KR20170110059A - Composition comprising GDF11 and uses thereof - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for skin regeneration, a pharmaceutical composition for improving wrinkles, a pharmaceutical composition for wound healing, a quasi drug composition for skin regeneration, a quasi drug composition for wrinkle improvement, a wound treatment composition containing GDF11 or a human- A cosmetic composition for improving skin appearance, a composition for improving wound healing, a composition for culturing fibroblasts, a method for culturing fibroblasts using the medium composition, and a method for producing GDF11 by culturing the stem cells . Since GDF11 provided in the present invention has an effect of promoting the proliferation of fibroblasts by being contained in a human adult stem cell culture solution, it can be widely used for development of a product for skin regeneration, wrinkle improvement or wound healing will be.

Description

Composition comprising GDF11 and uses thereof

The present invention relates to a composition comprising GDF11 and its use, and more specifically, the present invention relates to a pharmaceutical composition for skin regeneration comprising GDF11 or a human-derived adult stem cell culture solution comprising the same, a pharmaceutical composition for improving wrinkles, and for treating wounds. A pharmaceutical composition, a quasi-drug composition for skin regeneration, a quasi-drug composition for wrinkle improvement, a quasi-drug composition for wound treatment, a cosmetic composition for skin regeneration, a cosmetic composition for wrinkle improvement, a cosmetic composition for wound improvement, a medium composition for fibroblast culture, and the medium composition It relates to a method of culturing fibroblasts by using and a method of producing GDF11 by culturing the stem cells.

Recently, modern people are increasingly interested in healthy life, and with the improvement of living standards, the increase in women's advancement to society, and changes in an aging society, consumers' desire for cosmetics is gradually emphasized from the use of simply to beautify the cosmetics. As interest increases, research is being actively conducted to find natural substances that are harmless to the human body. Skin aging is a complex biological phenomenon and can be divided into two main factors: natural aging that progresses over time (endogenous aging) and photoaging that is rare in the external environment, especially ultraviolet rays. Since the skin is always exposed to oxygen and the solar tube, the oxidative stress caused by it promotes skin aging. Since the skin is always in contact with various environmental factors, it is directly exposed to the attack of oxidative stress factors.When the skin is exposed to a large amount of ultraviolet rays, reactive oxygen species (ROS) are excessively generated in the skin, preventing antioxidants. The system collapses, eventually accelerating aging.

Elastase, which is present in the neutrophil granulocytes of the human body, is an enzyme that degrades elastin, a matrix protein important for maintaining skin elasticity in the dermis, and is a non-specific hydrolytic enzyme capable of decomposing collagen, another important matrix protein. The elastase inhibitor has an effect of improving skin wrinkles, and ursolic acid is used as an elastase inhibitor, but ursolic acid is difficult to formulate because it has a property that is not well soluble in solvents such as water or oil. There is a problem that is difficult to use.

Collagen is mostly present in the dermal layer of the skin and accounts for about 70-80% of the total dry weight of the skin. Collagen, a major component of the extracellular matrix, is a major matrix protein produced by fibroblasts of the skin. Collagen synthesis and decomposition are properly regulated, but their synthesis decreases as aging progresses, and the expression of collagenase, an enzyme that decomposes collagen, is promoted by UV irradiation, which is known to be closely related to the formation of wrinkles in the skin.

In addition, the deficiency of the matrix protein is one of the important factors in photoaging, and when the synthesis of collagen and elasticity, which are components filling the cells by ultraviolet light, decreases, the expression of various matrix proteolytic enzymes increases. Therefore, it is decomposed by collagenase and elastase, which are the main causes of skin aging, collagen and elastin-degrading enzymes, and the dermal layer determines the physicochemical properties of the skin and plays an important role in supplying nutrients to the capillaries and epidermis. Therefore, it is closely related to the aging of the skin.

Conventionally, in order to take advantage of the wrinkle-improving effect of collagen, products containing collagen in external skin compositions such as cosmetics or ointments have been released, but these products apply collagen itself to the skin surface, making it difficult for the transdermal absorption of collagen, a polymer material. It could not show the essential wrinkle improvement effect. In order to solve this problem, interest in collagen synthesis promoting substances has increased, and conventionally known collagen synthesis promoting substances include vitamin C, retinoic acid, transforming growth factor (TGF), and animal placenta-derived protein (JP8). -231370), betulinic acid (JP8-208424), and chlorella extract (JP9-40523, JP10-36283, promoting fibroblast proliferation).

In recent years, as interest in stem cells has increased, it has been reported that stem cells having differentiation ability or extracts thereof can inhibit skin aging, and accordingly, research on a method of inhibiting skin aging using stem cells is actively progressing. Has become. For example, Korean Patent Application Publication No. 2009-0116659 discloses a cosmetic composition for improving wrinkles, whitening or preventing aging, including a stem cell culture solution. However, since such a stem cell culture solution contains a myriad of components, it has not been confirmed yet what components exhibit a substantial wrinkle improvement effect. If an active ingredient capable of exhibiting such wrinkle improvement effects is identified, it is expected that a product that can more effectively improve wrinkles will be developed, but there is no significant achievement yet.

Under this background, the present inventors have made intensive research efforts to discover an active ingredient capable of improving skin wrinkles from human-derived adult stem cell culture liquids known to have an effect of improving skin wrinkles. As a result, GDF11 contained in the culture liquid It not only promotes proliferation of blast cells, but also increases the level of synthesis of collagen that can improve skin wrinkles, inhibits the activity of collagenase, confirms that it is an active ingredient showing the effect of improving skin wrinkles, and completes the present invention. I did.

One object of the present invention is to provide a pharmaceutical composition for skin regeneration comprising GDF11 or a human-derived adult stem cell culture solution containing the same.

Another object of the present invention is to provide a pharmaceutical composition for improving wrinkles comprising GDF11 or a human-derived adult stem cell culture solution containing the same.

Another object of the present invention is to provide a pharmaceutical composition for treating wounds comprising GDF11 or a human-derived adult stem cell culture solution containing the same.

Another object of the present invention is to provide a quasi-drug composition for skin regeneration comprising GDF11 or a human-derived adult stem cell culture solution comprising the same.

Another object of the present invention is to provide a quasi-drug composition for improving wrinkles comprising GDF11 or a human-derived adult stem cell culture solution comprising the same.

Another object of the present invention is to provide a quasi-drug composition for wound improvement comprising GDF11 or a human-derived adult stem cell culture solution comprising the same.

Another object of the present invention is to provide a cosmetic composition for skin regeneration comprising GDF11 or a human-derived adult stem cell culture solution comprising the same.

Another object of the present invention is to provide a cosmetic composition for improving wrinkles comprising GDF11 or a human-derived adult stem cell culture solution containing the same.

Another object of the present invention is to provide a cosmetic composition for improving wounds comprising GDF11 or a human-derived adult stem cell culture solution containing the same.

Another object of the present invention is to provide a medium composition for culturing fibroblasts comprising GDF11 or a human-derived adult stem cell culture solution comprising the same.

Another object of the present invention is to provide a method of culturing fibroblasts using the medium composition.

Another object of the present invention is to provide a method for producing GDF11 comprising the step of culturing the human-derived adult stem cells.

The inventors of the present invention have conducted various studies to discover an active ingredient capable of improving skin wrinkles from human-derived adult stem cell culture fluid known to have an effect of improving skin wrinkles, while GDF11 (which is known to have an effect of preventing or treating dementia) ( Growth differentiation factor 11) was paid attention. The GDF11 is known as a protein that restores motor ability and regenerates degenerated cerebrovascular cells. It is a human-derived adult stem cell culture fluid that promotes fibroblast proliferation, improves mobility, and increases the expression of matrix proteins. It was confirmed that the GDF11 was detected, and as a result of comparing it with the culture medium of other stem cells, it was confirmed that it was contained in a large amount in the culture medium obtained by culturing human umbilical cord blood-derived mesenchymal stem cells among various adult stem cells. Accordingly, as a result of verifying the effect of GDF11, it was confirmed that when the expression of GDF11 is decreased, the proliferation rate of human-derived adult stem cells decreases, and the expression level of type 3 collagen expressed therefrom decreases. Various growth factors (EGF, bFGF, TGF-beta1 or vEGF) are involved in the expression, as well as GDF11 treatment of fibroblasts promotes the proliferation of fibroblasts and increases the expression of collagen and elastin in the fibroblasts. Sikkim was confirmed. Therefore, the GDF11 was analyzed to be able to promote the wound treatment effect, skin regeneration effect, wrinkle improvement effect, etc. that can be caused by promoting the proliferation of fibroblasts.

As such, the fact that GDF11 is involved in the proliferation of fibroblasts and the resulting skin wound treatment, wrinkle improvement, and skin regeneration was not known at all until now, and was first identified by the present inventors.

In order to achieve the above object, the present invention provides a pharmaceutical composition for skin regeneration, wrinkle improvement, or wound treatment comprising GDF11 or a human-derived adult stem cell culture solution containing the same as one embodiment.

The term "GDF11 (growth differentiation factor 11)" of the present invention, also referred to as "BMP-11 (bone morphogenetic protein 11)", refers to a protein expressed by the GDF11 gene present in human chromosome 12. The GDF11 is known as a myostatin analog protein, and is known to act as an inhibitor to inhibit the growth of nervous tissue, and recently, it has an effect of preventing or treating dementia, which has an effect of restoring exercise capacity and regenerating degenerative cerebrovascular blood vessels. It has been reported. The sequence information of GDF11 of the present invention can be obtained from a known database such as the National Center for Biotechnology Information (NCBI). For example, it may be a human-derived GDF11 gene (NM_005811), a human-derived GDF11 protein (NP_005802), a mouse-derived GDF11 gene (NM_010272), a mouse-derived GDF11 protein (NP_034402), and the like.

In the present invention, the GDF11 may exhibit effects such as promoting skin regeneration, promoting wrinkle improvement, and promoting wound treatment through promoting the proliferation of fibroblasts, and thus can be used as an active ingredient of a composition exhibiting these effects.

The term "human-derived adult stem cell culture medium" of the present invention refers to a culture obtained by culturing human adult stem cells or a culture supernatant from which stem cells are removed. The culture medium obtained by culturing the adult stem cells contains various substances secreted while culturing the adult stem cells (eg, GDF1, etc.), and the use of the human-derived adult stem cell culture solution promotes the proliferation of fibroblasts. It can exhibit effects such as promoting skin regeneration, promoting wrinkle improvement, and promoting wound healing.

In the present invention, the human-derived adult stem cell culture medium can be interpreted as a culture supernatant obtained by culturing human adult stem cells, and the adult stem cells used therein are specifically limited thereto as long as it can secrete GDF11 into the culture medium. However, as an example, it may be derived from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amniotic membrane or placenta, and in the present invention, a culture medium of human umbilical cord blood-derived mesenchymal stem cells is used as a human-derived mesenchymal stem cell culture solution. Was used.

According to an embodiment of the present invention, as a result of confirming the effect of the stem cell culture solution on the proliferation ability of fibroblasts, the human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM) is a fibroblast culture solution (HDF CM) or human adipose-derived It was confirmed that the proliferation of fibroblasts can be promoted and the total amount of proteins secreted from the cells can be increased compared to the stem cell culture solution (AD-MSC CM) (Fig. 1), and it has been confirmed that it promotes the mobility of fibroblasts (Fig. 2). ). In addition, it was confirmed that the expression level of various matrix proteins (collagen, fibronectin, elastin, etc.) expressed in fibroblasts was improved to a relatively high level (Figs. 3a and 3b), and showed relatively excellent wound treatment effects in animal models. (Figures 4a and 4b).

As a result of analyzing the effect of GDF11 (growth differentiation factor 11) as an active ingredient of human-derived adult stem cell culture medium that exhibits such effects, it was confirmed that GDF11 was contained in a large amount in the human umbilical cord blood-derived mesenchymal stem cell culture medium. (Fig. 5 a and b), it was confirmed that the GDF11 is secreted at a higher level in human cord blood-derived mesenchymal stem cells compared to human bone marrow and adipose-derived mesenchymal stem cells (Fig. 6 a and b). In addition, as a result of analyzing the effect of the GDF11 on human umbilical cord blood-derived mesenchymal stem cells, inhibition of the expression level of GDF11 inhibited the proliferation of human umbilical cord blood-derived mesenchymal stem cells, and the expression of type 3 collagen expressed therefrom. The level was decreased (Fig. 7 a and b), and it was confirmed that EGF, bFGF, TGF-beta1 and vEGF are involved in the expression of GDF11 (Fig. 8).

As a result of analyzing the effect of GDF11 on fibroblasts, it was confirmed that it promotes the proliferation of fibroblasts and increases the expression of collagen and elastin in the fibroblasts.

Therefore, the GDF11 was analyzed to be able to promote the wound treatment effect, skin regeneration effect, wrinkle improvement effect, etc. that may be caused by promoting the proliferation of fibroblasts.

The pharmaceutical composition of the present invention may be prepared in the form of a pharmaceutical composition for skin regeneration, wrinkle improvement, and wound treatment, further comprising an appropriate carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions. Specifically, the pharmaceutical composition is formulated in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories, and sterile injectable solutions according to a conventional method. I can. In the present invention, the carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like. In the case of formulation, it can be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient, such as starch, calcium carbonate, in the extract and its fractions, It can be prepared by mixing sucrose or lactose, gelatin, and the like. In addition, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include various excipients, such as humectants, sweeteners, fragrances, and preservatives, in addition to water and liquid paraffin, which are simple diluents commonly used for suspensions, liquid solutions, emulsions, syrups, etc. have. Formulations for parenteral administration may include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like may be used.

The content of GDF11 included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 1 X 10 ^-9 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition. have.

The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount, and the term "pharmaceutically effective amount" of the present invention is used to treat or prevent a disease at a reasonable benefit/risk ratio applicable to medical treatment or prevention. It means a sufficient amount, and the effective dose level is the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the rate of excretion. The duration, factors including drugs used in combination or co-use with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with known pharmaceutical compositions for skin regeneration, wrinkle improvement, and wound treatment. Considering all of the above factors, it is important to administer an amount capable of obtaining the maximum effect in a minimum amount without side effects.

The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art in consideration of the purpose of use, the degree of addiction to the disease, the age, weight, sex, history, or type of substance used as an active ingredient. For example, the pharmaceutical composition of the present invention can be administered in an amount of about 0.1 ng to about 100 mg/kg, preferably 1 ng to about 10 mg/kg per adult, and the frequency of administration of the composition of the present invention is specifically Although not limited, it may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way.

As another aspect, the present invention provides a method for skin regeneration, wrinkle improvement, or wound treatment comprising administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than a human suffering from cancer.

The term "individual" of the present invention may include, without limitation, mammals, farmed fish, etc., including mice, livestock, etc., which require skin regeneration, wrinkle improvement or wound treatment, but excluding humans among the subjects in which the disease is invented. do.

The term "treatment" of the present invention means that a pharmaceutical composition containing GDF11 of the present invention as an active ingredient is administered to an individual in need of skin regeneration, wrinkle improvement, or wound treatment to perform or benefit from skin regeneration, wrinkle improvement, or wound treatment. It means all the actions you do.

The route of administration of the pharmaceutical composition for skin regeneration, wrinkle improvement or wound treatment of the present invention may be administered through any general route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but the route of intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, rectal administration, etc. Can be administered through. However, since the GDF11 may be denatured or decomposed by gastric acid when administered orally, the oral composition should be coated with an active agent or formulated to protect it from decomposition in the stomach. In addition, the composition can be administered by any device capable of moving the active substance to the target cell.

The present invention provides a quasi-drug composition for skin regeneration, wrinkle improvement, or wound treatment comprising the GDF11 or a human-derived adult stem cell culture solution comprising the same as another aspect.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter related to the condition being treated, for example a symptom.

The term "quasi-drug" as used in the present invention refers to items that are less effective than medicines among items used for the purpose of diagnosing, treating, improving, alleviating, treating, or preventing diseases of humans or animals. According to this, quasi-drugs are products excluding items used for pharmaceutical purposes, textiles and rubber products used for the treatment or prevention of diseases of humans and animals, and those that have mild or no direct action on the human body, and are similar to those that are not instruments or machines. This includes sterilization and insecticides to prevent infectious diseases. The type or formulation of the quasi-drug composition of the present invention is not particularly limited, but preferably, it may be a disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.

In another aspect, the present invention provides a cosmetic composition for skin regeneration, wrinkle improvement, or wound improvement comprising the GDF11 or a human-derived adult stem cell culture solution containing the same.

As described above, since GDF11 can promote the proliferation of fibroblasts, it can be used in the manufacture of functional cosmetics exhibiting skin regeneration, wrinkle improvement, or wound improvement effect caused by the proliferation of fibroblasts.

The term "functional cosmetics (cosmedical, cosmeceutical)" of the present invention is a product that has a professional function that emphasizes physiologically active efficacy and effect unlike general cosmetics by introducing the professional treatment function of pharmaceuticals into cosmetics. It refers to cosmetics prescribed by the Ordinance of the Ministry of Health and Welfare among products that help, products that help improve skin wrinkles, and products that help to burn the skin finely or protect the skin from UV rays.

In the present invention, the functional cosmetic refers to a product that helps prevent skin aging by showing skin regeneration, wrinkle improvement, and wound improvement effect among various functional cosmetic products, and as an example, GDF11 or a human-derived adult stem cell culture solution containing the same It may be a functional cosmetic containing as an active ingredient, but is not particularly limited thereto, and the content of GDF11 included in the functional cosmetic is also not particularly limited.

The functional cosmetic of the present invention includes the GDF11 or a human-derived adult stem cell culture solution containing the same as an active ingredient, and may additionally contain a cosmetic that is commonly used, for example, glycerol or propylene glycol for water-soluble skin formulation. , 1,3-butylene glycol, sorbitol, polyethylene glycol, carboxyvinyl polymer, xanthan gum, carboxymethylcellulose, hydroxyethylcellulose, hydroxymethylcellulose, locust bean gum, allantoin, carrageenan, etc. may be added; Beeswax, paraffin wax, stearyl alcohol, carnauba wax, candelilla wax and calcium stearate, aluminum stearate, zinc stearate, witchhazel, and the like as viscosity and hardness control agents; Butylmethoxydibenzoylmethane, octylmethoxycinnamate, etc. may be used as the ultraviolet absorber; Pigments include extender pigments such as titanium dioxide, fine particles of dithanum dioxide, kaolin, nylon powder, talc, sericite, mica, polymethyl methacrylate, and yellow iron oxide, black iron oxide, red iron oxide, ultramarine, chromium oxide, chromium hydroxide, etc. It is possible to use colored pigments of; As a moisturizing agent, natural moisturizing substances such as 1,3-butylene glycol, concentrated glycerin, ethylene glycol, etc., such as chitin, chitosan, hyaluronic acid, hyaluronic acid, lactic acid, and glycolic acid, may be used; As a preservative, paraoxybenzoic acid esters, imidazolidinyl urea, and the like can be used, as well as one or two or more kinds of the above ingredients according to product characteristics.

The functional cosmetics of the present invention can be prepared in any formulation conventionally prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oil, powder foundation, emulsion foundation, wax foundation, and may be formulated as a spray, but is not limited thereto. In more detail, it may be prepared in the form of a flexible lotion, nutritional lotion, nutritional cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component. I can.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component. In particular, in the case of a spray, additionally chlorofluorohydrocarbon, propane / May contain propellants such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, and crystallites Sex cellulose, aluminum metahydroxide, bentonite, agar or tracanth, and the like can be used.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters may be used.

In another aspect, the present invention provides a fibroblast culture medium composition comprising the GDF11 or a human-derived adult stem cell culture medium containing the same, and a method of culturing fibroblasts using the medium composition.

As described above, since GDF11 or a human-derived adult stem cell culture medium containing the same provided by the present invention can promote the proliferation of fibroblasts, the GDF11 or a human-derived adult stem cell culture medium containing the same is used to cultivate fibroblasts. It can be used as an active ingredient of a culture medium composition for, and fibroblasts can be cultured using a culture medium composition for culturing the fibroblasts.

Meanwhile, the method of culturing fibroblasts provided in the present invention includes inoculating and culturing fibroblasts in the medium composition for culturing fibroblasts.

In another aspect, the present invention provides a method for producing GDF11 comprising the step of culturing the human-derived adult stem cells.

Specifically, the method for producing GDF11 provided by the present invention includes the steps of: (a) culturing human-derived adult stem cells, and obtaining a culture supernatant; And (b) recovering GDF11 from the obtained culture supernatant.

The term "cultivation" of the present invention refers to an overall action of growing cells in an appropriately artificially controlled environmental condition.

For the purposes of the present invention, the culturing is performed for the purpose of culturing the stem cells provided in the present invention to prepare GDF11 secreted into the culture supernatant, and such culturing method is not particularly limited, and a method widely known in the art is used. It can be done by using.

The culture conditions are not particularly limited thereto, but a basic compound (such as sodium hydroxide, potassium hydroxide, or ammonia) or an acidic compound (such as phosphoric acid or sulfuric acid) is used to provide an appropriate pH (pH 5 to 9, preferably pH 6 to 8). , Most preferably pH 6.8) can be adjusted, oxygen or an oxygen-containing gas mixture can be introduced into the culture to maintain aerobic conditions, and the culture temperature is not particularly limited thereto, but as an example, 30 to 40°C, As another example, it may be 33 to 37°C, and as another example, it may be 37°C, and the incubation time is not particularly limited thereto, but as an example, 5 to 15 days, as another example, 7 to 10 days, as another example, 7 days Can be

In addition, the culture medium used is a carbon source such as sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molase, starch and cellulose), fats and fats (e.g., soybean oil, sunflower seeds). Oil, peanut oil and coconut oil), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol), and organic acids (e.g. acetic acid) can be used individually or in combination. ; Nitrogen sources include nitrogen-containing organic compounds (e.g. peptone, yeast extract, broth, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds (e.g. ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, and Ammonium nitrate) or the like may be used individually or in combination; As the phosphorus source, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, corresponding sodium-containing salts and the like may be used individually or in combination; Other metal salts (such as magnesium sulfate or iron sulfate), amino acids, and essential growth-promoting substances such as vitamins may be included.

In addition, various growth factors such as EGF, bFGF, vEGF, and TGF-β1 may be additionally included in the medium to promote smooth proliferation of stem cells.

In addition, the step of recovering GDF11 from the culture may be performed by a method known in the art. Specifically, the known GDF11 recovery method is not particularly limited thereto, but centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g. For example, it is preferable to use methods such as ion exchange, affinity, hydrophobicity and size exclusion).

GDF11 provided by the present invention is contained in human-derived adult stem cell culture medium to promote the proliferation of fibroblasts, so it can be widely used in the development of more diverse skin regeneration, wrinkle improvement, or wound treatment products. will be.

1 is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) on the proliferation ability of fibroblasts It is a photograph showing the result of comparing the effect (A), a graph showing the level of absorbance (B), a graph showing the cell number level (C), and a graph showing the level of the total protein expression (D).
Figure 2 is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) effects on the mobility of fibroblasts. This is a photomicrograph showing the results of comparing the effects.
Figure 3a is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM), or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) various expressions in fibroblasts This is a Western blot analysis picture showing the result of comparing the effect of the matrix protein on the protein expression level.
Figure 3b is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM), or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) various expressions in fibroblasts This is a picture of RT-PCR analysis showing the result of comparing the effect on the expression level of the substrate protein.
Figure 4a is a comparison of the wound treatment effect of the control (CTL), fibroblast culture (HDF CM), human adipose-derived stem cell culture (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture (UCB-MSC CM) These are graphs and photos showing the results.
Figure 4b is a control (CTL), fibroblast culture (HDF CM), human adipose-derived stem cell culture (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture (UCB-MSC CM) of the treated wound animal model This is a tissue photo showing the result of comparing the area of the wound.
5A shows the level of GDF11 mRNA expressed in fibroblasts (HDF), human adipose-derived stem cells (AD-MSC), or human umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) by performing RT-PCR and realtime qPCR. It is a graph showing the result of the comparison, and b of FIG. 5 is a photograph showing the result of performing RT-PCR.
6A is a human bone marrow-derived stem cell culture solution (BM-MSC CM) used as a control (CTL), a human adipose-derived stem cell culture solution (AD-MSC CM), or a human cord blood-derived stem cell culture solution (UCB-MSC CM). It is a Western blot analysis picture showing the result of comparing the level of GDF11 contained in FIG. 6B is a graph of quantitative analysis of the western blot analysis result.
7A is a graph showing the effect of GDF11 on the proliferation of human umbilical cord blood-derived mesenchymal stem cells, and FIG. 7B is a comparison of the change in the expression level of collagen according to the inhibition of the expression of GDF11 in the human umbilical cord blood-derived mesenchymal stem cells. This is a picture showing the result.
8 is a control (a), EGF (b), bFGF (c), TGF-beta1 (d) or vEGF (e) in human umbilical cord blood-derived mesenchymal stem cells treated with the expression level of GDF11 according to treatment time and concentration It is a graph showing the result of comparing the change of.
FIG. 9 shows the level of proliferation of fibroblasts in fibroblasts treated with GDF11 (FIG. 9 a ), the level of expression of type 1 collagen expressed in the fibroblasts (FIG. 9 b and f ), and the expression in the fibroblasts. The expression level of type 3 collagen (Fig. 9 c and f), the expression level of elastin expressed in the fibroblasts (Fig. 9 d and f), and the expression level of MMP1 expressed in the fibroblasts (Fig. 9 e And f) are graphs and photographs showing the comparison result.

Hereinafter, the present invention will be described in more detail through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example 1: Acquisition of stem cell culture solution

Example 1-1: Obtaining human umbilical cord blood-derived mesenchymal stem cell culture solution

Human cord blood stem cells (1.89 X 10 ⁵ cells) isolated from cord blood were inoculated into EGM-2 (endothelial growth medium) containing 10% FBS, and _{cultured at 37° C. and 5% CO 2} for 48 hours, Cultured cells were obtained. The obtained cells were again inoculated into H1 medium, cultured for 96 hours, and then a culture solution of human umbilical cord blood-derived mesenchymal stem cells was obtained. At this time, as the H1 medium, a DMEM medium containing EGF, bFGF, vEGF and TGF-β1 was used.

Example 1-2: Obtaining a human adipose-derived stem cell culture solution

The inhaled adipose tissue was washed with PBS, 1 µl/ml of primocin and 1 mg/ml of type 1 collagenase were added, followed by reaction at 37°C for 2 hours. After the reaction was completed, the precipitated cells were obtained by centrifugation (2000 rpm, 5 minutes), and the cells were suspended in a culture solution (DMEM medium containing 0.2% primocin, 1% glutamax, and 10% FBS) and filtered. Then, centrifugation was performed again (1000 rpm, 5 minutes) to obtain precipitated cells. The obtained cells were placed in a lysis buffer (ACK lysing buffer, Gibco) and reacted for 1 minute. Subsequently, the cells (1.89 X 10 ⁵ cell count) were washed with PBS, and keratinocyte-SFM media containing 0.5% K-NAC medium (5% FBS, 20mM ascorbic acid 1% and 400mM N-acetyl-L-cysteine 0.5%) ) And cultured for 48 hours at 37° C. and 5% CO ₂ conditions, and cultured cells were obtained. The obtained cells were again inoculated into a serum-free medium (H1 media, DMEM medium), cultured for 96 hours, and then a human adipose-derived stem cell culture solution was obtained.

Example 2: of fibroblasts On the ability to proliferate Effect of cell culture medium on

Fibroblasts (HDFs) were inoculated in a 96-well plate at ^{a density of 1×10 3} cells per well, and cultured for 24 hours. Then, the fibroblast culture solution, the human umbilical cord blood-derived mesenchymal stem cell culture solution obtained in Example 1-1, or the human adipose-derived stem cell culture solution obtained in Example 1-2 were added and cultured for 72 hours. At this time, as a control, H1 medium was added and cultured was used. After the cultivation was completed, 10 µl of CCK-8 included in the CCK-8 kit was added to the culture, reacted for 3 hours, and then absorbance was measured at 450 nm to measure and compare the proliferative capacity of each fibroblast (Fig. One).

1 is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) on the proliferation ability of fibroblasts It is a photograph showing the result of comparing the effect (A), a graph showing the level of absorbance (B), a graph showing the cell number level (C), and a graph showing the level of the total protein expression (D). As shown in Figure 1, human cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM) promotes the proliferation of fibroblasts than the fibroblast culture solution (HDF CM) or human adipose-derived stem cell culture solution (AD-MSC CM), It was confirmed that the total amount of protein secreted from cells can also be increased.

Example 3: Analysis of fibroblast mobility

Fibroblasts were inoculated into 6-well plates at ^{a density of 2×10 5} cells per well, and cultured for 48 hours. Then, the medium was removed, a scratch was formed on the bottom of the culture vessel, and then washed with PBS. Subsequently, the fibroblast culture solution, the human umbilical cord blood-derived mesenchymal stem cell culture solution obtained in Example 1-1, or the human adipose-derived stem cell culture solution obtained in Example 1-2 were added and cultured for 72 hours. At this time, as a control, H1 medium was added and cultured was used. After the culture was completed, the level of fibroblasts moved to the scratch site was confirmed with a microscope (FIG. 2).

Figure 2 is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) effects on the mobility of fibroblasts. This is a photomicrograph showing the results of comparing the effects. 2, human cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) promotes fibroblast mobility than fibroblast culture medium (HDF CM) or human adipose-derived stem cell culture medium (AD-MSC CM). Confirmed.

Example 4: of fibroblasts Matrix protein Expression analysis

Example 4-1: Western Blot analysis

Fibroblasts were inoculated into 6-well plates at ^{a density of 2×10 5} cells per well, and cultured for 24 hours. Then, the fibroblast culture solution, the human umbilical cord blood-derived mesenchymal stem cell culture solution obtained in Example 1-1, or the human adipose-derived stem cell culture solution obtained in Example 1-2 was added and cultured for 24 hours, and then each of the cultured cells Western blot analysis was performed on fibroblasts using antibodies against type 1 collagen I, type 4 collagen IV, fibronectin, or elastin (FIG. 3A). At this time, GAPDH was used as an internal control.

Figure 3a is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM), or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) various expressions in fibroblasts This is a Western blot analysis picture showing the result of comparing the effect of the matrix protein on the protein expression level. As shown in FIG. 3A, it was confirmed that fibronectin and elastin were expressed at a relatively highest level in fibroblasts treated with human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM).

Example 4-2: RT- PCR analysis

Total RNA was obtained from each fibroblast cultured in Example 4-1, and each cDNA was synthesized using RT-Premix (Bioneer). Using the synthesized cDNA as a template, PCR was performed using the following primers, and the expression levels of type 1 collagen, type III collagen, or fibronectin were compared at the mRNA level. (Fig. 4b). At this time, GAPDH was used as an internal control.

collagen type I F: 5'-tcaaggtttccaaggacctg-3' (SEQ ID NO: 1)

collagen type I R: 5'-tcaaggtttccaaggacctg-3' (SEQ ID NO: 2)

collagen type III F: 5'-aaaggggagctggctacttc-3' (SEQ ID NO: 3)

collagen type III R: 5'-gcgagtaggagcagttggag-3' (SEQ ID NO: 4)

fibronectin F: 5'-tgaagaggggcacatgctga-3' (SEQ ID NO: 5)

fibronectin R: 5'-gtgggagttgggctgactcg-3' (SEQ ID NO: 6)

Figure 3b is a control (CTL), fibroblast culture medium (HDF CM), human adipose-derived stem cell culture medium (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture medium (UCB-MSC CM) various expressions in fibroblasts This is a picture of RT-PCR analysis showing the result of comparing the effect on the expression level of the substrate protein. As shown in FIG. 3B, it was confirmed that type 3 collagen and fibronectin were expressed at a relatively highest level in fibroblasts treated with human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM).

Example 5: Analysis of the therapeutic effect of the wound animal model

First, a 6mm full-layer skin peeling wound was induced on the back of a 5-week-old nude mouse using a biopsy punch, and a wound animal model was produced.

After 24 hours elapsed, 200 µl of each fibroblast culture solution obtained in the wound site of the produced wound animal model, the human umbilical cord blood-derived mesenchymal stem cell culture solution obtained in Example 1-1 or the obtained in Example 1-2 Human adipose-derived stem cell culture solution was applied, and each of the culture solutions was fixed to the wound area using a silicone band. The same operation was repeated after 72 hours. At this time, as a control, a wound animal model coated with H1 medium was used. Finally, the applied wound animal model was bred for 7 days, and then the reduced level of wound size was compared (FIG. 4A).

Figure 4a is a comparison of the wound treatment effect of the control (CTL), fibroblast culture (HDF CM), human adipose-derived stem cell culture (AD-MSC CM), or human umbilical cord blood-derived mesenchymal stem cell culture (UCB-MSC CM) These are graphs and photos showing the results. As shown in Figure 4a, it was confirmed that the human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM) showed the best wound treatment effect.

Meanwhile, the wound area of the animal model was excised, and the cross section of the wound area was compared (FIG. 4B).

Figure 4b is a control (CTL), fibroblast culture (HDF CM), human adipose-derived stem cell culture (AD-MSC CM) or human umbilical cord blood-derived mesenchymal stem cell culture (UCB-MSC CM) of the treated wound animal model This is a tissue photo showing the result of comparing the area of the wound area. As shown in Figure 4b, it was confirmed once again that the human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM) exhibited the most excellent wound treatment effect.

Example 6: GDF11 (growth differentiation factor 11) on For analysis

Example 6-1: To GDF11 For RT- PCR analysis

Each total RNA was obtained from fibroblasts cultured using H1 medium, human umbilical cord blood-derived mesenchymal stem cells obtained in Example 1-1, or human adipose-derived stem cells obtained in Example 1-2, from which Each cDNA was synthesized. Each of the synthesized cDNAs was used as a template, and the mRNA levels of GDF11 were compared by performing realtime qPCR and PCR using the following primers (Fig. 5a and b). At this time, RPL13A was used as an internal control group.

GDF11 F: 5'-gatcctggacctacacgacttc-3' (SEQ ID NO: 7)

GDF11 R: 5'-ggccttcagtacctttgtgaac-3' (SEQ ID NO: 8)

RPL13A F: 5'-gcacgaccttgagggcagcc-3' (SEQ ID NO: 9)

RPL13A R: 5'-catcgtggctaaacaggtactg-3' (SEQ ID NO: 10)

5A shows the level of GDF11 mRNA expressed in fibroblasts (HDF), human adipose-derived stem cells (AD-MSC), or human umbilical cord blood-derived mesenchymal stem cells (UCB-MSC) by performing RT-PCR and realtime qPCR. It is a graph showing the result of the comparison, and b of FIG. 5 is a photograph showing the result of performing RT-PCR. 5A and 5B, it was confirmed that GDF11 was expressed in a large amount in human umbilical cord blood-derived mesenchymal stem cells (UCB-MSC).

Example 6-2: To GDF11 About Western Blot analysis

Fibroblast culture solution, human umbilical cord blood-derived mesenchymal stem cell culture solution obtained in Example 1-1, or human adipose-derived stem cell culture solution obtained in Example 1-2 was filtered (0.22 um syringe filter), and each of these culture solutions was After concentration, a Western blot analysis using an antibody against GDF11 was performed on each concentrated culture medium (Fig. 6a and b).

6A is a human bone marrow-derived stem cell culture solution (BM-MSC CM) used as a control (CTL), a human adipose-derived stem cell culture solution (AD-MSC CM), or a human cord blood-derived stem cell culture solution (UCB-MSC CM). It is a Western blot analysis picture showing the result of comparing the level of GDF11 contained in FIG. 6B is a graph of quantitative analysis of the western blot analysis result. 6A and 6B, it was confirmed that a relatively high level of GDF11 was contained in the human umbilical cord blood-derived mesenchymal stem cell culture solution (UCB-MSC CM).

Example 7: In human umbilical cord blood-derived mesenchymal stem cells Of GDF11 Functional analysis

Example 7-1: Effect on the proliferation of human umbilical cord blood-derived mesenchymal stem cells Of GDF11 Functional analysis

Human cord blood stem cells were inoculated into a 6-well plate at ^{a density of 2×10 5} cells per well, and cultured for 24 hours. Then, the cultured cells were treated with 25 nM of control siRNA or siRNA against GDF11 (siGDF11), respectively, and cultured for 72 hours. Subsequently, after subculturing each of the cells, siRNAs under the same conditions were each treated and cultured. After the cultivation was completed, the cells were obtained ^{, inoculated in a 24-well plate at a density of 4 X 10 4} cells per well, and then cultured for 2 days, according to the method of Example 2, the proliferative capacity of these cells. (Fig. 7a), according to the method of Examples 4-1 and 6-1, mRNA of GDF11, type 1 collagen I, and type 3 collagen III expressed in each of the cells The levels were compared (Fig. 7 b). At this time, RPL13A was used as an internal control group.

7A is a graph showing the effect of GDF11 on the proliferation of human umbilical cord blood-derived mesenchymal stem cells, and FIG. 7B is a comparison of the change in collagen expression level according to inhibition of the expression of GDF11 in the human umbilical cord blood-derived mesenchymal stem cells. This is a picture showing the result. As shown in FIG. 7A, when the expression of GDF11 is decreased, it was confirmed that the proliferation rate of human umbilical cord blood-derived mesenchymal stem cells decreased. As shown in FIG. 7b, when the expression of GDF11 is decreased, human umbilical cord blood-derived mesenchymal stem It was confirmed that the expression level of type 3 collagen in the cells was decreased.

Example 7-2: in human umbilical cord blood-derived mesenchymal stem cells Of GDF11 Mechanism of expression analysis

^{Human cord blood stem cells were inoculated at a density of 5 X 10 5} cells in a 100 mm culture vessel, and when the saturation was 80 to 90%, a 6-well plate was inoculated at a density of ^{2 X 10 5 cells per well, and 24} Incubated for hours. Then, it was replaced with a DMEM medium containing 100X glutamax, and EGF, bFGF, vEGF or TGF-beta1 were treated at a concentration of 1 or 10 ng/ml, respectively, and cultured for 1, 3 and 6 days. After the cultivation was completed, total RNA was obtained from each cell, cDNA was synthesized therefrom, and then real-time PCR was performed using a SYBR Green PCR device (Fig. 8 a to e). At this time, as a control, cells treated with H1 medium were used.

8 is a control (a), EGF (b), bFGF (c), TGF-beta1 (d) or vEGF (e) in human umbilical cord blood-derived mesenchymal stem cells treated with the expression level of GDF11 according to treatment time and concentration It is a graph showing the result of comparing the change of. As shown in FIG. 8, in each case, it was confirmed that GDF11 expression was increased, and the highest expression was confirmed on day 6. In addition, it was confirmed that the expression was further increased when the above four factors were simultaneously treated.

These results were analyzed to suggest that the four factors are involved in the regulation of GDF11 expression in umbilical cord blood-derived mesenchymal stem cells.

Example 8: in fibroblasts Of GDF11 Functional analysis

While culturing fibroblasts, 0, 0.01, 0.1 or 0.2 µg/ml of GDF11 was treated, and after the cultivation was completed, the proliferation level of fibroblasts, the level of expression of type 1 collagen expressed in the fibroblasts, the fibers The expression level of type 3 collagen expressed in blast cells, the expression level of elastin expressed in the fibroblasts, and the expression level of MMP1 expressed in the fibroblasts were compared and analyzed (FIG. 9).

9 shows the proliferation level of fibroblasts in fibroblasts treated with GDF11 (FIG. 9 a ), the expression level of type 1 collagen expressed in the fibroblasts (FIG. 9 b and f ), and the expression in the fibroblasts. The expression level of type 3 collagen (Fig. 9 c and f), the expression level of elastin expressed in the fibroblasts (Fig. 9 d and f), and the expression level of MMP1 expressed in the fibroblasts (Fig. 9 e And f) are graphs and photographs showing the results of comparison. As shown in Fig. 9a, as the concentration of GDF11 increases, the proliferation of fibroblasts is promoted, and as shown in Figs. 9b and f, as the concentration of GDF11 increases, the expression of type 1 collagen is promoted. However, when excessively increased, it was confirmed that the expression was rather suppressed.As shown in FIG. 9 c and f, as the concentration of GDF11 increased, the expression of type 3 collagen was promoted, as shown in FIG. 9 d and f. As the concentration of GDF11 increased, the expression of elastin was promoted, but it was confirmed that expression of elastin was suppressed when the concentration of GDF11 increased. As shown in e and f of FIG. 9, as the concentration of GDF11 increased, the expression of MMP1 Is suppressed, but it was confirmed that when excessively increased, the expression was rather increased.

From the above results, it was found that GDF11 exhibits an effect of promoting skin regeneration and wrinkle improvement in which the collagen and elastin are involved.

<110> Kang Stem Biotech CO., LTD. <120> Composition comprising GDF11 and uses thereof <130> KPA160091-KR-D1 <160> 10 <170> KopatentIn 2.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 tcaaggtttc caaggacctg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 tcaaggtttc caaggacctg 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 aaaggggagc tggctacttc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gcgagtagga gcagttggag 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 tgaagagggg cacatgctga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 gtgggagttg ggctgactcg 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 gatcctggac ctacacgact tc 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 ggccttcagt acctttgtga ac 22 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 gcacgacctt gagggcagcc 20 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 catcgtggct aaacaggtac tg 22

Claims (7)

A growth differentiation factor 11 (GDF11), and a human adult adult stem cell culture fluid, for skin regeneration or wounds treatment by fibroblast proliferation.
The pharmaceutical composition according to claim 1, wherein the GDF11 is isolated from a human-derived adult stem cell or a culture thereof.
The pharmaceutical composition of claim 1, further comprising a pharmaceutically acceptable carrier, excipient or diluent.
A composition for quiescence for skin regeneration or wound healing by fibroblast proliferation, comprising GDF11 (growth differentiation factor 11) and human-derived adult stem cell culture fluid.
5. The quasi-drug composition according to claim 4, wherein the GDF11 is isolated from a human-derived adult stem cell or a culture thereof.
A growth differentiation factor 11 (GDF11), and a human adult adult stem cell culture fluid, for skin regeneration or wound healing by fibroblast proliferation.
The cosmetic composition according to claim 6, wherein the GDF11 is isolated from human adult stem cells or a culture thereof.

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