JP2021001166A - Composition for improving skin comprising neural stem cell culture solution - Google Patents

Abstract

To provide a cosmetic composition for improving skin that shows the effects of reduced damage, improved wrinkles, regenerated skin, increased resilience of skin, skin-whitening, antiinflammation, antioxidation, skin moisture retention, or reinforced skin barrier.SOLUTION: A cosmetic composition contains, as an active ingredient, a neural stem cell culture solution comprising at least one protein selected from the group consisting of activin RIA/ALK-2, adiponectin-1, Axl, BIK, chordin-like 1, Csk, EDA-A2, epidermal growth factor (EGF), endostatin, basic fibroblast growth factor (bFGF), galectin-3, growth differentiation factor-3 (GDF3), GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2 (ICAM-2), insulin-like growth factor binding protein 2(IGFBP-2), IGFBP-3, IGFBP-7 and the like.SELECTED DRAWING: None

Description

The present invention relates to a composition for skin improvement containing a neural stem cell culture solution as an active ingredient.

Stem cells are known to be involved in biological actions through microenvironmental regulation of damaged tissues such as promotion of angiogenesis, anti-inflammatory action, and immunomodulatory action in the human body. These biological actions occur when various anti-inflammatory cytokines and cell death suppressors that promote the protection and regeneration of damaged tissues and cell survival promoters are released from stem cells. These are called the paracrine effect.

On the other hand, Korean Registered Patent No. 10-0848056 discloses a method for inhibiting melanin synthesis using an adipose-derived stem cell culture solution which is a mesenchymal stem cell, and Korean Patent No. 10-2009-0116659. Discloses a whitening cosmetic composition containing a mesenchymal stem cell culture solution derived from umbilical cord blood, which relieves skin wounds, improves skin wrinkles, and regenerates skin in an outer lung lobe-derived neural stem cell culture solution. It is not known about the effects of increasing skin elasticity, skin whitening, skin anti-inflammatory, skin antioxidant, skin moisturizing or skin barrier strengthening.

An object of the present invention is a neural stem cell culture that exhibits the effects of wound relief, wrinkle improvement, skin regeneration, skin elasticity increase, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or skin barrier strengthening. It is an object of the present invention to provide a cosmetic composition for skin improvement, which comprises a liquid as an active ingredient.

Another object of the present invention is to provide a quasi-drug composition for skin improvement containing the neural stem cell culture solution as an active ingredient.

Another object of the present invention is a pharmaceutical composition containing the neural stem cell culture solution as an active ingredient for treating skin wounds, skin regeneration, skin anti-inflammatory, or treatment or prevention of autoimmune diseases. To provide.

As a result of diligent efforts to develop a skin improving composition containing a neural stem cell culture solution, the neural stem cell culture solution derived from brain tissue has been used to relieve wounds, improve wrinkles, and regenerate skin. The present invention has been confirmed to have effects of increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or strengthening of skin barrier, and confirming that it contains a large number of proteins involved in these effects. completed.

In order to achieve the above object, one aspect of the present invention includes neural stem cell culture solution as an active ingredient, which includes wound relief, wrinkle improvement, skin regeneration, increased skin elasticity, skin whitening, anti-inflammatory, and anti-inflammatory. Provided are cosmetic compositions for oxidizing, moisturizing the skin, or strengthening the skin barrier.

Another aspect of the present invention provides a quasi-drug composition for skin improvement, which comprises the neural stem cell culture solution as an active ingredient.

Another aspect of the present invention is a pharmaceutical composition comprising the neural stem cell culture solution as an active ingredient for treating skin wounds, skin regeneration, skin anti-inflammatory, or treatment or prevention of autoimmune diseases. Provide things.

Another aspect of the invention is actibin RIA / ALK-2, adiponectin-1, Axl, BIK, cordin-like 1, Csk, EDA-A2, Epidermal growth factor (EGF), endostatin, basic. Fibroblast growth factor (bFGF), galectin-3, growth differentiation factor-3 (GDF3), GDF5, GDF-15, gripecan 3, GRO, GLO-1, intercellular attachment molecule 2 (ICAM-2), insulin-like Growth factor binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15 Rα, IL-20 Rβ, insulin, inactive TGF- β bp1, monocytic growth factor-1 (MCP1), MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1 (PECAM-1), S100A10, sFRP-4, sgp130 , Mothers against decapentaplegic homolog 1 (Smad1), Smad4, Sonic hedgehog (Shh N-terminal), tissue factor pathway inhibitor (TFPI), TGF-β5, thrombospondin-1, tyrosine-protein phosphorylase receptor (Tyrosine-) Neurostem cell culture containing one or more proteins selected from the group consisting of protein kinase receptor, Tie-2), metalloprotease inhibitor 1 (TIMP-1), vascular endothelial growth factor (VEGF) and VEGF-C Provide cosmetics, non-pharmaceutical products or pharmaceutical compositions containing liquids.

Another aspect of the invention is a cosmetic, quasi-drug or pharmaceutical composition obtained by culturing immortalized neural stem cells (imNSCs) for the uses described above. Provide things.

The neural stem cell culture solution according to the present invention exhibits the effects of wound relief, wrinkle improvement, skin regeneration, skin elasticity increase, skin whitening, anti-inflammatory, antioxidant, skin moisturizing or skin barrier strengthening. It can be usefully used for cosmetics, non-pharmaceutical products and pharmaceuticals. In addition, by utilizing immortalized neural stem cells, even if the neural stem cells are repeatedly cultured, the composition and concentration of useful factors in the culture medium are constant, so that functional cosmetics having uniform efficacy or It can be usefully used as a raw material for pharmaceuticals.

FIG. 1a is a micrograph of a neural stem cell according to an embodiment of the present invention at × 40 magnification and × 100 magnification. FIG. 1b is a graph of the growth rate of neural stem cells. FIG. 1c is a graph showing the time point of diploid formation in each subculture of neural stem cells. FIG. 2a is a result of flow cytometric analysis showing the expression levels of nestin, PAX6 and SOX2 in neural stem cells according to an example of the present invention. FIG. 2b is a fluorescence micrograph of neural stem cells expressing Nestin, PAX6 and SOX2. FIG. 3 shows the results of separating the mRNA of the neural stem cell according to the embodiment of the present invention and confirming the expression of the neural stem cell-specific gene marker for each subculture (p8, p10, p12). .. FIG. 4 shows specificity in each differentiated cell after differentiating the neural stem cell according to an embodiment of the present invention into a neural cell (neuron), an astrocyte (astrocyte) and an oligodendrocyte (oligodendrocyte), respectively. It is a micrograph which confirmed the presence or absence of the expression of a marker by an immunofluorescent staining method. 5a and 5b show human epidermal cells (HaCaT) and human dermal fibroblasts (HS68) according to the treatment concentrations of the neural stem cell culture medium according to one example of the present invention and the adipose stem cell culture medium according to a comparative example. It is a graph which showed the cell viability of. 6a to 6d show human epidermal cells (HaCaT) and human dermal fibroblasts (HS68) according to the treatment concentrations of the neural stem cell culture medium according to one example of the present invention and the adipose stem cell culture medium according to a comparative example. It is a micrograph (6a, 6c) showing the recovery of the wound in the above and the graph (6b, 6d) comparing the recovery rate of the wound. FIG. 7a is a photograph of electrophoresis in which the presence or absence of COL1A1 expression was confirmed after treating human dermal fibroblasts (HS68) with different concentrations of the neural stem cell culture medium according to the embodiment of the present invention. FIG. 7b is a graph comparing the contents of COL1A1. FIG. 7c is a photograph of electrophoresis confirming the presence or absence of COL3A1 expression according to the treatment concentration of the neural stem cell culture solution according to one example of the present invention and the adipose stem cell culture solution according to a comparative example. FIG. 7d is a graph comparing the contents of COL3A1. FIG. 8 is a graph comparing the PICP expression levels in human dermal fibroblasts (HS68) according to the treatment concentrations of the neural stem cell culture medium according to one example of the present invention and the adipose stem cell culture medium according to a comparative example. is there. FIG. 9 is a graph comparing the elastase inhibitory activity in human dermal fibroblasts (HS68) according to the treatment concentration of the neural stem cell culture solution according to one example of the present invention and the adipose stem cell culture solution according to a comparative example. is there. 10a to 10d show the melanin synthesis inhibitory effect according to the treatment concentration of the neural stem cell culture solution according to the embodiment of the present invention and the adipose stem cell culture solution according to the comparative example. 10a and 10b show the case where only the neural stem cell culture medium and the adipose stem cell culture medium are treated, and FIGS. 10c and 10d show the neural stem cell culture medium and the adipose stem cell culture medium after the treatment with the melanin stimulating hormone. It is the result of doing. FIG. 11 shows the activity of tyrosinase, which is a main enzyme involved in the melanin biosynthesis process, according to the treatment concentration of the neural stem cell culture solution according to one example of the present invention or the adipose stem cell culture solution according to a comparative example. It is a graph comparing the rates. 12a to 12d show human dermal fibroblasts (HS68) treated with lipopolysaccharide (LPS) to induce an inflammatory reaction, and then shown in the neural stem cell culture medium and comparative example according to one embodiment of the present invention. This is the result of treating the adipose stem cell culture medium according to the concentration. FIG. 12a is a photograph of electrophoresis showing the presence or absence of expression of IL-6, IL-1β and COX-2, FIG. 12b shows the expression level of COX-2, and FIG. 12c shows the expression of IL-1β. FIG. 12d is a graph comparing the expression levels of IL-6. FIG. 13 shows changes in the concentration of intracellular reactive oxygen species (ROS) in human dermal fibroblasts (HS68) according to the treatment concentrations of the neural stem cell culture solution and the adipose stem cell culture solution according to the embodiment of the present invention. It is a graph which compared DCF-DA fluorescence values for measurement. FIG. 14 is a result of comparing the antioxidant capacity of Trolox equivalent in human dermal fibroblasts (HS68) according to the treatment concentration of the neural stem cell culture solution and the adipose stem cell culture solution according to the embodiment of the present invention. .. 15a and 15b show the results of treating human epidermal cells (HaCaT) with the neural stem cell culture medium and retinoic acid according to an example of the present invention. FIG. 15a is a photograph of electrophoresis showing whether or not loricrin, involucrin, HAS-2 and HAS-3 are expressed, and FIG. 15b compares the expression levels of loricrin, involucrin, HAS-2 and HAS-3. It is a graph. FIG. 16 is a result of analyzing the components of the protein secreted in the neural stem cell culture medium according to the embodiment of the present invention. FIG. 17 is a graph showing the expression intensity of the protein secreted in the neural stem cell culture medium according to the embodiment of the present invention.

The present invention, on the one hand, is a composition for skin improvement containing a nerve stem cell culture solution as an active ingredient, wherein the skin improvement includes relief of wounds, improvement of wrinkles, regeneration of skin, increase of skin elasticity, and skin. It relates to compositions that are whitening, anti-inflammatory, anti-oxidant, moisturizing the skin or strengthening the barrier of the skin.

In one embodiment of the invention, the neural stem cell can be an immortalized neural stem cell. Specifically, it is obtained by transducing an L-Myc expression vector into adult neural stem cells (NSCs) isolated from the brain tissue of a dead fetus that has passed 10 to 14 weeks. It can also be an immortalized neural stem cell.

The neural stem cells are preferably cultured in a medium having a clear composition and concentration, and the culture medium can be obtained by centrifuging and filtering in the process of culturing the neural stem cells.

The process of immortalizing and culturing the neural stem cells and then separating them is not limited to the method according to the present invention, and can be carried out by a method commonly practiced in the art. The neural stem cell culture medium can be obtained by centrifuging the neural stem cells in the process of subculturing the neural stem cells and then filtering the supernatant, and the neural stem cells are removed and homogenzized. It can be obtained through the process.

As a result of analyzing the secretome of the nerve stem cell culture solution according to the present invention, actibin RIA / ALK-2, adiponectin-1, Axl, BIK, cordin-like 1, Csk, EDA-A2, epidermal growth factor. , EGF), endostatin, basic fibroblast growth factor (bFGF), galectin-3, growth differentiation factor-3 (GDF3), GDF5, GDF-15, gripecan 3, GRO, GLO-1, intercellular attachment molecule 2 (ICAM-2), insulin-like growth factor binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15 Rα, IL-20 Rβ, insulin, inactive TGF-β bp1, monocytic growth factor-1 (MCP1), MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1 (PECAM-1) ), S100A10, sFRP-4, sgp130, Mothers against decapentaplegic homolog 1 (Smad1), Smad4, Sonic Hedgehog (Shh N-terminal), Tissue factor pathway inhibitor (TFPI), TGF-β5, Thrombospondin-1, Tyrosine- It was confirmed to contain protein phosphorylase receptor (Tyrosine-protein kinase receptor, Tie-2), metalloprotease inhibitor 1 (TIMP-1), vascular endothelial growth factor (VEGF) and VEGF-C.

In one aspect of the invention, the neural stem cell culture medium is a protein associated with skin regeneration and anti-aging: actibin RIA / ALK-2, adiponectin-1, cordin-like 1, Csk, EDA-A2, EGF, bFGF. , GDF3, GDF5, GDF15, GLO-1, IGFBP-2, IGFBP-3, IGFBP-7, Latent TGF-βbp1, MFG-E8, MMP-20, PDGF-AA, PECAM-1, Smad1, Smad4, Sonic Hedge Hog (Shh N-terminal), TGF-β5, thrombospondin-1, Tie-2, VEGF or VEGF-C can be included.

In another aspect of the present invention, the neural stem cell culture medium is a protein involved in promoting collagen synthesis and improving wrinkles, MCP-1, TIMP-1, bFGF, insulin, Latent TGF-β bp1, Smad1, Smad4. , TGF-β5 or thrombospondin-1 can be included.

In another aspect of the present invention, the neural stem cell culture medium can contain TGF-β5, a protein involved in the whitening effect.

In another aspect of the invention, the neural stem cell culture medium can contain proteins Axl, GRO, thrombospondin-1 or TGF-β5 that are required for anti-inflammatory effects and prevention of autoimmune diseases.

In one embodiment of the present invention, the neural stem cell culture solution can regenerate skin cells to reduce and treat the damage when the skin tissue is damaged.

In one embodiment of the present invention, the neural stem cell culture medium can suppress elastin degradation and prevent skin aging by increasing collagen synthesis and inhibiting elastase activity. Specifically, the neural stem cell culture medium wrinkles the skin at the mRNA level and / or the protein level, for example, by increasing the amount of procollagen type IC-terminal protein secreted and increasing collagen synthesis. It can improve the skin's elasticity, regenerate the skin, and suppress the aging of the skin.

In one embodiment of the present invention, the neural stem cell culture medium has a skin whitening effect. Specifically, the neural stem cell culture medium reduces the melanin content in the skin by suppressing melanin synthesis in the skin, inhibiting the activity of tyrosinase, and suppressing the conversion of tyrosine to melanin. can do.

In one embodiment of the present invention, the neural stem cell culture medium has an antioxidant effect of removing intracellular reactive oxygen species (ROS).

In one embodiment of the present invention, the neural stem cell culture medium has an anti-inflammatory effect that alleviates the inflammatory reaction. Specifically, when an inflammatory reaction of skin cells or immune cells occurs, Cyclooxygenase-2 (COX-2), interleukin-1β (interleukin-1β; IL-1β) and interleukin-6 (interleukin-6; The expression level of IL-6) can be reduced and the inflammatory reaction can be alleviated.

In one embodiment of the present invention, the neural stem cell culture medium has the effects of moisturizing the skin and strengthening the skin barrier. Specifically, the neural stem cell culture medium strengthens the skin barrier by increasing the expression levels of loricrin and involucrin and promoting differentiation into keratinocytes, and expresses HAS-2 and HAS-3. The moisturizing power of the skin can be improved by promoting the synthesis of hyaluronic acid, which is a moisturizing factor.

Keratinocytes, which account for 95% of epidermal cells, proliferate in the basal layer and finally differentiate into flat-shaped nuclei-free keratinocytes via the upper stratum spinosum and stratum granulosum. In the process of such differentiation, Loricirin, which is a differentiation-promoting factor in the stratum granulosum and stratum corneum of the epidermis, and involucrin, which is a differentiation-promoting factor in the stratum granulosum and the stratum spinosum of the upper epidermis, are used. It is expressed and aggregates keratin microfibers in the stratum corneum to form a stratum corneum cell membrane, which contributes to strengthening the skin barrier.

In addition, loricrin and involucrin form natural moisturizing factors in the skin and play an important role in moisturizing the skin. In addition to the natural moisturizing factors, the skin is affected by various moisturizing factors such as hyaluronic acid (HA). Performs a moisturizing function on the skin. HA is mainly synthesized by hyaluronan acid synthase (HAS) in keratinocytes and fibroblasts and accumulated in the extracellular matrix.

The composition can, if desired, be formulated in a cosmetic dosage form commonly produced in the art.

The cosmetic composition includes, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing, oils, powder foundations, emulsion foundations, wax foundations. And can be emulsified into a spray or the like, but is not limited to this. Specifically, it can be formulated into a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a facial mask, a spray or a powder. When the dosage form of the cosmetic composition is a paste, cream or gel, animal oil, dietary oil, wax, paraffin, starch, tragacanth, cellulose derivative, polyethylene glycol, silicon, bentonite, silica. , Tarc, zinc oxide and a carrier component selected from the group consisting of mixtures thereof.

When the dosage form of the cosmetic composition is a solution or an emulsion, it may contain a carrier component selected from the group consisting of a solvent, a solvent agent, an emulsion agent and a mixture thereof. it can. Examples of these are water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, sorbitan fatty acid esters and mixtures thereof. And so on.

When the dosage form of the cosmetic composition is a suspension, water, a liquid diluent such as ethanol or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan. It can contain a carrier component selected from the group consisting of suspending agents such as esters, microcrystalline cellulose, aluminum metahydroxydos, bentonite, aga, tragacant and mixtures thereof.

The carrier component can be contained in an amount of about 1 to about 99.99% by weight, preferably about 80% by weight to about 90% by weight, based on the total weight of the cosmetic composition.

In another aspect, the present invention is a quasi-drug composition for skin improvement containing the nerve stem cell culture solution as an active ingredient, wherein the skin improvement includes wound relief, wrinkle improvement, skin regeneration, and skin. It relates to a quasi-drug composition which is an increase in elasticity, skin whitening, anti-inflammatory or anti-oxidation.

The neural stem cell culture medium is as described above.

In the present invention, the "quasi-drug" is a fiber, rubber product or similar used for the purpose of treating, alleviating, treating or preventing a human or animal disease, which has a weak effect on the human body or is applied to the human body. Articles that do not act directly and fall under any of the following: non-mechanical or mechanical or similar products, sterilization, insecticidal and similar applications to prevent infection, humans and animals. Items used for the purpose of diagnosing, treating, alleviating, treating or preventing the disease, which are not instruments, machines or devices, and are used for the purpose of quasi-drugs on the structure and function of humans and animals. It means an article that is not an instrument, machine or device, and includes external skin preparations and personal hygiene products.

When the composition according to the present invention is included in a quasi-drug for the purpose of skin improvement or the like, the composition can be used as it is or can be used together with other components of the quasi-drug. It can be used as appropriate depending on the method. The mixed amount of the active ingredient can be appropriately determined according to the purpose of use.

In another aspect, the present invention comprises a pharmaceutical composition comprising the neural stem cell culture solution as an active ingredient for treating skin wounds, skin regeneration, skin anti-inflammatory, or treatment or prevention of autoimmune diseases. It's about things.

The neural stem cell culture medium is as described above.

The pharmaceutical composition contains the neural stem cell culture solution as an active ingredient in an amount of about 0.1% by weight to about 90% by weight, specifically, about 0.5% by weight to about 90% by weight, based on the total weight of the composition. It can be contained in an amount of 75% by weight, more specifically, about 1% by weight to about 50% by weight.

The pharmaceutical composition can include conventional non-toxic, pharmaceutically acceptable additives that are formulated into the formulation by conventional methods. For example, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient.

The pharmaceutical composition can be applied to the skin. The dosage form of the pharmaceutical composition can be an external preparation for skin. The external preparation for skin is not particularly limited to this, and is manufactured and used in the form of, for example, an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a patch or a gel. Can be done.

The above contents will be described in more detail with reference to the following examples. However, the following examples are for exemplifying the present invention, and the scope of the examples is not limited to these.

Production example 1. Production of adipose stem cell culture medium
Adipose stem cells purchased from Promocell (Cat # C-12978) were subcultured 3 or 4 times, followed by DMEM / Low (Hyclone), 10% FBS, 10ng / mLbFGF (basic fibroblast growth factor). It was added and further cultured. When the confluency reached 70 to 80%, an adipose stem cell culture medium was obtained in the process of exchanging the culture medium with phenol red-free Low DMEM and culturing for 48 hours.

Production example 2. Preparation of Neural Stem Cells and Neural Stem Cell Culture Solution L-Myc expression vector [pMXs-hu-L-] was added to neural stem cells (NSC) City of Hope (LM-NSC008) extracted from fetal brain tissue at 10 to 14 weeks. Through the process of transfecting and selecting Myc], immortalized neural stem cells (imNSCs) were produced (Okita et al., 2013).

Phenol-free DMEM / F12 (Gibco), 2% supplement (reduced glutathione, superoxide dismutase, human transferase (holo), L-carnitine, D ⁺ -galactose, putrecin, linoleic acid, linolenic acid, retinyl acetate, DL-α A medium containing tocopherol (vitamin E), DL-α tocopherol acetate), 20 ng / mL bFGF and 20 ng / mL EGF (epidermal growth factor) was prepared and then used to culture immortalized neural stem cells. .. In the process of subculturing this, a culture solution was obtained, and the non-adhesive cells after culturing were removed to obtain neural stem cells. Further, the obtained culture solution was centrifuged, and the supernatant was filtered through a 0.22 μm filter to obtain a neural stem cell culture solution.

Experimental example 1. Characteristics analysis of neural stem cells Experimental example 1.1. Observation of Neural Stem Cell Growth Rate The neural stem cells obtained in Production Example 2 were observed under a microscope at × 40 and × 100. Then, the cell growth rate of neural stem cells and the time point at which they became diploid for each subculture were measured and shown in a graph. FIG. 1a is a photomicrograph, FIG. 1b is a graph of cell growth rate, and FIG. 1c is a graph showing the time point at which cells become diploid.

Experimental example 1.2. Confirmation of expression of neural stem cell-specific gene marker In order to confirm the gene expression aspect of the neural stem cell obtained in Production Example 2, flow cytometry analysis method and real-time polymerase chain reaction method (qRT-PCR) were used.

Experimental Example 1.2.1. Confirmation of gene expression using flow cytometry analysis When the density of neural stem cells obtained in Production Example 2 reaches 80 to 90%, the culture medium is removed and phosphate buffered saline (PBS) is removed. ) Was washed. Then, the cells were peeled off using Accutase, and the cells were washed with PBS. The number of cells was counted to 1x10 ⁶ cells / mL, and then the cells were fixed with 4% p-formaldehyde at 4 ° C. for 20 minutes. After the fixed cells are washed with PBS, the cells are treated with Triton X-100 (surfactant) diluted with 0.01% for antibody permeation, and the permeabilization process is performed. After 10 minutes at room temperature, the cells were washed with PBS.

Next, a FACS buffer was added to the cells to react them with nestin-PE, PAX6-PE, and SOX2-PE antibodies, and then a specific expression marker for neural stem cells was used using a cell sorter (FACS) for fluorescence flow cytometry analysis. The expression of Nestin, PAX6 and SOX2 was confirmed. FIG. 2a is a graph showing the expression levels of Nestin, PAX6 and SOX2, respectively, and FIG. 2b is a fluorescence micrograph of neural stem cells expressing Nestin, PAX6 and SOX2.

As a result, it was confirmed that the cells obtained in Production Example 2 clearly expressed nestin, PAX6 and SOX2.

Experimental Example 1.2.2. Confirmation of gene expression using qRT-PCR RNA was extracted from the neural stem cells obtained in Production Example 2 using phenol / chloroform. The extracted RNA was reverse transcribed to synthesize cDNA. The degree of gene expression of cDNA was analyzed by qRT-PCR on the Applied Biosystems 700 Sequence System (foster City, CA, USA). At this time, the synthesized cDNA was prepared using a primer set specific to each of them shown in Table 1 below. qPCR was repeated 30 times in a cycle of 95 ° C. for 10 minutes, 95 ° C. for 15 seconds and 56 ° C. for 1 minute. The mRNA levels were normalized by β-actin values and compared. The result is shown in FIG.

As a result, it was confirmed that SOX1, SOX2, PAX6 and nestin were clearly expressed from the neural stem cells.

As a result, since the neural stem cell-specific gene markers SOX1, SOX2, PAX6 and nestin are expressed, it was found that the cells obtained in Production Example 2 have the characteristics of neural stem cells.

Experimental example 2. Confirmation of differentiation ability of neural stem cells After differentiating the neural stem cells obtained in Production Example 2 into neural cells (neurons), astrocytes (astrocytes) and oligodendrocytes (oligodendrocytes), proteins related to their intracellular differentiation. In order to confirm the expression of, the following immunofluorescent staining method was performed.

First, the neural stem cells maintained on the 4-well chamber slide were fixed with 4% p-formaldehyde at 37 ° C. for 20 minutes, and then washed twice with PBS containing calcium ions and magnesium ions. did. Next, Triton X-100 (surfactant) was diluted to 0.1% in PBS, treated for 10 minutes, and then washed again with PBS. Bovine Serum Albumin (BSA) is diluted with 0.1% Triton X-100 / PBS at 5% and then added to the sample to prevent detection with non-specific antibodies. It was allowed to react for 1 hour.

As for the primary antibody, the type of antibody to be attached differs depending on the cell, and the target antibody and the dilution amount according to the protein are shown in Table 2 below. After attaching the primary antibody, the reaction was carried out in a shaker at 4 ° C. for 16 hours. The secondary antibody is selected and used according to the host of the primary antibody and the wavelength, and is used as a goat anti-mouse IgG-conjugate Alexa 488 (abcam, cat.no.ab150113, diluted 1,000 times), anti-mouse FITC. (EBioscience, cat.no.11-5790-81, diluted 1,000-fold) was used.

In addition, the nucleus was stained using DAPI (diluted 1,000 times). The sample after staining was photographed using a fluorescence microscope. Nerve cell nuclei are shown in DAPI (blue), nerve cell expression factor MAP2 (green), astrocyte expression factor GFAP (green), and oligodendrocyte expression factor O1 (green). It was. The result is shown in FIG.

As a result, it was confirmed that MAP2, GFAP and O1 were expressed from each of the differentiated neural stem cells, astrocytes and oligodendrocytes.

From this, it was found that the neural stem cells obtained in Production Example 2 have a resolution to nerve cells, astrocytes and oligodendrocytes.

Experimental example 3. Evaluation of cytotoxicity and cell proliferation effect of nerve stem cell culture medium In order to confirm the cytotoxicity and cell proliferation effect of the nerve stem cell culture medium obtained in Production Example 2, human epidermal cells (HaCaT) and human dermal fibroblasts ( The following experiments were carried out using HS68).

HaCaT and HS68 were dispensed into 96-well plates at 1 × 10 ³ cells / 100 μl per well, cultured for 24 hours, and then the concentration was 10% as a negative control group (NC; untreated group) and a comparative control group. , 25%, 50%, 100% adipose stem cell culture medium, and 10%, 25%, 50%, 100% neural stem cell culture medium as an experimental group were treated.

After the treatment of the culture solution, the absorbance was measured at 450 nm using the CCK8 (Dojindo, CK04-13) reagent at the same time every day for 1 day, 2 days and 3 days, and changes in cell activity were confirmed. The viability of human epidermal cells is shown in FIG. 5a, and the viability of human dermal fibroblasts is shown in FIG. 5b.

As a result, it was confirmed that most of human epidermal cells and human dermal fibroblasts showed excellent survival rates when treated with neural stem cell culture medium.

Experimental example 4. To ensure 24-well plates relaxation effect of skin wounds in neural stem cell culture, human epidermal cells (HaCaT) are each 3 × 10 ⁵ cells per well, and human dermal fibroblasts (HS68) is well per 2 × 10 ⁵ cells were dispensed and cultured so as to have a density of 100%. Scratch the middle of the well with a 1000 P white tip to produce a wound, then negative control group (NC), adipose stem cell culture (comparative control group; concentrations 10%, 25%, 50%, 100%) and neural stem cell cultures (experimental group; concentrations 10%, 25%, 50%, 100%) were treated.

For each of human epidermal cells and human dermal fibroblasts, the area of the wound after treatment with the culture medium and after 24 hours was measured, and the recovery rate was confirmed. At this time, the cells after 24 hours were also shown with micrographs stained with crystal violet reagent. 6a and 6b are photomicrographs and graphs showing the recovery rate of wounds in human epidermal cells (HaCaT), and FIGS. 6c and 6d show the recovery rate of wounds in human dermal fibroblasts (HS68). Micrographs and graphs.

As a result, it was confirmed that the wounds of human epidermal cells and human dermal fibroblasts healed faster when treated with the neural stem cell culture medium than with the negative control group and the adipose stem cell culture medium.

Experimental example 5. Confirmation of collagen synthesis effect in neural stem cell culture experimental example 5.1. The analysis 6-well plates of the presence or absence of expression in collagen gene using RT-PCR, and cultured for 24 hours aliquoted per well human dermal fibroblasts (HS68) by 1.0 × 10 ⁵ cells min. Negative control group (NC), adipose stem cell culture medium (comparative control group; concentrations 10%, 25%, 50%, 100%) and neural stem cell culture medium (experimental group; concentrations 10%, 25%, 50%, 100%) ) Was added and cultured for 24 hours, and then using each specific primer set shown in Table 3 below, Experimental Example 1.2.2. QRT-PCR was performed in the same manner as described in 1 to measure the presence or absence of expression in the collagen genes (COL1A1 and COL3A1) and the expression level.

7a and 7b are photographs of electrophoresis showing the presence or absence of expression of COL1A1 and graphs measuring the expression level thereof, and FIGS. 7c and 7d are photographs of electrophoresis showing the presence or absence of expression of COL3A1 and a graph thereof. It is a graph which measured the expression level.

As a result, it was confirmed that the expression levels of the collagen genes COL1A1 and COL3A1 were increased when the neural stem cell culture medium was treated as compared with the case where the negative control group and the adipose stem cells were treated.

Experimental Example 5.2. Evaluation 6-well plates ability to promote collagen synthesis using ELISA, and cultured for 24 hours aliquoted per well human dermal fibroblasts (HS68) by 1.0 × 10 ⁵ cells min. Negative control group (NC), positive control group (TGF-β), adipose stem cell culture medium (comparative control group; concentration 10%, 25%, 50%, 100%) and neural stem cell culture medium (experimental group; concentration 10%) , 25%, 50%, 100%), and after culturing for 48 hours, the cultured medium was centrifuged to obtain a supernatant. The ability to promote collagen synthesis was confirmed by analyzing the degree of procollagen synthesis using the Procollagen Type IC-peptide (PICP) EIA Kit (Takara, Cat. # MK101). The result is shown in FIG.

As a result, when neural stem cells were treated, the PICP expression level was higher than when the negative control group and the adipose stem cell culture medium (comparative control group; 10%, 25%, 50%, 100%) were treated. The PICP expression level was higher than that of the conventionally known positive control group.

From this, it was found that the neural stem cell culture solution is effective in improving the wrinkles of the skin and increasing the elasticity of the skin.

Experimental example 6. Confirmation of Elastase Activity Inhibitory Effect of Neural Stem Cell Culture Solution The degree of inhibition of elastase activity by treatment of the neural stem cell culture solution was measured through an elastase inhibition assay.

More specifically, 6-well plate, and cultured for 24 hours aliquoted per well human dermal fibroblasts (HS68) by 1.0 × 10 ⁵ cells min. Next, add 50 μl of 0.6 unit / mL porcine spleen elastase (PPE; in 0.2 M Tris-buffer pH 8.0), 50 μl of each sample, and 100 μl of 1 mg / mL N-Succ- (Ala) 3-nitroanilide (SANA; DMSO). The absorbance was measured at 410 nm within 10 minutes. At this time, the untreated group was a negative control group (NC), each sample was a positive control group using phosphoramidon disodium salt (PPDS, 2.5 mM), and adipose stem cells (concentrations 10%, 25%, 50%) as a comparative control group. 100%), and neural stem cell cultures (concentrations 10%, 25%, 50%, 100%) were used as the experimental group. The result is shown in FIG.

As shown in FIG. 9, the elastase inhibitory activity increased in a concentration-dependent manner when the neural stem cell culture medium was treated, as compared with the negative control group and the comparative control group treated with the adipose stem cell culture medium.

From this, it was found that the neural stem cell culture medium is effective in increasing the elasticity of the skin by suppressing the decomposition of elastin.

Experimental example 7. Confirmation of skin whitening effect of neural stem cell culture solution Experimental example 7.1. The melanin synthesis inhibiting effect of 6-well plates of neural stem cell cultures, and dispensed in 1.0 × 10 ⁵ cells of B16F10 melanoma cells in rodents per well min, nonadherent cells the next day with PBS After washing and removal, the negative control group (NC), the positive control group, Albutin 10 Mm, which has an intracellular melanin production inhibitory effect, and the comparative control group, the adipose stem cell culture medium (ASC; concentrations 10%, 25%, 50%, 100%) and nerve stem cell cultures (NSC; concentrations 10%, 25%, 50%, 100%) were treated as experimental groups. After 48 hours, the cells were washed with PBS and cells were obtained with trypsin-EDTA.

Next, 1 N NaOH + 50% DMSO solution was added 1 ml per well to dissolve the cells at 95 ° C., and then the melanin absorbance was measured at 405 nm using a micro plate reader to measure melanin. The amount of secretion and the intracellular content of FIG. 10a shows a photograph showing the presence or absence of melanin synthesis in melanoma cells when the adipose stem cell culture solution or the neural stem cell culture solution was treated, and FIG. 10b shows a graph showing the intracellular melanin content.

After treating 100 nm of melanocyte-stimulating hormone (α-MSH), the amount of melanin secreted and the intracellular content were measured by the same method as described above. FIG. 10c shows a photograph showing the presence or absence of melanin synthesis in melanoma cells when the adipose stem cell culture solution or the neural stem cell culture solution was treated after α-MSH treatment, and a graph showing the intracellular melanin content is shown. It is shown in FIG. 10d.

As a result, when the neural stem cell culture medium was treated, the melanin synthesis inhibitory effect was exhibited in proportion to the concentration, and the melanin synthesis inhibitory effect was more excellent than when the negative control group and the adipose stem cell culture medium were treated. I confirmed that it would appear.

Experimental Example 7.2. Inhibitory effect on tyrosinase activity of neural stem cell culture solution In a 96-well plate, 220 μl of 0.1 M phosphate buffer (pH 6.8, 0.1% PMSF) and 20 μl of neural stem cell culture solution were placed, and 1.5 mM tyrosine (Tyrosine) was added. ) 50 μl and 20 μl of Tyrosinase were added in order to react, and then the absorbance at 492 nm was measured using a microplate reader. At this time, the untreated group was used as a negative control group (NC), the positive control group was used as an albumin 1 mM, the comparative control group was used as an adipose stem cell culture medium (concentrations 10%, 25%, 50%, 100%), and the experimental group was used. Neural stem cell cultures (concentrations 10%, 25%, 50%, 100%) were treated. The result is shown in FIG.

As a result, it was confirmed that the effect of suppressing the tyrosinase activity was more excellent when the neural stem cell culture medium was treated than in the negative control group and the comparative control group treated with the adipose stem cell culture medium.

From this, it was found that the neural stem cell culture medium has a skin whitening effect by suppressing melanin synthesis and tyrosinase activity.

Experimental example 8. Anti-inflammatory confirmation 6-well plates of the effects of neural stem cell cultures, dispensed per well human dermal fibroblasts (HS68) by each 2.5 × 10 ⁵ cells, as density is 80% After culturing, it was replaced with a serum-free medium. Lipopolysaccharide (LPS; 20 μg / mL) that induces an inflammatory reaction after 24 hours, negative control group (NC), adipose stem cell culture solution (concentration 10%, 25%, 50%, 100%) ), And the neural stem cell culture medium (concentration 10%, 25%, 50%, 100%) of the experimental group was treated.

After 24 hours, RNA was separated from the cells and cDNA was synthesized. Experimental Example 1.2.2. The expression levels of COX-2, IL-1β and IL-6 were confirmed by performing qRT-PCR by the same method as described in. FIG. 12a is a photograph of electrophoresis showing the presence or absence of expression of IL-6, IL-1β and COX-2, and FIGS. 12b to 12d show the expression levels of COX-2, IL-1β and IL-6. It is a graph shown.

It was confirmed that the expression levels of COX-2, IL-1β and IL-6 were lower when the neural stem cell culture medium was treated than when the negative control group and the adipose stem cells were treated.

From this, it was found that the neural stem cell culture medium has an effect of suppressing skin inflammation.

Experimental example 9. Confirmation of Antioxidant Effect of Neural Stem Cell Culture Solution Experimental Example 9.1. Effect of scavenging intracellular reactive oxygen species (ROS) ROS detection containing carboxy-H2DCFDA to know the effect of neural stem cell culture medium on the production of intracellular reactive oxygen species (ROS) Using the kit (Abcam), we experimented as follows.

DCFH-DA easily penetrates the cell membrane, diffuses into the cell, is hydrolyzed to DCFH that has lost fluorescence by intracellular esterase, and is later rapidly oxidized to DCF that exhibits high fluorescence in the environment where ROS is present. To. Therefore, the fluorescence intensity of DCF is proportional to the amount of intracellular ROS.

Human dermal fibroblasts (HS68), in 24-well microplates, and dispensed into 2.5 × 10 ⁴ cells per well min, media and 37 ° C. containing 10% FBS, 5% CO ₂ conditions The cells were cultured in an incubator for 24 hours. After that, untreated group as a negative control group, 250 μM ascorbic acid (vitamin C) as a positive control group, hydrogen peroxide as a comparative control group, and neural stem cell culture medium (concentration 10%, 25%, 50%) as an experimental group. , 100%) was added and cultured for 24 hours. Subsequently, 25 μM DCFDA was added simultaneously and reacted at 37 ° C. for 45 minutes, followed by treatment with 50 μM TBHP (Tert-butyl hydrogen peroxide solution) and reacted at 37 ° C. for 1 minute to 5 minutes. Wash once with 1x phosphate buffered saline, add 100 μl of 1x phosphate buffered saline in each well, then use a fluorescent plate reader to excite 485 nm and emission wavelengths. (Emission) The fluorescence value at 528 nm was measured. The result is shown in FIG.

As a result, it was confirmed that when the neural stem cell culture medium was added, the ROS level was significantly lower than that in the oxidative damage induction group in which the ROS level was increased by hydrogen peroxide. It produces low levels of ROS despite being exposed to the same concentration of hydrogen peroxide due to increased activity of the antioxidant system in human dermal fibroblasts by pre-adding neural stem cell cultures. It means that it can be maintained.

Experimental example 9.2. Measurement of total antioxidant effect It was confirmed that in human dermal fibroblasts (HS68) to which neural stem cell culture medium was added in Experimental Example 9.1, cell death due to peroxide was reduced and intracellular ROS levels were also reduced. did. Therefore, in order to know the total antioxidant status of the neural stem cell culture medium, the negative control group (NC) and the adipose stem cell culture medium (concentration 10%, 25%, 50%, 100%), which are comparative control groups, are used. ), And the neural stem cell culture medium (concentration 10%, 25%, 50%, 100%) of the experimental group was measured by the Trolox equivalent antioxidant capacity method.

There are three categories of antioxidants: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbic acid, uric acid, GSH, vitamin E, etc.), and proteins (albumin, transferase, etc.). Trolox is used to standardize antioxidants, all other antioxidants are measured with Trolox equivalents. Measured using the Total Antioxidant Capacity Assay Kit, which can measure a combination of a small molecule antioxidant and either a protein or a small molecule, Cu ²⁺ ions are measured by both the small molecule and the protein, Cu ^+. Is converted to. Protein Mask can prevent the reduction of Cu ²⁺ by protein and analyze only low molecular weight antioxidants. The reduced Cu ⁺ ions are chelated with a colorimetric probe to provide a wide absorption peak of about 570 nm in proportion to the volume of the total antioxidant. Reduced 2,2'-azinobis (3-methylbenzothiazo-thiazoline-6-sulfonate (ABTS)), which is colorless at acidic pH, is available. Hydrogen peroxide oxidizes to bluish green ABTS. In the presence of antioxidants in the sample, ABTS discolors in proportion to their concentration, and the result of such a color change reaction is 570 nm. For the TAC measurement of the sample material, a standard curve was created using trolox as a standard reagent. Trollox is a typical widely used measure of total antioxidant capacity. Standard reagent, TAC activity is expressed as mM Trolox equivalent.

In a 96-well plate, mix Cu ²⁺ Reagent, sample and Protein mask, put in 200 μl, react with an orbital shaker for 90 minutes under cancer conditions, and then irradiate with absorbance at 570 nm to measure. did. The result is shown in FIG.

As a result, it was confirmed that the scavenging activity of ABTS radicals increased in a concentration-dependent manner when the neural stem cell culture medium was treated as compared with the negative control group and the adipose stem cell culture medium.

From this, it was found that the neural stem cell culture medium has an antioxidant effect on the skin.

Experimental example 10. Checking 6-well plates of the effect of moisturizing and barrier strengthening skin in neural stem cell cultures, after per well human epidermal cells (HaCaT), it was cultured dispensed at each 1.0 × 10 ⁶ cells / well, It was replaced with a serum-free medium. After 24 hours, the negative control group (NC), the positive control group retinoic acid (Retinoic acid, RA, sigma-aldrich, R2625, 1uM), and the experimental group neural stem cell culture medium (concentration 10%, 25). %, 50%, 100%) were treated.

After 24 hours, RNA was separated from the cells and cDNA was synthesized. Experimental Example 1.2.2. The expression levels of Loricrin, Involucrin, Hyaluronan synthase-2 (HAS-2), and HAS-3 were determined by performing qRT-PCR in the same manner as described in 1. confirmed. At this time, the synthesized cDNA was prepared using the primer sets specific to each of them shown in Table 4 below.

FIG. 15a is a photograph of electrophoresis showing the presence or absence of expression of loricrin, involucrin, HAS-2 and HAS-3, and FIG. 15b shows the expression levels of loricrin, involucrin, HAS-2 and HAS-3. It is a graph.

As a result, it was confirmed that the expression levels of loricrin, involucrin, HAS-2 and HAS-3 were higher when the neural stem cell culture medium was treated than when the negative control group was treated.

From this, it was found that the neural stem cell culture solution is effective in moisturizing the skin and strengthening the skin barrier.

Experimental example 11. Secretome analysis of neural stem cell culture medium
RayBio Human Cyokine / Growth Factor Antibody (RayBiotech, Noncross, GA, USA) was used to confirm the components of the neural stem cell culture medium in the absence of serum.

The Array membrane was incubated with blocking buffer for 30 minutes at room temperature, followed by treatment with 2 ml of neural stem cell culture medium for 1 hour. After washing the membrane 5 times, the biotin-conjugated antibody was treated at room temperature for 1-2 hours and 2 ml of the substrate HRP-conjugated streptavidin was added. After 2 hours, the detection buffer was treated for 2 minutes, and the components of the neural stem cell culture medium were confirmed by iBright (CL1000 Imaging system, Thermo Scientific). In addition, the signal intensity was shown in a graph in the secritome analysis of the neural stem cell culture medium. The results are shown in Table 4, FIG. 16 and FIG.

As a result, it was confirmed that the neural stem cell culture medium contained a large number of various growth factors and cytokines.

Specifically, activin RIA / ALK-2, adiponectin-1, cordin-like 1, Csk, EDA-A2, EGF, bFGF, GDF3, GDF5, GDF15, GLO, which are proteins involved in skin regeneration and skin aging prevention. -1, IGFBP-2, IGFBP-3, IGFBP-7, inactive TGF-β bp1, MFG-E8, MMP-20, PDGF-AA, PECAM-1, Smad1, Smad4, Sonic Hedgehog (Shh N end) ), TGF-β 5, thrombospondin-1, Tie-2, VEGF and VEGF-C were found to be specifically compounded. In addition, MCP-1, TIMP-1, bFGF, insulin, inactive TGF-β bp1, Smad1, Smad4, TGF-β5, and thrombospondin-1, which are proteins involved in promoting collagen synthesis and improving wrinkles, are used. It is specifically contained. It also contains TGF-β 5, a protein known to be involved in whitening effects, and GLO-1, a protein associated with antioxidant effects. It was also found to contain proteins such as Axl, GRO, thrombospondin-1, and TGF-β 5, which are necessary for anti-inflammatory effect and prevention of autoimmune diseases.

Claims (12)

A cosmetic composition for skin improvement containing a culture solution of neural stem cells as an active ingredient.
The cosmetic composition, wherein the skin improvement is wound relief, wrinkle improvement, skin regeneration, increased skin elasticity, skin whitening, anti-inflammatory, anti-oxidant, skin moisturizing or skin barrier strengthening. The cosmetic composition according to claim 1, wherein the neural stem cells are derived from fetal brain tissue at 10 to 14 weeks. The cosmetic composition according to claim 1, wherein the neural stem cells are transduced with the MYC gene. The nerve stem cell culture medium includes actibin RIA / ALK-2, adiponectin-1, Axl, BIK, cordin-like 1, Csk, EDA-A2, epidermal growth factor (EGF), endostatin, and basic fibroblast. Cell growth factor (bFGF), galectin-3, growth differentiation factor-3 (GDF3), GDF5, GDF-15, gripecan 3, GRO, GLO-1, intercellular adhesion molecule 2 (ICAM-2), insulin-like growth factor Binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15 Rα, IL-20 Rβ, insulin, inactive TGF-β bp1 , Monosphere Growth Protein-1 (MCP1), MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, Thrombocytopenic Cell Adhesion Molecular 1 (PECAM-1), S100A10, sFRP-4, sgp130, Mothers against decapentaplegic homolog 1 (Smad1), Smad4, Sonic Hedgehog (Shh N-terminal), tissue factor pathway inhibitor (TFPI), TGF-β5, thrombospondin-1, tyrosine-protein phosphorylase receptor (Tyrosine-protein kinase) It comprises one or more proteins selected from the group composed of receptor, Tie-2), metalloprotease inhibitor 1 (TIMP-1), vascular endothelial growth factor (VEGF) and VEGF-C. The cosmetic composition according to claim 1. The cosmetic composition according to claim 1, which promotes collagen synthesis in skin cells. The cosmetic composition according to claim 1, which inhibits the elastase activity of skin cells. The cosmetic composition according to claim 1, which inhibits melanin synthesis in skin cells. The cosmetic composition according to claim 1, which inhibits the tyrosinase activity of skin cells. The cosmetic composition according to claim 1, which suppresses the production of reactive oxygen species (ROS) in skin cells. The cosmetic composition according to claim 1, which promotes the differentiation of keratinocytes and the synthesis of hyaluronic acid. A quasi-drug composition for skin improvement containing a culture solution of neural stem cells as an active ingredient.
The skin improvement is a wound relief, wrinkle improvement, skin regeneration, increased skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing or skin barrier strengthening. .. A pharmaceutical composition containing a culture solution of neural stem cells as an active ingredient for the treatment of skin wounds, skin regeneration, skin anti-inflammatory, treatment or prevention of atobi or autoimmune diseases.