Composition for use in promoting digit formation, composition for use in promoting digit extension, method for promoting digit formation, and method for promoting digit extension

Abstract

To provide a composition capable of promoting digit formation.SOLUTION: A composition for use in promoting digit formation according to the present disclosure comprises an animal tissue-derived proteoglycan.SELECTED DRAWING: Figure 5

Description

Technical Field

[0001] The present disclosure relates to a composition for promoting finger formation, a composition for promoting finger extension, a method for promoting finger formation, and a method for promoting finger extension.

Background Art

[0002] Proteoglycan is a molecule that constitutes the extracellular matrix together with collagen, hyaluronic acid, etc. The proteoglycan is known to have various physiological functions such as promoting neurogenesis and angiogenesis. In addition, various studies have been conducted on the expression of the proteoglycan associated with the development of limb buds (primordia of the limbs) during the development process.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0004] Therefore, the present disclosure aims to provide a composition or the like that can promote finger formation.

Means for Solving the Problems

[0005] To achieve the above object, the composition for promoting finger formation of the present disclosure contains a proteoglycan derived from animal tissue.

[0006] The composition for promoting finger extension of the present disclosure contains a proteoglycan derived from animal tissue.

[0007] The method for promoting finger formation of the present disclosure uses the composition for promoting finger formation of the present disclosure.

[0008] The method for promoting finger extension of the present disclosure uses the composition for promoting finger extension of the present disclosure.

[0009] According to the present disclosure, for example, it is possible to provide a composition or the like that can promote finger formation.

Brief Description of the Drawings

[0010]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0011] Hereinafter, the present disclosure will be specifically described with examples. Hereinafter, unless otherwise specified, each disclosure can incorporate the description of other disclosures.

[0012] <Definition> As used herein, "proteoglycan" means a molecule (glycoprotein) in which a protein (core protein) and a glycosaminoglycan (GAG, also referred to as "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists, for example, as an extracellular matrix such as skin, organs, and cartilage. The glycosaminoglycan is usually known as a sugar chain having a long-chain structure without a branched structure. Examples of the proteoglycan include the following. · Aggrecan family (also referred to as the lectican family or the hyalectan family): Aggrecan, Versican, Neurocan, Brevican, etc. · Small Leucine Rich Proteoglycans (SLRPs) family: Biglycan, Decorin, Fibromodulin, Lumican, PG-Lb (Epiphycan), Keratocan, Mimecan, etc. · Proteoglycans of the basement membrane: Perlecan, Agrin, Bamacan, etc. · Other proteoglycans: Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc. The proteoglycan can be classified, for example, into chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, heparan sulfate proteoglycan, or keratan sulfate proteoglycan according to the type of GAG bound to the protein.

[0013] The GAGs include, for example, chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The chondroitin includes an O-type sugar chain having a disaccharide structure of glucuronic acid and N-acetylgalactosamine as a main disaccharide structure, and an iO-type sugar chain having a disaccharide structure of iduronic acid and N-acetylgalactosamine as a main structure (hereinafter, also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO", respectively). The chondroitin sulfate (CS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of glucuronic acid and N-acetylgalactosamine is repeated. The chondroitin sulfate (CS) includes, for example, chondroitin sulfate A (type A) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate C (type C) having a disaccharide structure of glucuronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. The dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which a disaccharide of iduronic acid and N-acetylgalactosamine is repeated. The dermatan sulfate includes, for example, chondroitin sulfate iA (iA type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 4-sulfate as a main disaccharide structure, chondroitin sulfate iC (iC type) having a disaccharide structure of iduronic acid and N-acetylgalactosamine 6-sulfate as a main disaccharide structure, and the like. Each chondroitin sulfate has, for example, a disaccharide structure shown in FIG. 1 as a main disaccharide structure. In FIG. 1, the sulfate group (sulfonyl group) is bonded to a hydrogen atom, but the present disclosure is not limited thereto, and the sulfate group of the GAG may be, for example, ionized by desorption of a hydrogen atom, or may form a salt.

[0014] As used herein, "finger formation" means the generation or regeneration of fingers. The above-mentioned "generation of fingers" means that the interdigital cells of the limb bud, that is, the cells in the part corresponding to the webbing between fingers (interdigital part) undergo apoptosis, and independent fingers are formed. The above-mentioned "regeneration of fingers" means promoting the reformation of tissue structure at the site of finger defect or injury. Also, as used herein, "promotion of finger formation" means that the generation or regeneration of fingers is promoted.

[0015] As used herein, "finger length" means the straight-line distance from the tip of the finger to the wrinkle at the base of the finger (when there are multiple wrinkles, the one closest to the body).

[0016] As used herein, "finger elongation" means that the length of the finger increases.

[0017] Hereinafter, the present disclosure will be described with examples. However, the present disclosure is not limited to the following examples and can be arbitrarily changed and implemented. Also, each description in the present disclosure and each embodiment can be mutually incorporated unless otherwise specified. In addition, in this specification, when the expression "~" is used, it is used in the sense of including the numerical values or physical values before and after it. Further, in this specification, the expression "A and / or B" includes "only A", "only B", and "both A and B".

[0018] <The first composition> In one aspect, the present disclosure provides a composition capable of promoting finger formation. The composition capable of promoting finger formation of the present disclosure contains a proteoglycan derived from an animal.

[0019] As a result of intensive research, the inventors of the present invention have found that a proteoglycan derived from an animal exhibits an effect of promoting finger formation, and thus have established the present disclosure. Therefore, according to the composition capable of promoting finger formation of the present disclosure, for example, it is expected to promote finger formation.

[0020] Examples of the proteoglycan include proteoglycans derived from animals. The animals are not particularly limited, and examples thereof include mammals such as pigs (mammals); avian animals such as chickens (birds); fish such as flounder of the family Paralichthyidae, salmon such as rainbow trout and Atlantic salmon, and sharks (e.g., including dogfish); etc. The animals are preferably, for example, salmon, pigs, birds, flounder, and sharks, etc.

[0021] The proteoglycan is preferably, for example, aggrecan.

[0022] The peak top molecular weight of the proteoglycan is, for example, 300,000 to 1,300,000, or 400,000 to 1,200,000. The peak top molecular weight is measured, for example, by the GPC (Gel Permeation Chromatography) method. The GPC method can be carried out, for example, under the following conditions. The standard sample (molecular weight marker, pullulan) is individually injected into an HPLC system to obtain a molecular weight calibration curve for calculation.

[0023] (Measurement conditions for peak top molecular weight (Mp)) HPLC system: LC-20AD (manufactured by Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8 mm × 300 mm (manufactured by Tosoh Corporation) Eluent: phosphate buffer at pH 6.8 Flow rate: 0.5 mL / min Column temperature: 40 °C Detector: differential refractive index detector (RID-20A manufactured by Shimadzu Corporation) Injection volume: 50 μL Molecular weight marker: Shodex STANDARD P-82 (pullulan) The peak top molecular weight (Mp) and weight average molecular weight (Mw) / number average molecular weight (Mn) of the molecular weight marker are as follows. STD P-800: Mp: 739,000, Mw / Mn: 1.24 STD P-400: Mp: 348,000, Mw / Mn: 1.33 STD P-200: Mp: 216,000, Mw / Mn: 1.22 STD P-100: Mp: 107,000, Mw / Mn: 1.12 STD P-50: Mp: 49,400, Mw / Mn: 1.08 STD P-20: Mp: 22,000, Mw / Mn: 1.08 STD P-10: Mp: 9,800, Mw / Mn: 1.07 STD P-5: Mp: 6,300, Mw / Mn: 1.09

[0024] Examples of the proteoglycan include proteoglycans derived from tissues containing proteoglycans. Examples of the tissues include epithelial tissues such as skin; cartilage tissues such as cartilage; digestive organs; circulatory organs; respiratory organs; and placenta. Specific examples of the tissues include, for example, cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.

[0025] Examples of the sugar chain of the proteoglycan include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The sugar chain is preferably chondroitin sulfate.

[0026] For the proteoglycan, self-prepared proteoglycan or commercially available proteoglycan may be used. When using the self-prepared proteoglycan, the proteoglycan may be a proteoglycan purified by acetic acid extraction or a proteoglycan purified by guanidine extraction. When performing the acetic acid extraction, the proteoglycan can be purified with reference to the method of Japanese Patent No. 3731150. When performing the guanidine extraction, the proteoglycan can be purified with reference to the methods of References 1 to 2 below. Reference 1: Kakizaki I, Tatara Y, Majima M, Kato Y, Endo M. Identification of proteoglycan from salmon nasal cartilage. Arch Biochem Biophys. 2011 Feb 1;506(1):58-65. doi: 10.1016 / j.abb.2010.10.025. Epub 2010 Nov 5. PMID: 21056541. Reference 2: Kakizaki I, Mineta T, Sasaki M, Tatara Y, Makino E, Kato Y. Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan. Carbohydr Polym. 2014 Mar 15;103:538-49. doi: 10.1016 / j.carbpol.2013.12.083. Epub 2014 Jan 7. Erratum in: Carbohydr Polym. 2015 Jan 22;115:805. PMID: 24528764.

[0027] The first composition of the present disclosure may contain, for example, other components. Examples of the other components include carriers, growth factors, and the like.

[0028] In the first composition of the present disclosure, the proteoglycan may be supported on a substrate or a carrier. Examples of the substrate or the carrier include gels. The gel is usually biocompatible, can contain a large amount of water, and can easily diffuse and transfer substances necessary for cell survival such as oxygen, water, nutrients, and enzymes. The form of the gel is not particularly limited, and examples thereof include particulate, granular, film-like, tubular, disk-like, net-like, mesh-like, porous, suspension, or dispersion forms. Examples of the gel include hydrogels such as gelatin hydrogel; collagen gel, Matrigel (registered trademark), gelatin gel, hyaluronic acid gel, Matrigel, and the like. The gel may contain, for example, laminin, type IV collagen, entactin / nidogen, a predetermined growth factor, and the like.

[0029] The evaluation of the promotion of finger formation can use, for example, the length of the finger as an evaluation. Specifically, when the first composition of the present disclosure is used for the development of limb buds, it can be evaluated based on whether the length of the finger has increased as compared to the case in the absence of the first composition of the present disclosure. In the evaluation, when the increase rate of the finger length in the control group in which the first composition of the present disclosure is not used for the development of limb buds is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, the composition can be evaluated as having an effect of promoting finger formation.

[0030] The first composition of the present disclosure can be used, for example, for promoting the formation of finger structures in the development of limb buds, or for promoting finger formation at finger defect or injury sites, such as for finger regeneration applications. Specifically, the first composition of the present disclosure may be used, for example, for the treatment of congenital finger hypoplasia or deformities. Examples of the congenital finger hypoplasia or deformities include polydactyly (polydactylism), syndactyly (syndactylism), brachydactyly (brachydactylism), and the like.

[0031] The first composition of the present disclosure in vitro may be used in in vivoIt may also be used. The first composition of the present disclosure can be used, for example, as a research reagent or as a pharmaceutical. In the former case, the first composition of the present disclosure can also be referred to as a test reagent or a test kit.

[0032] When the first composition of the present disclosure is in vitro used, the administration target can be, for example, cells, tissues, organs, etc. The cells can be, for example, cells collected from a living body, cultured cells, etc. The tissue or organ can be, for example, a tissue (biological tissue) or an organ collected from a living body. The cells can be, for example, limb bud mesenchymal cells (LBM), chondrogenic progenitor cells (ATDC5), vascular endothelial progenitor cells, etc.

[0033] When the first composition of the present disclosure is in vivo used, the administration target can be, for example, a human or a non-human animal excluding humans. Examples of the non-human animal include a mouse, a rat, a rabbit, a dog, a sheep, a horse, a cat, a goat, a monkey, a guinea pig, etc.

[0034] The usage conditions (administration conditions) of the first composition of the present disclosure are not particularly limited, and for example, the administration form, administration timing, administration amount, etc. can be appropriately set according to the type of the administration target, etc.

[0035] When the first composition of the present disclosure is in vitro used, the first composition of the present disclosure can be used, for example, by adding it to the medium of the target cells. The first composition of the present disclosure may be added to the maintenance medium used when maintaining the cells, for example.

[0036] When the first composition of the present disclosure is in vivo used, it can be appropriately determined, for example, according to the type, symptoms, age, administration method, etc. of the administration target.

[0037] The dosage form of the first composition of the present disclosure is not particularly limited. When administering the first composition of the present disclosure in vivo it may be administered orally or parenterally. Examples of the parenteral administration include intravenous injection (intravenous administration), intramuscular injection (intramuscular administration), transdermal administration, subcutaneous administration, intradermal administration, enteral administration, rectal administration, vaginal administration, nasal administration, pulmonary administration, intraperitoneal administration, topical administration, etc.

[0038] The dosage of the first composition of the present disclosure is an amount that can promote finger formation in the administration subject, that is, an effective dosage. The dosage can be appropriately determined depending on, for example, the age, weight, symptoms, etc. of the administration subject.

[0039] The number of administrations of the first composition of the present disclosure is one or more times. The plurality of times is, for example, 2 times, 3 times, 4 times, 5 times or more. The number of administrations may be appropriately determined while confirming the finger formation promoting effect in the administration subject. When administering multiple times, the administration interval can be appropriately determined while confirming the finger formation promoting effect in the administration subject, and examples include once a day, once a week, once every two weeks, once a month, once every three months, once every six months, etc.

[0040] The dosage form of the first composition of the present disclosure is not particularly limited and can be appropriately determined, for example, according to the administration form. Examples of the dosage form include liquid or solid.

[0041] The first composition of the present disclosure may further contain a pharmaceutically acceptable carrier. Examples of the carrier include a suspending agent, solubilizing agent, stabilizer, isotonic agent, preservative, anti-adsorption agent, surfactant, diluent, medium, pH adjuster, soothing agent, buffer, sulfur-containing reducing agent, antioxidant, etc. for administering the active ingredient, and can be appropriately added within a range that does not interfere with the effects of the present disclosure.

[0042] The suspending agent is not particularly limited, and examples thereof include methylcellulose, polysorbate 80, hydroxyethylcellulose, gum arabic, tragacanth powder, sodium carboxymethylcellulose, polyoxyethylene sorbitan monolaurate, and the like.

[0043] The solubilizing agent is not particularly limited, and examples thereof include polyoxyethylene hydrogenated castor oil, polysorbate 80, nicotinamide, polyoxyethylene sorbitan monolaurate, macrogol, ethyl ester of castor oil fatty acid, and the like.

[0044] The stabilizer is not particularly limited, and examples thereof include dextran 40, methylcellulose, gelatin, sodium sulfite, sodium metabisulfite, and the like.

[0045] The isotonic agent is not particularly limited, and examples thereof include D-mannitol, sorbitol, and the like.

[0046] The preservative is not particularly limited, and examples thereof include methyl paraben, ethyl paraben, sorbic acid, phenol, cresol, chlorocresol, and the like.

[0047] The anti-adsorption agent is not particularly limited, and examples thereof include human serum albumin, lecithin, dextran, ethylene oxide-propylene oxide copolymer, hydroxypropylcellulose, methylcellulose, hydrogenated castor oil, polyethylene glycol, and the like.

[0048] The sulfur-containing reducing agent is not particularly limited, and examples thereof include those having a sulfhydryl group such as N-acetylcysteine, N-acetylhomocysteine, thioxoic acid, thiodiglycol, thioethanolamine, thioglycerol, thiosorbitol, thioglycolic acid and its salts, sodium thiosulfate, glutathione, thioalkanoic acids having 1 to 7 carbon atoms, and the like.

[0049] The antioxidant is not particularly limited, and examples thereof include erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-tocopherol, tocopherol acetate, L-ascorbic acid and its salts, L-ascorbyl palmitate, L-ascorbyl stearate, sodium bisulfite, sodium sulfite, triamyl gallate, propyl gallate, or chelating agents such as sodium ethylenediaminetetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate, etc.

[0050] The first composition of the present disclosure may further appropriately contain commonly added components such as inorganic salts such as sodium chloride, potassium chloride, calcium chloride, sodium phosphate, potassium phosphate, sodium hydrogen carbonate; organic salts such as sodium citrate, potassium citrate, sodium acetate; saccharides such as glucose; and the like.

[0051] <Second Composition> In another aspect, the composition for promoting finger extension of the present disclosure (hereinafter, also referred to as the "second composition") contains a proteoglycan derived from an animal. The second composition of the present disclosure is characterized by containing a proteoglycan derived from an animal, and other configurations and conditions are not particularly limited. According to the second composition of the present disclosure, finger extension can be promoted. The description of the first composition of the present disclosure can be incorporated by reference for the second composition.

[0052] The evaluation of the promotion of finger extension can use, for example, the finger length, etc. as an evaluation. Specifically, when the second composition of the present disclosure is used for limb bud development, it can be evaluated based on whether the finger length has increased compared to the absence of the second composition of the present disclosure. In the above evaluation, when the increase rate of the finger length in the control group without using the second composition of the present disclosure for limb bud development is 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, the composition can be evaluated as having an effect of promoting finger extension.

[0053] <Method for promoting finger formation> In another aspect, the present disclosure provides a method for promoting finger formation. The method for promoting finger formation of the present disclosure (hereinafter, also referred to as "formation promotion method") uses a composition for use in promoting finger formation of the present disclosure. The formation promotion method of the present disclosure is characterized by using the first composition of the present disclosure, and other steps and conditions are not particularly limited. According to the formation promotion method of the present disclosure, finger formation can be promoted. The formation promotion method of the present disclosure can incorporate the description of the first composition of the present disclosure.

[0054] The formation promotion method of the present disclosure in vitro may be carried out, in vivo or may be carried out. The formation promotion method of the present disclosure includes, for example, an administration step of administering the first composition of the present disclosure to an administration subject in the presence of the first composition of the present disclosure.

[0055] The formation promotion method of the present disclosure in vitro When carried out, the formation promotion method of the present disclosure includes, for example, a formation step of forming fingers in the presence of the first composition of the present disclosure.

[0056] <Method for promoting finger extension> In another aspect, the present disclosure provides a method for promoting finger extension. The method for promoting finger extension of the present disclosure (hereinafter, also referred to as "extension promotion method") uses a composition for use in promoting finger extension of the present disclosure. The extension promotion method of the present disclosure is characterized by using the second composition of the present disclosure, and other steps and conditions are not particularly limited. According to the extension promotion method of the present disclosure, finger extension can be promoted. The extension promotion method of the present disclosure can incorporate the description of the second composition of the present disclosure.

[0057] The extension promotion method of the present disclosure, for example, in vitro may be carried out, in vivoIt may also be implemented. The elongation promotion method of the present disclosure includes, for example, an administration step of administering the second composition of the present disclosure to an administration subject in the presence of, for example, the second composition.

[0058] The elongation promotion method of the present disclosure in vitro When implemented, the elongation promotion method of the present disclosure includes, for example, an elongation step of extending a finger in the presence of the second composition of the present disclosure.

[0059] <Use> In another aspect, the present disclosure is the use of a composition for promoting finger formation and / or a composition for promoting finger elongation. The use of the composition for promoting finger formation and / or the composition for promoting finger elongation of the present disclosure can refer to, for example, the description of the composition for promoting finger formation, the composition for promoting finger elongation, the method for promoting finger formation, and / or the method for promoting finger elongation.

Example

[0060] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used based on their protocols unless otherwise indicated. Note that "mol / l" may also be denoted as "M".

[0061] [Example 1] It was confirmed that proteoglycan contributes to finger formation and elongation.

[0062] (1) Examination of the localization of proteoglycan during finger formation In the developmental stage, fingers are formed by apoptosis of cells in the interdigital region. The localization of proteoglycans in the process of finger formation was examined by immunostaining. Specifically, fingers of the forelimbs were collected from normal chickens at E (Embryonic day) 6, E7, or E8, and the fingers were fixed using 4% paraformaldehyde under conditions of 24 hours. After the fixation, washing was performed a total of 2 times for 5 minutes on ice using 1×PBS. After the washing, sucrose substitution was performed using a 10% sucrose solution, a 20% sucrose solution, and a 30% sucrose solution in sequence to obtain an embedded sample. In the substitution, treatment was performed with the 10% sucrose solution for 30 minutes, then with the 20% sucrose solution for 1 hour, and finally with the 30% sucrose solution for 1 hour. After the sucrose substitution, the sample was dipped twice in OCT (Optimal Cutting Temperature Compound, manufactured by Sakura Finetek Japan Co., Ltd.) to remove the sucrose around the sample. After the removal, approximately 1 / 3 of the bottom of Tissue-Tek (registered trademark) Cryomold No. 1 added with OCT was frozen using dry ice. Thereafter, the sample was set in the cryomold as parallel as possible. After the setting, the sample was placed in a box together with dry ice, and the sample was frozen. After the freezing, storage of the frozen sample was performed under conditions of -80°C.

[0063] Next, the sample was sliced into 100 μm sections. After slicing, blocking was performed using PBS(-) containing 10% donkey serum (manufactured by Merck) at 4°C overnight. After blocking, primary staining was performed using primary antibodies at 4°C for two nights. The primary antibodies used were Anti-PECAM-1 Antibody (clone 2H8, Cat No: MAB1398Z, manufactured by Merck, diluted 200-fold) as an antibody against CD31, Anti-Aggrecan antibody (Cat No: ab3778, manufactured by abcam, diluted 200-fold), and Anti-Human / Mouse Active Caspase-3 Affinity Purified PAb (Cat No: AF835-SP, manufactured by R&D Systems, Inc., diluted 200-fold). After primary staining, the sample was washed twice on ice for 10 minutes each using cold 1×PBS (approximately 4°C). After washing, secondary staining was performed using secondary antibodies at 4°C overnight. The secondary antibodies used were Anti-GOLDEN SYRIAN & ARMENIAN HAMSTER IgG (H&L) (GOAT) (Min X MOUSE and RAT Serum Proteins), DyLight 649 (Cat No: 620-143-440, manufactured by R&D Systems, Inc., diluted 500-fold), Donkey anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Cat No: A21202, manufactured by Invitrogen, diluted 500-fold), and Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor(™) 594 (Cat No: A-21207, manufactured by Thermo Fisher scientific, diluted 500-fold). After secondary staining, the sample was washed twice on ice for 10 minutes each using cold 1×PBS (approximately 4°C). After washing, a border was drawn around the sample on the slide glass with a pap pen, and 1×PBS was added inside the border. After adding, the sample was set inside the border.After the above setting, 1×PBS was removed, and a mounting medium containing DAPI (DAPI-Fluoromount-G (registered trademark), manufactured by Cosmo Bio Co., Ltd.) was added. After the addition, a cover glass was placed, and nail polish was applied around the cover glass to enclose the sample. After the enclosure, the sample was observed using a confocal microscope (Olympus Corporation: confocal laser scanning microscope FV3000). These results are shown in FIGS. 2 to 4.

[0064] FIG. 2 is a photograph showing the results of immunostaining of fingers collected from E6 chickens. FIG. 2(A) shows a photograph taken using a 10× objective lens, and FIG. 2(B) shows a photograph taken using a 20× objective lens. In FIG. 2(A), the scale bar indicates 200 μm, and in FIG. 2(B), the scale bar indicates 100 μm. As shown in FIG. 2, in the fingers of E6 chickens, aggrecan and CD31 were co-localized in the interdigital region (the region surrounded by the dashed line).

[0065] FIG. 3 is a photograph showing the results of immunostaining of fingers collected from E7 chickens. FIG. 3(A) shows a photograph taken using a 10× objective lens, and FIG. 3(B) shows a photograph taken using a 20× objective lens. In FIG. 3(A), the scale bar indicates 200 μm, and in FIG. 3(B), the scale bar indicates 100 μm. As shown in FIG. 3, in the fingers of E7 chickens, similar to E6, aggrecan and CD31 were co-localized in the interdigital region (the region surrounded by the dashed line). Also, in the fingers of E7 chickens, no expression of Caspase-3 was observed.

[0066] FIG. 4 is a photograph showing the results of immunostaining of fingers collected from E8 chickens. FIG. 4(A) shows a photograph taken using a 10× objective lens, and FIG. 4(B) shows a photograph taken using a 20× objective lens. In FIG. 4(A), the scale bar indicates 200 μm, and in FIG. 4(B), the scale bar indicates 100 μm. As shown in FIG. 4, in the fingers of E8 chickens, expression of Caspase-3 was observed between the co-localized parts of aggrecan and CD31 in the interdigital region (the region surrounded by the dashed line).

[0067] From these results, it was found that apoptosis occurs at sites other than the localization site of aggrecan in the interdigital region during finger formation in chickens.

[0068] (2) Examination of the finger formation process Next, it was examined whether the deficiency of aggrecan affects finger formation. Specifically, the finger formation in the development process of CMD (cartilage matrix deficiency) mice, which are spontaneous knockout mice of the aggrecan gene (ACAN) encoding aggrecan, was examined. At E17.5, wild-type mice (WT mice), ACAN heterozygous knockout mice (HT mice), and ACAN knockout mice (KO mice) were observed with a compact grino stereomicroscope (ZEISS Stemi 305), and the middle finger of the forelimb was measured using Image J. These results are shown in Fig. 5.

[0069] Fig. 5 is a photograph and graph showing the results of observing the fingers of E17.5 mice. Fig. 5(A) shows photographs of the fingers of WT mice, HT mice, and KO mice from left to right. Also, in Fig. 5(A), the scale bar indicates 500 μm. In Fig. 5(B), the vertical axis indicates the relative length (%) of the middle finger when the length of the middle finger of WT mice is set to 100%, and the horizontal axis indicates the type of mouse. As shown in Fig. 5, it was found that the relative length of the middle finger of HT mice was shorter than that of WT mice, and the relative length of the middle finger of KO mice was even shorter. From these results, it was found that aggrecan promotes finger formation and finger elongation. Also, it was suggested that the deficiency of aggrecan causes an increase in apoptosis and a shortening of the relative length of the middle finger.

[0070] As described above, the present disclosure has been described with reference to the embodiments and examples, but the present disclosure is not limited to the above embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0071] Patents, patent applications, and documents cited in this specification are incorporated herein by reference in their entirety as if their contents were specifically set forth herein.

[0072] <Supplementary Note> Some or all of the above-described embodiments and examples may be described as follows, but are not limited thereto. <First Composition> (Supplementary Note 1) A composition for promoting finger formation, comprising a proteoglycan derived from animal tissue. (Supplementary Note 2) The composition according to Supplementary Note 1, wherein the proteoglycan is aggrecan. (Supplementary Note 3) The composition according to Supplementary Note 1 or 2, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000. (Supplementary Note 4) The composition according to any one of Supplementary Notes 1 to 3, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, chicken, and skate. (Supplementary Note 5) The composition according to any one of Supplementary Notes 1 to 4, wherein the proteoglycan is supported on a substrate. (Supplementary Note 6) The composition according to Supplementary Note 5, wherein the substrate is a gel. <Second Composition> (Supplementary Note 7) A composition for promoting finger extension, comprising a proteoglycan derived from animal tissue. (Supplementary Note 8) The composition according to Supplementary Note 7, wherein the proteoglycan is aggrecan. (Supplementary Note 9) The composition according to Supplementary Note 7 or 8, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000. (Supplementary Note 10) The composition according to any one of Supplementary Notes 7 to 9, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, chicken, and skate. (Supplementary Note 11) The proteoglycan is a composition according to any one of Appendices 7 to 10, supported on a substrate. (Appendix 12) The substrate is a gel, and it is a composition according to Appendix 11. <Method for promoting finger formation> (Appendix 13) A method for promoting finger formation, which uses a composition for promoting finger formation according to any one of Appendices 1 to 6. <Method for promoting finger extension> (Appendix 14) A method for promoting finger extension, which uses a composition for promoting finger extension according to any one of Appendices 7 to 12.

Industrial applicability

[0073] As described above, according to the present disclosure, it is possible to promote finger formation and the like. Therefore, the present disclosure can be said to be extremely useful, for example, in the medical field.

Claims

1. A composition for promoting finger formation, comprising a proteoglycan derived from animal tissue.

2. The composition according to claim 1, wherein the proteoglycan is aggrecan.

3. The composition according to claim 1 or 2, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000.

4. The composition according to any one of claims 1 to 3, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, domestic chicken, and skate.

5. The composition according to any one of claims 1 to 4, wherein the proteoglycan is supported on a substrate.

6. The composition according to claim 5, wherein the substrate is a gel.

7. A composition for promoting finger extension, comprising a proteoglycan derived from animal tissue.

8. The composition according to claim 7, wherein the proteoglycan is aggrecan.

9. The composition according to claim 7 or 8, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000.

10. The composition according to any one of claims 7 to 9, wherein the animal is selected from the group consisting of salmon, pig, chicken, flounder, domestic chicken, and skate.

11. The composition according to any one of claims 7 to 10, wherein the proteoglycan is supported on a substrate.

12. The composition according to claim 11, wherein the substrate is a gel.

13. A method for promoting finger formation, using the composition for promoting finger formation according to any one of claims 1 to 6.

14. A method for promoting finger extension, using the composition for promoting finger extension according to any one of claims 7 to 12.

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