクチニダインを、塩化ナトリウムの濃度が2000mMである透析外液に対して透析した。その後、透析外液を蒸留水に変えて透析を続けてアクチニダインを得た。なお、アクチニダインとしては、周知の方法にて精製したものを利用した(例えば、非特許文献2参照)。アクチニダインを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、アクチニダインを溶解し、90分間、25℃にて静置した。
透析チューブにアクチニダインを入れ、当該アクチニダインを、塩化ナトリウムの濃度が2000mMである透析外液に対して透析した。その後、透析外液を蒸留水に変えて透析を続けてアクチニダインを得た。なお、アクチニダインとしては、周知の方法にて精製したものを利用した(例えば、非特許文献2参照)。アクチニダインを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、アクチニダインを溶解し、90分間、25℃にて静置した。
透析チューブにアクチニダインを入れ、当該アクチニダインを、塩化ナトリウムの濃度が2000mMである透析外液に対して透析した。その後、透析外液を蒸留水に変えて透析を続けてアクチニダインを得た。アクチニダインを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、アクチニダインを溶解し、90分間、25℃にて静置した。
<4>と同様にカテプシンKを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、カテプシンKを溶解し、45分間、25℃にて静置した。
透析チューブにアクチニダインを入れ、当該アクチニダインを、塩化ナトリウムの濃度が2000mMである透析外液に対して透析した。その後、透析外液を蒸留水に変えて透析を続けてアクチニダインを得た。アクチニダインを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、アクチニダインを溶解し、90分間、25℃にて静置した。
上述したコラーゲンの分解物(塩濃度200mMにおける、ブタ由来のα1鎖の分解物)と接触させた、市販の培養用プレートを試験に用いた。
上述したコラーゲンの分解物(塩濃度200mMにおける、ブタ由来のα1鎖の分解物)と接触させた、市販の培養用プレートを試験に用いた。
上述した<1>に記載したコラーゲンの分解物と接触させた、市販の培養用プレート(マイクロ・ディッシュ35mm(μ-Dish 35mm)未コーティング,Cat.#:ib81151,日本ジェネティックス株式会社)を、試験に用いた。
上述した<1>に記載したコラーゲンの分解物と接触させた、市販の培養用プレートを、試験に用いた。
上述した<1>に記載したコラーゲンの分解物と接触させた、市販の培養用プレート(ibidi μ-Dish(未コーティング),日本ジェネティックス株式会社)を、試験に用いた。
本実施例では、in vivoにおける、上述した<1>に記載したコラーゲンの分解物の骨形成能力を試験した。以下に、試験方法および試験結果について説明する。
上述したコラーゲンの分解物(塩濃度200mMにおける、ブタ由来のコラーゲンの分解物)と接触させた、市販の培養用プレートを試験に用いた。マウス線維芽細胞(NIH/3T3)を10%CS DMEM培地中に懸濁した後、上述した培養用プレート上に播種し、37℃、5%CO2の条件下で培養した。
透析チューブにアクチニダインを入れ、当該アクチニダインを、塩化ナトリウムの濃度が2000mMの透析外液に対して透析した。その後、透析外液を蒸留水に変えて透析を続けてアクチニダインを得た。なお、アクチニダインとしては、周知の方法にて精製したものを利用した(例えば、非特許文献2参照)。アクチニダインを活性化するため、10mM ジチオスレイトールを含む50mM リン酸緩衝液(pH6.5)に対し、アクチニダインを溶解し、90分間、25℃にて静置した。
Claims (4)
- コラーゲンまたはアテロコラーゲンの分解物を含む、細胞の分化を誘導するための分化誘導用組成物であって、
上記分解物は、上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインの少なくとも一部分を含んでいることを特徴とする、分化誘導用組成物。 - 上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(1)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、または、X6とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメイン内の下記(2)にて示されるアミノ酸配列の、X1とX2との間の化学結合、X2とGとの間の化学結合、GとX3との間の化学結合、X4とGとの間の化学結合、X6とGとの間の化学結合、GとX7との間の化学結合、または、X14とGとの間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、または、
上記コラーゲンまたはアテロコラーゲンのトリプルヘリカルドメインのアミノ末端の下記(3)にて示されるアミノ酸配列の、Y1とY2との間の化学結合が切断されている、コラーゲンまたはアテロコラーゲンの分解物、を含む、請求項1に記載の分化誘導用組成物:
(1)-G-X1-X2-G-X3-X4-G-X5-X6-G-;
(2)-G-X1-X2-G-X3-X4-G-X5-X6-G-X7-X8-G-X9-X10-G-X11-X12-G-X13-X14-G-;
(3)-Y1-Y2-Y3-G-Y4-Y5-G-Y6-Y7-G-Y8-Y9-G-;
(但し、Gは、グリシンであり、X1~X14およびY1~Y9は、任意のアミノ酸である)。 - 上記(1)または(2)にて示されるアミノ酸配列は、上記トリプルヘリカルドメインのアミノ末端のアミノ酸配列であることを特徴とする、請求項2に記載の分化誘導用組成物。
- 上記(1)~(3)の何れかにて示されるアミノ酸配列における切断が、上記コラーゲンまたはアテロコラーゲンのα1鎖内およびα2鎖内の少なくとも一方で行われていることを特徴とする、請求項2に記載の分化誘導用組成物。
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