KR101527880B1 - Composition for skin-regeneration, skin-whitening, anti-oxidation or wound healing comprising culture medium of adipose-derived stem cell-T cell - Google Patents

Abstract

The present invention relates to a composition for skin regeneration, whitening or antioxidation, which comprises a culture medium of ADSC-T cells into which T antigen has been introduced into adipose derived stem cells as an active ingredient. The culture medium of the adipose-derived stem cell line (ADSC-T) according to the present invention shows an excellent effect on skin regeneration and an effect of increasing collagen elasticity and wound contraction in collagen culture, inhibiting tyrosinase activity and melanin activity, And exhibits excellent effect on DPPH radicals. Thus, it is excellent in antioxidative effect by the inhibition of DPPH radicals. Further, since it has a remarkable effect on wound healing by increasing cell mobility of fibroblasts, it can be usefully used for skin regeneration, whitening, antioxidation or wound treatment .

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for skin regeneration, whitening, antioxidation or wound treatment, which comprises a culture medium of ADSC-T cells into which adipose stem cells are introduced, oxidation or wound healing-containing culture medium of adipose-derived stem cell-T cell}

The present invention relates to a composition for skin regeneration, whitening, antioxidation or wound treatment, which comprises a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which adipose stem cells .

By improving the quality of medicine and life in the 21st century, human life expectancy is 100 years old. As human life prolongs, there is growing interest in disease prevention and prevention of aging. As the human body's skin over time, wrinkles, stains and regenerative power are reduced by external stimuli and aging of the skin. This is an element that can be used to confirm the physical aging, and the interest in prevention of aging of the skin is very great, and people use therapeutic agents and cosmetics to prevent skin aging. With this interest, cosmetics companies are constantly exploring new substances.

As for the existing whitening and antiaging effect substances, arbutin and ascorbic acid derivative selina are used as the whitening functional substance first.

Skin pigmentation occurs in relation to melanin production in melanocytes. This increases melanin production by external stimuli (UV, inflammation) and active oxygen in the body. The process of melanogenesis is a process in which tyrosine is oxidized to dopaquinone via dopa to form 5,6-dihydroxyindole-2-carboxylic acid in Dopachrome, carboxylic acid to produce the final eumelanin. The tyrosinase enzyme, which acts on the formation of dopaquinone and the formation of 5,6-dihydroxyindole-2-carboxylic acid, is the most important enzyme in melanin production. Thus, inhibition of tyrosinase is a major target in the search for whitening effect substances.

Anti-aging substances include retinoids, silicic acid, Mevalonolactone (Mevalonic acid, MA), adenosine, and retinyl palmitate, which regulate skin cell regeneration , Control of collagen production, and improvement of wrinkles. In order to prevent skin aging, there are activation of skin cell regeneration, promotion of collagen formation, and antioxidative action. Skin is composed of epidermis, dermis, subcutaneous tissue, and dermis fibroblast plays a big role in skin aging. Cell senescence due to a decrease in the number of fibroblasts causes damage to the skin tissue.

In addition , fibroblast migration, proliferation, and contraction of wound sites are important factors in the wound healing process. Fibroblasts are involved in collagen production , and collagen is composed of more than 90% of the dermis, which affects skin moisturization and elasticity.

In general, adipose stem cell culture fluids are known to have whitening, wound healing, wrinkle improvement, and anti-aging functions, and their commercial value as pharmaceuticals and cosmetics has recently been rising. Adipose stem cells are known to produce a number of growth factors and cytokines, and materials produced by adipose stem cells have been reported to contain substances that are effective for skin whitening, regeneration, and antioxidation.

Stem cells, on the other hand, are cells that are capable of differentiating into all types of cells that constitute the body, such as nerves, blood, There are two ways to obtain such stem cells. First, stem cells obtained from embryos (embryonic stem cells), and second stem cells (adult stem Cells). Although there are functional differences, embryonic stem cells and adult stem cells all have differentiation characteristics into different types of cells. Embryonic stem cells have excellent differentiation ability and telomeres have long advantages, but they have ethical problems and it is difficult to acquire a large number of cells. However, adult stem cells can obtain a large number of cells, The risk of infection or the ability to differentiate is relatively low.

Despite the above disadvantages, adult stem cells are very safe for medical application. In addition, the fat-derived stem cells can be easily obtained from the inhaled fat, thus there is no ethical problem and the advantage is that it is easy to obtain.

Primary adipocytes (ADSCs) derived from fat have a great disadvantage in that it is not easy to mass-produce stem cell culture fluids because of their slow growth rate and short life span.

In order to overcome the disadvantages described above, the present inventors produced ADSC (Adipose-derived stem cell) -T cell line injected with T antigen of Simian virus (SV40). The cell line showed a threefold increase in proliferation rate and cell longevity by about 50 times as compared with the primary ADSC cell line. Accordingly, the present invention complements the limitations in the mass production of the culture medium of the first ADSC cell line through the production of the ADSC-T cell line.

Accordingly, the present inventors have conducted studies to solve the problems in skin regeneration, whitening, antioxidation and wound healing, and found that adipose-derived stem cell (ADSC-T) T) cell line and confirming the effect of skin regeneration, whitening, antioxidation and wound healing of the ADSC-T cell culture fluid, thereby completing the present invention.

An object of the present invention is to provide a cosmetic composition for skin regeneration, whitening, antioxidation or the like, which comprises a culture medium of ADSC-T cells (adipose-derived stem cell-T cell) into which adipose- To provide a food composition.

Another object of the present invention is to provide a pharmaceutical composition for wound healing, which comprises a culture medium of adipose-derived stem cell-T cells (ADSC-T cells) into which T antigen has been introduced into adipose-derived stem cells as an active ingredient.

In order to solve the above-mentioned problems, the present invention provides a method for regenerating skin, for whitening, for skin, and the like, which comprises a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which adipose- An antioxidant cosmetic composition or a food composition.

The present invention provides a pharmaceutical composition for the treatment of wounds, which comprises a culture medium of adipose-derived stem cell-T cells (ADSC-T cells) into which T antigen has been introduced into adipose derived stem cells as an active ingredient.

The culture medium of the adipose derived stem cell line (ADSC-T) according to the present invention has an effect of increasing collagen elasticity and wound contraction in collagen culture, showing excellent effect on skin regeneration, inhibiting tyrosinase activity and melanin production, It exhibits excellent effect on whitening, inhibits DPPH radical activity, and is excellent in antioxidative effect. Furthermore, since it has a remarkable effect on wound healing by increasing cell mobility of fibroblast, it is useful for skin regeneration, whitening, antioxidation or wound treatment .

FIG. 1A is a microscopic view of all cells isolated from the inhaled adipose tissue, and FIG. 1B is a microscopic view showing the cells adhered to the plate only after removing the floating cells (FIG. x100 magnification).
FIG. 2 shows the result of staining with adipocyte stem cells (ADSC) into adipocytes and staining with Oil-Red O (pre-differentiation induction (A), 30 days after induction of differentiation (B) After 30 days of induction of differentiation, the result of staining with Oil-Red O (C)].
FIG. 3 is a microscopic observation of morphological changes of ADSC-T cells (primary ADSCs (A)), high density cell clusters of adipose-derived stem cells formed by the introduction of the pEF321β-T plasmid high-density focus (B, C), ADSC-T cells (D) established by culturing cells obtained from high-density cell clusters] (each x100 magnified).
FIG. 4 is a chart of SV40 T antigen expressed in ADSC-T and COS-1 cells used as a control group by fluorescent antibody staining.
FIG. 5 is a graph showing Western blot analysis results of T antigen of ADSC-T cells using monoclonal antibody of T antigen [primary adipose-derived stem cell (A), ADSC-T (B, C, D), COS-1 (E)].
6 is a graph showing the proliferation rate of primary adipogenic stem cells (primary ADSC) and three cells of ADSC-T (ADSC-T-1, ADSC-T-2 and ADSC-T-3).
7 is a diagram showing the morphology of 3Y1 cells in collagen gel. 3Y1 cells were cultured on collagen gels in media containing 50% CM of ADSC-T or ADSC for each indicated time.
8 is a graph showing the shrinking effect of ADSC-T-CM on a Fibroblast-populated collagen lattice (FPCL) model. 3Y1 cells were cultured on collagen gel for 8 days using 50% culture medium (CM) of ADSC-T or ADSC to calculate collagen area.
Figure 9 shows the effect of ADSC-T and ADSC culture media on the mobility of fibroblasts. A shows the mobility of 3Y1 cells for 48 hours after treatment of each culture solution and B is a graph showing cell migration distance per hour (value = mean ± standard deviation, n = 0.3) [* p <0.05]
10 is a view showing collagen production by 3Y1 cells after treatment with culture medium (CM). Cells were cultured for 5 days using 50% CM of ADSC-T, ADSC and 3Y1 cells themselves. Cell lysate and culture medium of 3Y1 cells were used for western blotting of collagen type I.
Figure 11 shows the cell pellet of B16F10 after treatment with CM of ADSC-T or ADSC. The cells were incubated for the indicated time each, and then centrifuged. The cell pellet was then photographed with a digital camera.
12 shows the effect of ADSC-T-CM or ADSC CM on melanin content in B16F10 cells. The melanin content in cells (A) and medium (B) was measured with a spectrophotometer at 492 nm (value = mean value + standard deviation, n = 3) [* P < 0.05]
Figure 13 shows the effect of ADSC-T-CM and ADSC CM on tyrosinase activity in B16F10 cells. B16F10 cells were cultured using a medium containing 50% CM of ADSC-T and ADSC, and the tyrosinase activity was measured as a cell lysate after 48 hours (mean = standard deviation, n = 3). [* P < 0.05]
14 is a graph showing DPPH free radical scavenging activity of ADSC-T CM. The ADSC-T CM and 0.13 mM DPPH were mixed in an equal volume and the free radical scavenging activity was measured at OD 515 nm (mean = standard deviation, n = 3) after each reaction for the indicated time. [* P < 0.05]

The present invention relates to a method for skin regeneration and whitening comprising an adipose-derived stem cell (ADSC-T) cell line prepared by injecting a T antigen of Simian virus (SV40) into adipose stem cells , Antioxidants or wound healing compositions.

The composition comprises a cosmetic composition, a food composition or a pharmaceutical composition.

Hereinafter, the present invention will be described in detail.

Based on the expression of SV40 T antigen prolongs the lifespan of various cells, the present inventors isolated adult stem cells (Adipose-Derived Stem Cells, ADSC) from the inhaled fat and determined the proliferation rate of isolated adipose stem cells And to prolong the proliferative lifespan by introducing a vector expressing the SV40 T antigen, the prolongation of the fat-derived stem cell life would be expected. To confirm this, the pEF321β-T plasmid, which is a vector expressing T antigen of SV40, was introduced into adipose-derived stem cells. As a result, the survival time of fat-derived stem cells expressing SV40 T antigen was prolonged, .

In the present invention, &quot; Simian Virus 40 (SV40) &quot; is a virus belonging to polyoma virus, 40 kinds of polyoma virus and simian virus, having a cyclic double-stranded genome composed of 5245 bases, It has a broad host range and is used as a transformation vector for various cells.

The &quot; T antigen &quot; of SV40 is an early protein synthesized at the early stage of infection of SV40, a tumor-inducing virus, and is a protein contributing to tumorization of infected cells. T antigen is involved in the immortalization of various cells due to the action of inhibiting its activity by binding to a cancer-suppressing gene product (such as p53) on the cell side when the cell is transformed.

The culture medium of ADSC-T cells, which is an active ingredient in the composition of the present invention,

(a) separating and culturing adipose derived stem cells by treating the inhaled adipose tissue with a collagenase enzyme and then centrifuging;

(b) preparing adipose-derived stem cells (ADSC-T) expressing SV40 T antigen by transfecting adipose-derived stem cells cultured in step (a) with a plasmid expression vector (pEF321? -T); And

(c) culturing the prepared ADSC-T cells to obtain a culture solution.

The process for producing the ADSC-T cell culture of the present invention will be described in detail as follows.

The step (a) is a step of isolating and culturing the adipose tissue-derived stem cells from the inhaled adipose tissue. First, the collagenase enzyme is mixed with the collagenase enzyme at a weight ratio of 1: 1, And then centrifuged to separate fat-derived stem cells.

The &quot; collagenase enzyme &quot; is an enzyme that promotes the hydrolysis of collagen, and serves to separate collagen of adipose tissue to separate adipose stem cells. The centrifugation can be performed at 500 to 1000G for 2 to 5 minutes, preferably at 800G for 3 minutes, and the lipid and adipocyte layers suspended in the supernatant are removed, and then the cellular residues are removed using a filter. The filter is preferably a 100 μm filter, but is not limited thereto.

After removing the cellular residue, physiological saline is added and centrifugation is repeated to wash the cells. The centrifugation may be performed at 150 to 500 G for 2 to 5 minutes, preferably at 300 G for 3 minutes.

The separated fat-derived stem cells (ADSC) to 10% FBS (Fetal Bovine Serum) , 100units / ml penicillin and 100㎍ / ml streptomycin the (Dulbecco's Modified Eagle Media) was added DMEM 37 ℃ in a culture medium, 5% CO ₂ The cells are cultured in an incubator and subcultured when the confluence of the cells reaches 80-90%. As the culture method, a cell culture method known in the art can be applied.

In step (b), the adipose-derived stem cell (ADSC-T) expressing the SV40 T antigen is produced. The plasmid expression vector (pEF321β-T) To obtain ADSC-T cells expressing SV40 T antigen.

The &quot; vector &quot; is a DNA capable of introducing a desired DNA fragment into a host cell or the like in a DNA recombination experiment. The vector DNA is cleaved with a restriction enzyme or the like, inserted into a target DNA fragment, Cells. The vector DNA that connects the target DNA fragments is inserted into the chromosomal DNA of the host cell and is distributed to each cell along with the division of the host to maintain the desired DNA fragment for generations.

The plasmid expression vector &quot; pEF321? -T &quot; means a plasmid containing a nucleic acid sequence encoding the T antigen of SV40.

The term &quot; transfection &quot; means introducing a gene DNA, a plasmid DNA, a viral DNA or RNA into a cell through a suspension of a cell culture or a cell to induce gene introduction and infection. Specifically, the transfection of the expression vector can be performed using all available methods including calcium phosphate transfection, electroporation, microinjection, and liposome injection, which are well known in the art. Transfection methods can be used. DNA can also be introduced into eukaryotic cells using viruses or bacteria as carriers.

For example, the method of introducing the pEF321? -T plasmid by electroporation can be carried out by mixing adipocyte stem cells with a plasmid in a serum-free medium, preferably serum-free DMEM, The plasmid can be introduced using an electroporation protocol, which is not limited, and is known in the art.

Cells transfected with the foreign gene should induce the expression of the introduced gene through culture for a certain period of time and verify their expression. The culture for expression of the transgene is preferably, but not exclusively, cultured in a 5% CO ₂ incubator at 37 ° C. The gene introduced into the present invention is a gene expressing the T antigen of SV40. The expression of the T antigen is detected by the antigen-antibody binding reaction using an antibody specifically binding to the T antigen, and the expression of T It can be verified by detecting the antigen. Specifically, it can be verified using conventional enzyme immunoassay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, antigen-antibody aggregation and the like, and Western blotting and fluorescent antibody staining And the expression of T antigen can be verified.

The step (c) comprises culturing ADSC-T cells to obtain a culture solution, culturing ADSC-T cells at 1 × 10 ⁵ cells / ml each; A step of subculturing after the cell density reaches 80%; And collecting the culture medium after 2 days after the density of the cells became 70 to 90%.

The culture solution prepared by the above method is centrifuged to remove cell debris and only the supernatant is obtained. The centrifugation is performed at 500 to 1000 G for 2 to 5 minutes, preferably at 800 G for 3 minutes.

The medium for the ADSC-T cell culture can be used without limitation in a basic medium known in the art. The basic medium may be prepared by artificial synthesis, or a commercially prepared medium may be used. Examples of commercially produced media include Dulbecco's Modified Eagle's Medium, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Essential Medium, G-MEM, and Isocove's Modified Dulbecco's Medium. In addition, one or more auxiliary components may be added to the cell culture medium, if necessary. Examples of such auxiliary components include serum such as fetal calf, horse or human, penicillin G for preventing microbial contamination, Antibiotics such as streptomycin sulfate and gentamycin, antifungal agents such as amphotericin B and nystatin, and mixtures thereof. You can use more than one.

The culture medium of ADSC-T cells of the present invention produced by the above method has an effect of increasing collagen elasticity and wound contraction in collagen culture, showing an excellent effect on skin regeneration, inhibiting tyrosinase activity and melanin production It exhibits excellent effect on skin whitening, inhibits DPPH radical activity, and is excellent in antioxidative effect. Furthermore, since it has a remarkable effect on wound healing by increasing cell mobility of fibroblast, it is useful for skin regeneration, whitening, antioxidation or wound treatment Can be used.

The pharmaceutical composition of the present invention may contain a pharmaceutically acceptable carrier and may be formulated into a transdermal dosage form such as a liquid, a suspension, an emulsion, a lotion, and an ointment according to a conventional method. The pharmaceutically acceptable carriers include aqueous diluents or solvents such as phosphate buffered saline, purified water, sterilized water, etc., and include non-aqueous diluents or solvents such as propylene glycol and olive oil . Further, if necessary, it may contain a wetting agent, a fragrance, a preservative, and the like. The ADSC-T cell culture fluid contained in the pharmaceutical composition varies depending on the condition and the weight of the patient, the severity of the disease, the drug form, the administration route and the period, but can be appropriately selected by those skilled in the art. For example, the daily dose of the ADSC-T cell culture fluid may be 0.01 to 100 mg / kg, preferably 0.1 to 10 mg / kg per day, and the administration may be administered once a day or divided into several times .

The carrier (s) should be "acceptable" in terms of compatibility with the other ingredients of the formulation without being detrimental to the recipient. In this regard, the pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal active agents, isotonicity and absorption delaying agents, and the like, which are suitable for any pharmaceutical administration. The use of such media and actives for pharmaceutically active substances is well known in the art. Any conventional media or active agent is contemplated for use in the composition, except that it is not compatible with the active compound. Supplementary active compounds (known in the art and / or identified or designed in accordance with the present invention) can also be incorporated into the compositions. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the pharmaceutical / microbiology arts. In general, some formulations are prepared by mixing the compound with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product into a suitable formulation.

The pharmaceutical composition of the present invention should be formulated to be suitable for its intended route of administration. Routes of administration include oral, ears, eye, nose, or parenteral, such as intravenous, intradermal, inhalation, transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application may include the following components: sterile diluents such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, propylene glycol or other synthetic menstruum; Antibacterial agents such as benzyl alcohol or methyl paraben; Antioxidants such as ascorbic acid or sodium bisulfite; Chelating agents such as ethylenediaminetetraacetic acid; Buffers such as acetate, citrate or phosphate, and tension modifiers such as sodium chloride or dextrose, pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

Solutions useful for oral or parenteral administration may be prepared by any of the methods known in the art of pharmaceutical ramification and may be prepared, for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.), Mack Pub. )]. Formulations for parenteral administration may also include oral administration of glycocholate, rectal administration of methoxysalicylate, or vaginal administration of citric acid. The parenteral preparation may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Suppositories for rectal administration may also be prepared by mixing the drug with non-polar excipients such as cocoa butter, other glycerides, or other compositions that are solid at room temperature and liquid at body temperature. The formulations may also include, for example, polyalkylene glycols such as polyethylene glycols, vegetable oils, and hydrogenated naphthalenes. Formulations for direct administration may include glycerol and other high viscosity compositions. Other parenteral carriers that may be useful for such drugs include ethylene vinyl acetate copolymer particles, osmotic pumps, implantable injection systems, and liposomes. Formulations for inhalation administration may contain as an excipient, for example, lactose, or may be administered in the form of, for example, an aqueous solution containing polyoxyethylene-9-lauryl ether, glycolcholate and deoxycholate, Oily liquid, or gel applied in the nose. Stomach enema may also be used for rectal delivery.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, gelatin capsules, cachets, tablets, troches, or lozenges, each containing a predetermined amount of the drug; A powder or granular composition; Solutions or suspensions in aqueous or non-aqueous liquids; Or in the form of an oil-in-water emulsion or a water-in-oil emulsion. The drug may also be administered in bolus, soft or paste form. Tablets may be prepared by compressing or molding the drug, optionally with one or more accessory ingredients. Compressed tablets may be prepared by compressing the drug in a free-flowing form such as powder or granules, in a suitable machine, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be prepared by molding in a suitable machine, a mixture of a suitable carrier and a powdered drug, wetted with an inert liquid diluent.

Oral compositions generally include an inert diluent or an edible carrier. For oral therapeutic administration purposes, the active compound may be introduced with an excipient. Oral compositions made with a fluid carrier for use as an oral cleanser may contain a compound in a fluid carrier and may be applied orally to swish, spit, or swallow. Pharmaceutically compatible binding agents, and / or adjuvant materials may be included as part of the composition. Tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature: binders such as microcrystalline cellulose, gum tragacanth or gelatin; Excipients such as starches or lactose; Disintegrants such as alginic acid, Primogel, or corn starch; Lubricants such as magnesium stearate or stearothex; A fluidizing agent such as colloidal silicon dioxide; Sweeteners such as sucrose or saccharin; Or flavoring agents such as peppermint, methyl salicylate, or organic flavoring agents.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, pesticides, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier may be, for example, a solvent or dispersion medium containing water, ethanol, a polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, isotonic agents such as sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride are preferably included in the composition. Prolonged absorption of the injectable composition may be achieved by adding to the composition an active agent that delays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by introducing one or a combination of the above listed ingredients and the required amount of the active compound in the appropriate solvent followed by filtration sterilization, if necessary. In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other ingredients required of the above listed ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method includes a vacuum drying method and a freeze drying method in which a powder of the active ingredient is added from the previously sterilized filtered solution to any additional desired components.

Formulations suitable for intraarticular administration may be in the form of sterile aqueous preparations of the drug, which may be in the form of microcrystalline, for example, aqueous microcrystalline suspensions. Liposome formulations or biodegradable polymer systems can also be used to prepare drugs for both intra-articular and ocular administration.

Formulations suitable for topical administration, including eye treatment, include liquid or semi-liquid preparations such as powders, lotions, gels, coatings, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; Or solutions or suspensions such as drops. Formulations for topical administration to the skin surface are prepared by dispersing the drug in a dermatologically acceptable carrier such as a lotion, cream, ointment or soap. Particularly useful are carriers which are capable of forming a film or layer on the skin to topically apply and inhibit removal. For topical application to the internal tissue surface, the active agent may be dispersed in a liquid tissue adhesive or other material known to enhance adhesion to the tissue surface. For example, it is advantageous to use hydroxypropylcellulose or a fibrinogen / thrombin solution. Alternatively, tissue coating solutions, such as pectin-containing preparations, may be used.

For inhalation therapy, powder inhalation (self-propelled or sprayed) dispensed with a spray may be a nebulizer or an automizer may be used. Such formulations may be in the form of powders for pulmonary administration from powder inhalers or self-propelled powder-dispensing formulations. In the case of self-propelled solutions and spray formulations, the active ingredient is introduced by selection of a valve having the desired spray characteristics (i.e., a feature capable of producing a spray having a desired particle size) or as a suspended powder of controlled particle size, Effect can be achieved. For inhalation administration, the compound may also be delivered in the form of an aerosol spray from a pressurized container or dispenser containing a suitable propellant, for example, gas to carbon dioxide, or a nebulizer.

Systemic administration may also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art and include, for example, for transmucosal administration, detergents and bile salts. Transmucosal administration can be accomplished through the use of a nasal spray or suppository. For transdermal administration, the active compounds are formulated in ointments, gels, gels, or creams, as generally known in the art.

There is no particular limitation on the type of food contained in the food composition of the present invention. Examples of the food include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen and other noodles, gums and ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complex And includes all health foods in a conventional sense.

The food composition may comprise a health beverage composition, and may contain various flavors or natural carbohydrates, such as ordinary beverages, as an additional ingredient. Such natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. Examples of sweeteners include natural sweeteners such as tau Martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like. The ratio of the natural carbohydrate is generally 0.01 to 0.04 g, preferably about 0.02 g per 100 g of the culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which T antigen has been introduced into the adipose-derived stem cells of the present invention To 0.03 g.

The cosmetic composition can be prepared in various forms according to a conventional cosmetic preparation method using the ADSC-T cell culture liquid of the present invention. For example, the cosmetic composition may be prepared in the form of a perfume product containing the culture solution, a shampoo, a hair lotion, a hair cream, a hair gel, etc. It may be diluted with a usual cleansing solution, have. The cosmetic composition may also contain conventional adjuvants such as stabilizers, solubilizers, vitamins, pigments, and flavorings commonly used in the cosmetic composition arts. In the cosmetic composition, the content of the culture liquid may be an amount effective to achieve the effect of skin regeneration, whitening and antioxidation, for example, 0.001 to 10% by weight based on the total weight of the composition, 0.01 to 1% by weight.

For the statistical processing of experimental data in the embodiment of the present invention, the difference between the mean values of the experimental group and the control group using the SPSS 20 program was evaluated by the mann-whitney u test.

Hereinafter, the present invention will be described more specifically based on examples. It will be apparent to those skilled in the art that the embodiments are only for describing the present invention in more detail and that the scope of the invention is not limited by these embodiments in accordance with the gist of the present invention.

[Preparation Example 1] Preparation of ADSC-T cells (adipose-derived stem cell-T cell) culture medium in which T antigen was introduced into adipose derived stem cells

1. Isolation and Culture of Adipose-Derived Stem Cell from Adipose Tissue

The adipocyte stem cells (ADSC) were isolated from the adipose tissues after obtaining consent from the patients who underwent liposuction in plastic surgery. Specifically, the adipose tissue was washed with PBS (phosphate buffered saline), mixed with 0.075% collagenase (Sigma) at a weight ratio of 1: 1, and treated at 37 ° C for 45 minutes. After the enzymatic treatment, the supernatant was centrifuged at 800 G for 5 minutes, and the lipid and adipocyte layer suspended in the supernatant was removed, and the supernatant was removed by filtration through a 100 μm filter. After adding physiological saline to the filtrate, the cells were washed 2-3 times at 300G for 3 minutes to obtain adipose stem cells (ADSC).

The obtained adipose derived stem cells (ADSC) were cultured in DMEM (Dulbecco's Modified Eagle Media) medium supplemented with 10% FBS (Fetal bovine Serum), 100 units / ml penicillin and 100 μg / ml streptomycin at 37 ° C in a 5% CO ₂ incubator Lt; / RTI &gt; Isolated adipocyte stem cells (ADSC) are mixed with erythrocytes, and erythrocyte lysis buffer is used to remove these erythrocytes, but the number of adipose stem cells (ADSC) tends to decrease when using erythrocyte lysis buffer. Therefore, the isolated adipocyte stem cell (ADSC) cells and erythrocytes were cultured together during the initial culture. For this reason, adipose-derived stem cells (ADSC) were mixed with erythrocytes during the initial culture (FIG. 1A). One day after the initial culture, the adipocyte stem cells (ADSC) were judged to be attached to the plate, and the floating cells and erythrocytes were removed by washing twice with PBS. RBCs that were not completely removed were removed by trypsinization during subculture. When the confluence of the cells reached 80-90% of the culture, they were subcultured.

It was confirmed that adipogenic stem cells (ADSC) with fibroblast morphology appeared about 2-3 days after the initial culture. Then, the shape of adipose derived stem cells (ADSC) was observed after subculture with a 10 cm plate. As a result, it was found that adipose derived stem cells (ADSC) exhibit a fibroblast-like morphology like other adult stem cells (Fig. 1B).

2. Identification of adipocyte differentiation ability of adipocyte stem cell (ADSC)

In order to confirm that the adipose-derived stem cells (ADSC) obtained in the isolation and culture of the adipose-derived stem cells were differentiated into adipocytes, adipocyte stem cells (ADSC) were induced to induce differentiation for 4 weeks using adipocyte differentiation induction medium And then differentiated into adipocytes. The adipocyte differentiation medium used for the induction of differentiation of adipocytes was Preadipocyte Differentiation Medium, which was marketed by PromoCell, and Adipocyte Nutrition Medium, which is commercially available from PromoCell, was used for the adipocyte maintenance medium for differentiated adipocytes. Specifically, adipocyte stem cells (ADSC) were induced to differentiate by inducing differentiation in an adipocyte differentiation inducing medium for 10 days while changing the medium at intervals of 3 days. Thereafter, the adipocyte stem cells were further cultured for 20 days in an adipocyte maintenance medium . The cultured cells were fixed in 10% formalin and stained with a 60% Oil-Red O solution to visualize lipid droplets. The results are shown in Fig. 2. [Oil-red O staining result (C)] before induction of differentiation induction (A), 30 days after induction of differentiation induction (B), 30 days after induction of differentiation.

As shown in FIG. 2, adipocytes specific to adipocytes were observed in adipose derived stem cells (ADSC) 30 days after culturing, and it was confirmed that they were differentiated into adipocytes. In addition, after 30 days of incubation, the oil-red O staining showed that the lipids were reddish and differentiated into adipocytes. (C) The cells that did not induce differentiation were not stained (A). Thus, it can be seen that adipose-derived stem cells (ADSC) possess the ability to differentiate into normal adipocytes.

3. Establishment and characterization of ADSC-T cell line by expression of SV40 T antigen

(1) Transfection

To stably express SV40 T antigen in adipose derived stem cells (ADSC), pEF321β-T plasmid DNA expressing SV40 T antigen gene was introduced into adipose derived stem cells (ADSC) by electroporation. Specifically, 20 μg of the plasmid vector pEF321β-T and 1 × 10 6 adipose-derived stem cells were mixed in 800 μl of serum-free DMEM and placed in a 0.4 cm 2 cuvet for electric shock. The electric shock was performed using a Gene Pluser X cell Electroporation System (BIO-RAD) at 160 V and 15 m / sec. Derived stem cells having a high density focus introduced with the plasmid vector pEF321? -T are cloned into a penicillin cap to trypsinize the cells, and then the cells are cultured in a 24-well plate. After the cells are cultured in an incubator , And the cell line was named ADSC-T. The morphological changes of the ADSC-T cells are shown in Fig.

As shown in Fig. 3A, in primary adipocyte stem cells (primary ADSCs), proliferation is stopped by contact inhibition in the form of a large and long fibroblast, resulting in a cell monolayer . &Lt; / RTI &gt; 3B and 3C, the adipose-derived stem cells (ADSC-T) in which the pEF321? -T plasmid was introduced into primary adipose-derived stem cells and the SV40 T antigen was expressed were not contact-regulated, And it was confirmed that high-density focus cells with high cell density were formed. In addition, as shown in FIG. 3D, ADSC-T was found to have changed into a spindle shape with decreasing cell size. These changes in cell morphology of ADSC-T may be attributed to the expression of SV40 T antigen.

(2) T-antigen expression of ADSC-T cells - Fluorescent antibody staining

In order to confirm the expression of T antigen in ADSC-T cells, fluorescent antibody staining was performed using monoclonal antibody against SV40 T antigen and anti-mouse IgG labeled with FITC (Fluorescein isothiocyanate) T antigen was observed. In this case, COS-1 cells were used as a control, and COS-1 cells were prepared using SV40 T antigen, and the presence or absence of SV40 T antigen expression of ADSC-T cells can be confirmed.

Specifically, ADSC-T cells and COS-1 cells were fixed with a 1: 1 mixture of ethanol and acetone for 18 minutes. The immobilized cells were washed with PBS and incubated with monoclonal antibody of mouse specifically binding to SV40 T antigen at 37 DEG C for 1 hour. The cultured cells were washed with PBS and incubated with anti-mouse IgG of rabbit labeled with FITC for 1 hour at 37 ° C. The cultured cells were observed with a fluorescence microscope. The results of the above experiment are shown in FIG.

As shown in FIG. 4, ADSC-T cells isolated from high-density focus cells were stained with a monoclonal antibody against SV40 T antigen and expressed T antigen similarly to COS-1 cells .

3. Expression of T antigen by Western blotting of ADSC-T cells

Total cell extracts from three ADSC-T cells were prepared and Western blotting was performed to detect SV40 T antigen present in the cells. Specifically, an extract of each cell containing the same amount of protein was electrophoresed on a 10% SDS-polyacrylamide gel, and transferred to a nitrocellulose membrane. A mouse monoclonal antibody (IgG) that specifically binds to the SV40 T antigen was bound and then developed with peroxidase-conjugated anti-mouse IgG. The results are shown in Fig.

As shown in FIG. 5, Western blotting revealed that the T antigen was expressed in three cells (B, C, and D) of ADSC-T as positive control COS-1 (E) cells, The adipose-derived stem cells (primary ADSC) were found to be free of T antigen (A). From the above results, it was found that the isolated ADSC-T cell line stably expressed the T antigen, and the cell proliferation was increased by the T antigen and the cell shape was changed.

4. Propagation rate of ADSC-T cells established by SV40 T antigen

Cell proliferation rates of ADSC-T and primary adipogenic stem cells (primary ADSC) established by simian virus 40 (SV40) T antigen were measured and compared. When the density of the cells reached 80% at the time of cell culture, subculture was performed. Cell growth curves were generated by counting the cells in each passage. The results are shown in Fig.

As shown in Fig. 6, primary adipogenic stem cells (primary ADSCs) undergo cell division once every about 6-7 days, while all three ADSC-T cells undergo cell division . In addition, the primary adipocyte stem cells (primary ADSCs) showed progressive kinetics of apoptosis, but in the case of ADSC-T, there was a slight difference between the three cell days, Respectively. This proliferation rate continued until about 6 months (50 passages), after which the rate of cell proliferation decreased.

5. Preparation of ADSC-T cell culture

To prepare the cell culture of the present invention, ADSC-T and ADSC cells were cultured in a culture medium (Cultrued Medium: CM) medium for 3 days in medium containing 10% FBS in DMEM. The number of passages of the culture used in the experiment was the 22 passages of ADSC-T and the culture obtained from 6 passages of the first ADSC cells.

[Example 1] Skin regeneration and wound healing effect of ADSC-T cell culture fluid

1. Fibroblast-populated collagen lattice contraction assay.

To make a collagen solution, and prepare 5x DMEM (-NaHCO3) and antiseptics forming buffer _{(Sterile reconstitution Buffer) (2.2gNaHCO 3} , 0.05N NaOH, 200mM HEPES in 100ml). 7 volumes of collagen type (Nitta Gelatin, Japan), 2 volumes of 5X DMEM, and Buffer 1 were mixed on ice. Add the cells to the collagen solution after adjusting the cell number to 1 × 10 ⁵ per well and mix to minimize bubbles. 1.5 ml per well was dispensed into 6 wells and cultured at 37 ° C and 5% CO ₂ for 30 minutes. The hardened collagen was removed with a sterilized syringe at the edge, a sample medium to be treated was added at a concentration of 50%, and the collagen area was determined using Image J program by incubating for 8 days.

2. Skin contraction effect of ADSC-T culture in Fibroblast-populated collagen lattice (FPCL) culture experiment

Collagen accounts for 90% of the dermis, and collagen content in the skin is related to moisturizing and elasticity. In addition, when the skin is damaged, the healing process consists of hemostasis, inflammation, proliferation, and wound constriction, and the degree of collagen shrinkage during wound constriction is very important. This process is similar in the restoration process of skin tissue according to anti-aging and wound. This anti-aging (increase of elasticity) and wound contraction effect can be confirmed by the culture using FPCL (Fibroblast-populated collagen lattice) model in vitro . In this experiment, when FPCL was used to treat stem cell culture, collagen shrinkage Respectively. Collagen was mixed with fibroblast (3Y1) to form collagen and then cultured in a medium containing 50% concentration of stem cell culture medium. First, collagen cells were cultured for 2 days, 5 days, and 8 days, respectively, and then observed with a microscope. The results are shown in FIGS. 7 and 8. FIG.

As shown in Fig. 7, there was no significant difference in the case of 2 days, but after 2 days, the collagen cells treated with ADSC-T CM were found to form a network. The results are shown in FIG.

As shown in Fig. 8, the area of the collagen was measured at 8 days after the incubation. As a result, the control group (control) 9.6 cm 2, ADSC-T 4.3 cm 2 and ADSC 10.1 cm 2 showed ASDC-T Decreased by 2.4 times.

The cultures of ADSC-T were found to be excellent in shrinkage of wound area, and it could be used as a raw material for skin regeneration and anti-aging functional cosmetic and wound treatment medicine. The culture of the 1st ADSC (6th passage) showed little effect compared with the control group.

3. Analysis of wound healing effect of ADSC-T culture by cell migration distance

(1) To confirm the wound healing process, the movement distance of fibroblast 3Y1 was confirmed. 3Y1 was cultured to a confluence of 100%, and then cells were smeared with a sterilized tip, and the width thereof was adjusted to 1600 μm (± 100 μm). After that, stem cell culture solution was added, and as a control, 3Y1 culture solution was prepared as a stem cell culture solution for serum correction and used as a control group. The distance of cell movement was photographed at intervals of 24 hours and 48 hours, and then the distance of movement was measured using an Image J program.

(2) Fibroblasts play the most important role in skin elasticity, wrinkles and wound healing. The mobility of the cells increases the elasticity of the skin, and it has a quick wound recovery function. In order to confirm the wound healing effect of the stem cell culture solution, the mobility of rat fibroblast 3Y1 cells was confirmed. 3Y1 was cultured on a 100 mm plate (confluent on plate 0) and plated with a sterilized tip. Then, ADSC-T, primary ADSC culture was treated with 50% concentration of cells, and untreated 3Y1 was used as a control. The movement distance of the cells was measured at 24 hours and 48 hours by time, and the appearance of the cells was photographed at the time point of 48 hours. The results are shown in FIG.

As shown in Fig. 9A, it was confirmed that cells treated with the ADSC-T culture at the time of 48 hours were actively moving.

As shown in FIG. 9B, the travel distance of ADSC-T was shifted from 24 hours to 730 mm and from 48 hours to 1123 탆, and ADSC was shifted from 513 ㎛ to 857 ㎛. The wound healing effect of ADSC-T cell cultures was higher than that of the first passive ADSC 6 passages.

Therefore, it has been found that the culture solution of the ADSC-T cells of the present invention is remarkably effective in wound healing.

4. Confirmation of collagen production promoting effect of ADSC-T culture

(1) In order to measure intracellular collagen content and free collagen, 3Y1 cells were treated with 50% concentration of stem cell culture medium and cultured for 5 days. The stem cell culture was washed twice with PBS and centrifuged at 1,100 rpm for 3 minutes to remove the supernatant. Total lysis buffer (20 mM 7.0 Hepes, 25% Glycerol, 450 mM NaCl, 0.4 mM EDTA, The cells were suspended in 0.5 mM DTT and protease inhibitor (1% NP-40). After reaction for 1 hour on ice, supernatant was obtained by centrifugation at 12,000 rpm at 4 ° C for 15 minutes. The separated supernatant was subjected to western blotting after protein quantification using the Bradford method.

Electrophoresis (75V, 2 hours) was performed on 8% SDS polyacrylamide gel and electrotransfer (90V, 3 hours) to Hybond ECl (Amersham) membrane After this, the membrane was blocked with 5% skin-milk. The membrane was reacted with COL1A2 (SANTA CRUZ BIOTECHNOLOGY) primary antibody at room temperature for 2 hours, and the solution was incubated with a solution of Tris-buffered saline-tween (pH 7.6 Tris-HCl, 137 mM NaCl, 0.1% Tween- After washing, the cells were incubated with peroxidase-conjugated anti-Goat IgG secondary antibody for 1 hour at room temperature. The cells were washed three times with TBS-T solution, and then luminesced with ECL (Enhanced Chemiluminescence). The cells were sensitized to X-ray film and analyzed.

(2) Increased fibroblast proliferation and collagen production during skin wound repair process are important factors in wound healing process. The stem cell culture solution was treated with 3Y1 cells and compared with the untreated group, intracellular collagen protein production and extracellular collagen protein amount were confirmed by western blotting. The results are shown in FIG.

As shown in Fig. 10, it was confirmed that intracellular collagen protein increased when ADSC-T culture medium was treated (Fig. 10A). It was also found that collagen liberated in the medium was also increased compared to the untreated group (Fig. 10B). From the above results, it was found that the culture solution of ADSC-T of the present invention not only promotes collagen contraction but also collagen production of fibroblasts, thereby inducing wound healing and skin regeneration.

Therefore, the ADSC-T culture solution of the present invention proved the remarkable effect of wound healing agent and functional cosmetic raw material for skin regeneration, anti-aging and wrinkle improvement.

[Example 2] Verification of skin whitenig effect of ADSC-T cell culture

1. Identification of inhibitory effect of melanin on ADSC-T culture

(1) Melanoma 16F10 cells were cultured on a 100 mm plate for the measurement of melanin, and cultured at 50% concentration for 24 hours, 48 hours, and 72 hours. The cell numbers were adjusted to 3.5 x 10 ⁶ for each time zone, collected, washed with PBS, and centrifuged at 1100 rpm for 3 minutes to obtain a cell suspension. The cell pellet and culture medium were vortexed with 1N NaOH · DMSO solution and then treated at 80 ° C for 1 hour. Thereafter, the absorbance at 492 nm was measured, and the melanin content was expressed as the content of the untreated group.

(2) Mouse Melanoma B16F10 cells were treated with a stem cell culture medium to confirm inhibition of melanin formation. B16F10 cells were cultured using a medium containing 50% of ADSC-T and ADSC culture medium. Cells cultured with DMEM containing no culture medium were used as an untreated control group, and the amount of melanin produced in the untreated cells and the production The amount of melanin was compared. The amount of melanin in the cells and media was measured at 24 hours, 48 hours, and 72 hours. The cells were washed with PBS, and then the cells were precipitated and the color of the cell pellet was confirmed with a digital camera. The results are shown in Fig.

As shown in Fig. 11, melanogenesis was inhibited in the cell suspension of B16F10 treated with the culture solution of ADSC-T to such an extent that it was confirmed by eyes from 48 hours, and it was confirmed that the cell pellet color was different. Also, the difference in melanin production was more remarkable in the cells cultured for 72 hours.

In addition, 3 x 10 ⁵ B16F10 cell pellets were treated with 1N NaOH DMSO to dissolve the cells at 80 ° C and the melanin content therein was measured at 492 nm. The results are shown in Fig.

As shown in Fig. 12, it was confirmed that the content of melanin at 72 hours after treatment with the ADSC-T culture solution was reduced to 73% as compared with the untreated group (Fig. 12A). In order to measure the melanin released from the cells of B16F10 cells, the media collected by each time period were measured by the above method. As a result, there was no difference in the melanin content in the medium treated with the first ADSC culture solution, and the ADSC-T culture solution And melanin in the treated medium decreased to 83.9%, 76.1% and 50.5%, respectively, over time (FIG. 12B).

2. Inhibition of tyrosinase activity of ADSC-T culture

(1) After culturing B16F10, 50% stem cell culture was added and cultured for 48 hours and 72 hours. Cells were then washed twice in PBS and centrifuged at 1100 rpm for 8 minutes to obtain cells. 800 ㎕ of lysis buffer (20 mM sodium phosphate, pH 6.8, 1% Trion x-100, 1 mM protein inhibiton) was added and vortexing was repeated 5 times for 10 seconds. The culture supernatant obtained by centrifugation at 12,000 rpm at 4 ° C for 15 minutes was used as the tyrosinase enzyme solution. The digested enzyme solution was quantified using the Bradford method and the same amount was used. To confirm tyrosinase activity, 2.5 mM L-DOPA (3,4-Dihydroxy-L-phenylalanine, Sigma-Aldrich), 50 mM phosphate buffer (pH 6.8) The absorbance was measured at 475 nm after the reaction, and the tyrosinase activity was expressed as the activity of the untreated group.

(2) It was confirmed that the melanin production was inhibited when the ADSC-T culture medium was treated. Tyrosinase is the most important key enzyme in the process of melanogenesis and is involved in the first step of melanin formation. Therefore, inhibition of tyrosinase activity by the ADSC-T medium was examined to confirm once again the inhibitory action of melanin formation in the ADSC-T medium.

In order to measure the tyrosinase enzyme activity, B16F10 cells were treated with a culture solution at a concentration of 50%, and after 48 hours, the cells were collected and dissolved buffer was added thereto to obtain intracellular proteins. The solution was used for the measurement of tyrosinase activity. The tyrosinase protein was quantitated using the Bradford method and the extent of L-dopa becoming dopachrome by taking the same amount of protein was measured at an absorbance of 475 nm. The results are shown in Fig.

As shown in FIG. 13, the cultures of ADSC-T and ADSC were 26.5% and 84.9%, respectively, compared with the tyrosinase activity of the untreated control group. And the inhibitory effect was greater in the ADSC-T culture than in the ADSC.

Therefore, it was confirmed that the culture solution of ADSC-T of the present invention inhibits tyrosinase activity and reduces melanin synthesis, thus showing a whitening effect.

[Example 3] Evaluation of antioxidative effect of ADSC-T cell culture using DPPH assay

(1) The antioxidative effect of stem cell culture solution was confirmed by DPPH (2,2-Diphenyl-1-picrylhydrazyl) radical scavenging ability and 0.13 mM DPPH (2,2-Diphenyl-1-picrylhydrazyl, sigma-Aldrich) 0.13 mM DPPH was mixed with the sample in a weight ratio of 1: 1, and the reaction was carried out in a dark room for 10 minutes and 20 minutes, and a decrease in absorbance was confirmed at 515 nm.

X 100DPPH radical scavenging activity (%) =

X 100

: 무처리군

: No treatment group

: 처리군

: Treated group

(2) DPPH assays were used to determine the antioxidative effects of stem cell cultures. DPPH is a very stable free radical, itself a violet compound that exhibits light absorption at 515 nm. It is decolorized by a proton-radical scavenger and turns into yellow and can be observed with the naked eye. After the 0.13 mM DPPH solution was prepared and the ADSC-T cell culture was treated, the absorbance decreased. The ADSC-T culture solution was mixed with 0.13 mM DPPH solution at a weight ratio of 1: 1 and reacted in a dark room for 10 minutes and 20 minutes to confirm the decrease in absorbance at 515 nm. Stem cell cultures are produced in DMEM media. Vitamin E, an antioxidant, is added to the DMEM medium, and DPPH radicals are expected to be eliminated. The results are shown in Fig.

As shown in Fig. 14, the DPPH radical was also eliminated by the DMEM medium itself, but the scavenging activity decreased after 20 minutes from the addition of the medium. This suggests that the amount of DPPH radical scavenging substance in the medium is insignificant. In the case of the ADSC-T culture medium, after 10 minutes of reaction, the scavenging ability was larger than that of DMEM medium. When the reaction was carried out for 20 minutes, the scavenging ability was further increased and DPPH radical was inhibited about twice as much as DMEM.

Therefore, it was confirmed that the culture solution of ADSC-T of the present invention showed a remarkable antioxidative effect .

Hereinafter, pharmaceutical compositions, food compositions and cosmetic compositions of the present invention will be described, but the present invention is not intended to be limited thereto but is specifically described .

Formulation Example 1. Preparation of pharmaceutical preparations

1-1. Manufacture of Powder

20 mg of ADSC-T cell culture

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

1-2. Manufacture of tablets

10 mg of ADSC-T cell culture

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

1-3. Preparation of capsules

10 mg of ADSC-T cell culture

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

1-4. Injection preparation

10 mg of ADSC-T cell culture

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO _4? 2H ₂ O 26 mg

(2 ml) per 1 ampoule in accordance with the usual injection preparation method.

1-5. Manufacture of liquid agent

20 mg of ADSC-T cell culture

10 g per isomer

5 g mannitol

Purified water quantity

Each component was added and dissolved in purified water according to the usual liquid preparation method, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was added with purified water to adjust the total volume to 100 ml, And sterilized to prepare a liquid preparation.

Formulation Example 2. Preparation of food preparation

2-1. Manufacture of health food

100 mg of the culture medium of ADSC-T cells

Vitamin mixture quantity

Vitamin A acetate 70 μg

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

0.15 mg of vitamin B2

Vitamin B6 0.5 mg

Vitamin B12 0.2 μg

Vitamin C 10 mg

Biotin 10 μg

Nicotinic acid amide 1.7 mg

Folic acid 50 μg

Calcium pantothenate 0.5 mg

Mineral mixture quantity

1.75 mg of ferrous sulfate

0.82 mg of zinc oxide

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Secondary calcium phosphate 55 mg

Potassium citrate 90 mg

Calcium carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixture is comparatively mixed with a composition suitable for health food as a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional method for producing healthy foods , Granules can be prepared and used in the manufacture of health food compositions according to conventional methods.

2-2. Manufacture of health drinks

100 mg of the culture medium of ADSC-T cells

Vitamin C 15 g

Vitamin E (powder) 100 g

19.75 g of ferrous lactate

3.5 g of zinc oxide

Nicotinic acid amide 3.5 g

Vitamin A 0.2 g

Vitamin B1 0.25 g

Vitamin B2 0.3g

Water quantification

The above components were mixed according to a conventional health drink manufacturing method, and the mixture was stirred and heated at 85 DEG C for about 1 hour. The solution thus prepared was filtered and sterilized in a sterilized 2 liter container, It is used in the production of the health beverage composition of the invention.

Although the compositional ratio is relatively mixed with a component suitable for a favorite drink, it is also possible to arbitrarily modify the compounding ratio according to the regional or national preference such as the demand class, the demanding country, and the use purpose.

Formulation Example 3  : Manufacture of cosmetic preparations

1. Flexible lotion

0.5% by weight of the culture medium of ADSC-T cells,

Glycerin 5.0 wt%

3.0% by weight of 1,3-butylene glycol

5.0% by weight of ethanol

0.5% by weight of polyoxyethylene nonylphenyl ether

Fragrance quantity

Antiseptic

Purified water balance

2. converging lotion

0.5% by weight of the culture medium of ADSC-T cells,

Glycerin 3.0 wt%

Citric acid 0.1 wt%

Ethanol 10.0 wt%

Polyoxyethylene oleyl ether 1.0 wt%

Sorbitol 2.0 wt%

Fragrance quantity

Antiseptic

Purified water balance

3. Lotion (emulsion)

0.5% by weight of the culture medium of ADSC-T cells,

Glycerin 3.0 wt%

Butylene glycol 8.0 wt%

Squalane 10.0 wt%

2.0 wt% of polyoxyethylene sorbitan monooleate,

1.5% by weight triethanolamine

0.5% by weight of glyceryl stearate

0.2% by weight of stearyl glycyrrhetinate

Carboxyvinyl polymer 0.1 wt%

Arginine 0.1 wt%

Fragrance quantity

Antiseptic

Purified water balance

4. Cream

0.5% by weight of the culture medium of ADSC-T cells,

Glycerin 3.0 wt%

Stearic acid 8.0 wt%

Squalane 5.0 wt%

2.5% by weight of self emulsifying glycerin monostearate

1.5% by weight of polyoxyethylene sorbitan monostearate,

4.0% by weight of propylene glycol

0.2% by weight of stearyl glycyrrhetinate

Vaseline 2.0 wt%

Antioxidant amount

Fragrance quantity

Antiseptic

Purified water balance

Claims (10)

A cosmetic composition for improving skin regeneration comprising a culture medium of ADSC-T cells (adipose-derived stem cell-T cell) into which T antigen has been introduced into adipose-derived stem cells as an active ingredient.
The cosmetic composition for improving skin regeneration according to claim 1, wherein the T antigen is derived from simian virus 40 (SV40).
2. The method according to claim 1, wherein the culture medium of ADSC-T cells is
(a) separating and culturing adipose derived stem cells by treating the inhaled adipose tissue with a collagenase enzyme and then centrifuging;
(b) preparing an ADSC-T cell expressing SV40 T antigen by transfecting adipose-derived stem cells cultured in step (a) with a plasmid expression vector (pEF321? -T); And
(c) culturing the prepared ADSC-T to obtain a culture liquid.
The method according to claim 3, wherein the transfection in step (b) is carried out by at least one method selected from the group consisting of calcium phosphate transfection, electroporation, microinjection and liposome injection. Cosmetic composition.
A food composition for improving skin regeneration comprising a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which T antigen has been introduced into adipose-derived stem cells as an active ingredient.
A cosmetic composition for whitening comprising an adipose-derived stem cell-T cell (ADSC-T cell) culture medium into which T antigen has been introduced into adipose-derived stem cells as an active ingredient.
The present invention relates to a whitening food composition comprising adipose-derived stem cell-T cell (ADSC-T) cells into which T antigen has been introduced into adipose-derived stem cells as an active ingredient.
A cosmetic composition for anti-aging comprising a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which T antigen has been introduced into adipose-derived stem cells.
A food composition for anti-aging comprising a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which T antigen has been introduced into adipose-derived stem cells.
A pharmaceutical composition for wound healing, comprising a culture medium of ADSC-T cells (adipose-derived stem cell-T cells) into which T antigen has been introduced into adipose-derived stem cells.

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