CN112107535B - Composition for improving skin comprising neural stem cell culture liquid as active ingredient - Google Patents
Composition for improving skin comprising neural stem cell culture liquid as active ingredient Download PDFInfo
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- CN112107535B CN112107535B CN202010568825.9A CN202010568825A CN112107535B CN 112107535 B CN112107535 B CN 112107535B CN 202010568825 A CN202010568825 A CN 202010568825A CN 112107535 B CN112107535 B CN 112107535B
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Abstract
The present invention relates to a composition for improving skin comprising a neural stem cell culture broth as an active ingredient, the composition for improving skin according to the present invention exhibits effects of relieving wounds, improving wrinkles, skin regeneration, increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or enhancing skin barrier, and contains a plurality of proteins related thereto, thus improving skin with excellent effects, and by using immortalized neural stem cells, the composition and concentration of active factors in the culture broth are maintained constant even if the neural stem cells are repeatedly cultured, thus being effectively used as a raw material of functional cosmetics or medicines having uniform efficacy.
Description
Technical Field
The present invention relates to a composition for improving skin, which comprises a neural stem cell culture fluid as an active ingredient.
Background
Stem cells are known to participate in biological actions in humans by promoting microenvironment regulation of damaged tissues such as angiogenesis, anti-inflammatory actions, immunomodulatory actions, and the like. This biological effect is caused by liberating from stem cells various anti-inflammatory cytokines, apoptosis-inhibiting substances, and cell survival-promoting substances that promote the protection and regeneration of damaged tissues. This is known as paracrine effect.
Meanwhile, korean patent publication No. 10-0848056 discloses a method of inhibiting melanin synthesis using adipose-derived stem cell culture broth as mesenchymal stem cells, korean patent publication No. 10-2009-016659 discloses a whitening cosmetic composition comprising umbilical cord blood-derived mesenchymal stem cell culture broth, but has not been disclosed on the effects of relieving skin wound, improving skin wrinkles, skin regeneration, increasing skin elasticity, skin whitening, skin anti-inflammatory, skin antioxidant, skin moisturizing or enhancing skin barrier of ectodermal-derived neural stem cell culture broth.
Disclosure of Invention
Technical problem
The present invention is directed to a cosmetic composition for improving skin, which comprises a neural stem cell culture broth as an active ingredient, and which exhibits effects of relieving wounds, improving wrinkles, skin regeneration, increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or enhancing skin barrier.
Another object of the present invention is to provide a pharmaceutical external composition for improving skin, which comprises the neural stem cell culture broth as an active ingredient.
Still another object of the present invention is to provide a pharmaceutical composition for treating skin wounds, skin regeneration, skin anti-inflammatory, or treating or preventing autoimmune diseases, which comprises the neural stem cell culture broth as an active ingredient.
The inventors of the present invention have made a great deal of efforts to develop a composition for improving skin comprising a neural stem cell culture broth, and as a result, have confirmed that a culture broth of neural stem cells derived from brain tissue exhibits effects of relieving wounds, improving wrinkles, skin regeneration, increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or enhancing skin barrier, and contains a plurality of proteins related thereto, thereby completing the present invention.
Technical proposal
In order to achieve the above object, in one aspect, the present invention provides a cosmetic composition for relieving wounds, improving wrinkles, skin regeneration, increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or enhancing skin barrier, comprising a neural stem cell culture broth as an active ingredient.
In another aspect, the present invention provides a pharmaceutical external composition for improving skin, comprising the neural stem cell culture broth as an active ingredient.
In still another aspect, the present invention provides a pharmaceutical composition for treating skin wound, skin regeneration, skin anti-inflammation, or treating or preventing autoimmune disease, comprising the neural stem cell culture broth as an active ingredient.
In yet another aspect, the invention provides a cosmetic, pharmaceutical external or pharmaceutical composition comprising a neural stem cell culture broth comprising a polypeptide selected from the group consisting of Activin RIA/ALK-2 (Activin RIA/ALK-2), adiponectin 1 (adionectin-1), axl, BIK, tenascin-1 (chord-like 1), csk, EDA-A2, epidermal growth factor (Epidermal growth factor, EGF), endostatin, basic fibroblast growth factor (bFGF), galectin 3 (galectin-3), growth differentiation factor-3 (GDF-3), GDF5, GDF-15, glypican 3 (Glypican 3), GRO, GLO-1, intercellular adhesion molecule 2 (ICAM-2), insulin-like growth factor binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-7, IL-1F6, IL-1F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1 (Monocyte Chemoattractant Protein-1, MCP 1), MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1 (Platelet Endothelial Cell Adhesion Molecule, PECAM-1), S100A10, sFRP-4, sgp130, DPP 35, sod 1, soh pathway (Hedgehog pathway), soh 42, soh pathway 84, TFPI), TGF-beta 5, thrombospondin 1 (thrombin-1), tyrosine kinase receptor (Tyrosine-protein kinase receptor, tie-2), metalloprotease tissue inhibitor 1 (Metalloproteinase inhibitor, TIMP-1), vascular Endothelial Growth Factor (VEGF), and VEGF-C.
In yet another aspect, the present invention provides a cosmetic, pharmaceutical external or pharmaceutical composition for the above-described use obtained by culturing immortalized neural stem cells (immortalized Neural Stem Cells, imNSC).
Advantageous effects
The neural stem cell culture fluid according to the present invention exhibits effects of relieving wounds, improving wrinkles, skin regeneration, increasing skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturizing, or enhancing skin barrier, and thus can be effectively applied to cosmetics, external medicine, and medicines. In addition, by using immortalized neural stem cells, the composition and concentration of the effective factors in the culture fluid remain constant even if the neural stem cells are repeatedly cultured, and thus can be effectively used as a raw material for functional cosmetics or medicines having uniform efficacy.
Drawings
Fig. 1a shows a photomicrograph of an X40 magnification and an X100 magnification of a neural stem cell according to an embodiment of the present invention, fig. 1b shows a graph of a growth rate of the neural stem cell, and fig. 1c shows a graph of a time at which the neural stem cell becomes diploid under different subcultures.
Fig. 2a shows the results of flow cytometry analysis representing the expression amounts of nestin, PAX6, and SOX2 in neural stem cells according to one embodiment of the present invention, and fig. 2b shows a fluorescence micrograph of neural stem cells having the expression of nestin, PAX6, and SOX 2.
FIG. 3 shows the results of isolating mRNA from neural stem cells according to one embodiment of the present invention and confirming whether neural stem cell-specific gene markers are expressed under different subcultures (p 8, p10, p 12).
Fig. 4 shows a microscopic photograph of whether or not a specific marker is expressed in each differentiated cell by immunofluorescent staining after differentiating neural stem cells according to an embodiment of the present invention into neural cells (neuron), astrocytes (astrocyte), and oligodendrocytes (oligodendrocyte), respectively.
Fig. 5a and 5b show graphs representing cell viability of human epidermal cells (HaCaT) and human dermal fibroblasts (HS 68) at different treatment concentrations of neural stem cell culture fluid and adipose stem cell culture fluid according to one embodiment of the present invention.
Fig. 6a to 6d show photomicrographs (6 a,6 c) showing wound recovery in human epidermal cells (HaCaT) and human dermal fibroblasts (HS 68) and graphs (6 b,6 d) comparing wound recovery rates at different treatment concentrations of neural stem cell culture fluid and adipose stem cell culture fluid according to one embodiment of the present invention.
Fig. 7a shows an electrophoresis photograph for confirming whether COL1A1 is expressed after treating human dermal fibroblasts (HS 68) with different concentrations of the neural stem cell culture solution according to one embodiment of the present invention, fig. 7b shows a graph comparing COL1A1 content, fig. 7c shows an electrophoresis photograph for confirming whether COL3A1 is expressed at different treatment concentrations of the neural stem cell culture solution and the adipose stem cell culture solution according to one embodiment of the present invention, and fig. 7d shows a graph comparing COL3A1 content.
FIG. 8 shows a graph comparing PICP expression levels in human dermal fibroblasts (HS 68) at different treatment concentrations of neural stem cell culture fluid and adipose stem cell culture fluid according to an embodiment of the present invention.
FIG. 9 shows a graph comparing elastase inhibitory activity in human dermal fibroblasts (HS 68) at different treatment concentrations of neural stem cell culture fluid and adipose stem cell culture fluid according to one embodiment of the invention.
Fig. 10a to 10d illustrate effects of inhibiting melanin synthesis at different treatment concentrations of a neural stem cell culture fluid and an adipose stem cell culture fluid according to one embodiment of the present invention. Fig. 10a and 10b show the case of treatment with only the neural stem cell culture solution and the adipose stem cell culture solution, and fig. 10c and 10b show the results of treatment with the neural stem cell culture solution and the adipose stem cell culture solution after treatment with the melanocyte stimulating hormone.
FIG. 11 shows a graph comparing the activity rates of the primary enzyme involved in melanin biosynthesis process, i.e., tyrosinase (Tyrosinase), at different treatment concentrations of neural stem cell culture broth or adipose stem cell culture broth according to one embodiment of the present invention.
Fig. 12a to 12d show the results of treating human dermal fibroblasts (HS 68) with Lipopolysaccharide (LPS) to induce an inflammatory response, and then treating with a neural stem cell culture solution and an adipose stem cell culture solution at different concentrations according to one embodiment of the present invention. FIG. 12a is an electrophoresis photograph showing whether or not IL-6, IL-1β and COX-2 are expressed, FIG. 12b is a graph comparing the expression levels of COX-2, FIG. 12c is a graph comparing the expression levels of IL-1β, and FIG. 12d is a graph comparing the expression levels of IL-6.
FIG. 13 shows a graph comparing DCF-DA fluorescence values for measuring changes in the concentration of intracellular Reactive Oxygen Species (ROS) in human dermal fibroblasts (HS 68) at different treatment concentrations of neural stem cell culture solution and adipose stem cell culture solution according to one embodiment of the present invention.
Fig. 14 shows the results of comparing Trolox (water-soluble vitamin E) equivalent antioxidant capacity in human dermal fibroblasts (HS 68) at different treatment concentrations of neural stem cell culture broth and adipose stem cell culture broth according to one embodiment of the present invention.
Fig. 15a and 15b show the results of treatment of human epidermal cells (HaCaT) with neural stem cell culture broth and retinoic acid according to an embodiment of the present invention. FIG. 15a is an electrophoresis photograph showing whether or not pocket A protein, endo-coat protein, HAS-2 and HAS-3 are expressed, and FIG. 15b is a graph comparing the expression amounts of pocket A protein, endo-coat protein, HAS-2 and HAS-3.
Fig. 16 shows the analysis results of protein components secreted from the neural stem cell culture broth according to one embodiment of the present invention.
FIG. 17 shows a graph of measuring the expression intensity of a protein secreted from a neural stem cell culture broth according to one embodiment of the present invention.
Detailed Description
In one aspect, the present invention relates to a composition for improving skin comprising a neural stem cell culture broth as an active ingredient, wherein the improvement of skin is a relief of wounds, an improvement of wrinkles, skin regeneration, an increase of skin elasticity, skin whitening, anti-inflammatory, anti-oxidation, skin moisturizing, or an enhancement of skin barrier.
In one exemplary embodiment of the present invention, the neural stem cell may be an immortalized neural stem cell. Specifically, the neural stem cells may be immortalized neural stem cells obtained by transducing (transforming) an L-Myc expression vector to adult neural stem cells (Neural stem cells, NSCs) isolated from dead fetal brain tissue over 10 to 14 weeks.
The neural stem cells are preferably cultured in a medium having a definite composition and concentration, and the culture solution is obtained by centrifugation and filtration during the culture of the neural stem cells.
After immortalizing and culturing the neural stem cells, the process of isolation is not limited to the method of the present invention and may be performed by a method generally used in the art. The neural stem cell culture solution may be obtained by centrifuging and filtering a supernatant during the subculture of the neural stem cells, and the neural stem cells may be obtained by a process of homogenizing (homogenesis) after isolating the cells.
From the result of the secretome analysis of the neural stem cell culture broth according to the present invention, it was confirmed that it contained the Activin RIA/ALK-2 (Activin RIA/ALK-2), adiponectin 1 (Adiponectin-1), axl, BIK, tenascin-1 (chord-like 1), csk, EDA-A2, epidermal growth factor (Epidermal growth factor, EGF), endostatin, basic fibroblast growth factor (bFGF), galectin 3 (galectin-3), growth differentiation factor-3 (GDF-3), GDF 5, GDF-15, glypican 3 (Glypican 3), GRO, GLO-1, intercellular adhesion molecule 2 (ICAM-2), insulin-like growth factor binding protein 2 (IGFBP-2), IGFBP-3, IGFBP-7, IL-1F6, IL-1F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1 (Monocyte Chemoattractant Protein-1, MCP 1), MFG-E8, MMP-20, NRG1Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1 (Platelet Endothelial Cell Adhesion Molecule, PECAM-1), S100A10, sFRP-4, sgp130, DPP 35, sod 1, soh pathway (Hedgehog pathway), soh 42, soh pathway 84, TFPI), TGF-beta 5, thrombospondin 1 (thrombin-1), tyrosine kinase receptor (Tyrosine-protein kinase receptor, tie-2), metalloprotease tissue inhibitor 1 (Metalloproteinase inhibitor, TIMP-1), vascular Endothelial Growth Factor (VEGF) and VEGF-C.
In one aspect of the invention, the neural stem cell culture broth may comprise proteins associated with skin regeneration and aging resistance, i.e., may comprise activin RIA/ALK-2, adiponectin 1, tenascin-like protein 1 (chord-like 1), csk, EDA-A2, EGF, bFGF, GDF3, GDF5, GDF15, GLO-1, IGFBP-2, IGFBP-3, IGFBP-7, latent TGF-beta binding protein 1, MFG-E8, MMP-20, PDGF-AA, PECAM-1, smad4, sonic hedgehog (Shh N-terminus), TGF-beta 5, thrombospondin 1, tie-2, VEGF, or VEGF-C.
In another aspect of the invention, the neural stem cell culture broth may comprise proteins involved in promoting collagen synthesis and improving wrinkles, i.e., may comprise MCP-1, TIMP-1, bFGF, insulin, latent TGF-beta binding protein 1, smad4, TGF-beta 5, or thrombospondin 1.
In still another aspect of the present invention, the neural stem cell culture broth may include TGF- β5 as a protein involved in the whitening effect.
In yet another aspect of the invention, the neural stem cell culture broth may comprise a protein required for anti-inflammatory effects and prevention of autoimmune diseases, i.e., may comprise Axl, GRO, thrombospondin 1, or TGF-beta 5.
In one exemplary embodiment of the present invention, when a wound occurs in skin tissue, the neural stem cell culture fluid may alleviate and treat the wound by regenerating skin cells.
In an exemplary embodiment of the present invention, the neural stem cell culture fluid may increase collagen synthesis and inhibit degradation of elastin by inhibiting elastase activity, thereby preventing skin aging. Specifically, the neural stem cell culture fluid increases collagen synthesis at the mRNA level and/or protein level, for example, by increasing the secretion amount of C-terminal protein of type I procollagen, thereby improving skin wrinkles, enhancing skin elasticity and regenerating skin, to inhibit skin aging.
In one exemplary embodiment of the present invention, the neural stem cell culture fluid has a skin whitening effect. Specifically, the neural stem cell culture fluid inhibits conversion of tyrosine to melanin by inhibiting synthesis of melanin in skin and inhibiting tyrosinase activity, thereby reducing the content of melanin in skin.
In one exemplary embodiment of the invention, the neural stem cell culture broth has an antioxidant effect of removing intracellular reactive oxygen species (reactive oxygen species, ROS).
In one exemplary embodiment of the invention, the neural stem cell culture broth has an anti-inflammatory effect of alleviating an inflammatory reaction. Specifically, when an inflammatory reaction occurs in skin cells or immune cells, the inflammatory reaction can be alleviated by reducing the expression levels of Cyclooxygenase 2 (COX-2), interleukin-1β (IL-1β), and interleukin-6 (IL-6 ).
In an exemplary embodiment of the present invention, the neural stem cell culture fluid has effects of moisturizing skin and enhancing skin barrier. Specifically, the neural stem cell culture solution can enhance skin barrier by increasing expression levels of paphiopedilum and endocape proteins to promote differentiation into keratinocytes, and can improve skin moisturization by increasing expression levels of HAS-2 and HAS-3 to promote synthesis of hyaluronic acid as a moisturizing factor.
Keratinocytes, which account for 95% of the epidermal cells, proliferate in the basal layer and pass through the upper stratum spinosum and stratum granulosum, eventually differentiating into flat-form, coreless keratinocytes. In this differentiation process, keratin microfibers are aggregated to form a keratin cell membrane by expressing papilin (loriciin) as a differentiation promoting factor in the granular layer and the stratum corneum of the epidermis, endo-protein (involucrin) as a differentiation promoting factor in the granular layer and the upper part of the stratum spinosum of the epidermis, and the like, thereby contributing to the enhancement of the barrier of the skin.
In addition, papilins, endo-proteins, and the like form natural moisturizing factors in the skin, thereby playing an important role in skin moisturizing, and the skin plays a role in skin moisturizing through various moisturizing factors such as Hyaluronic Acid (HA) and the like in addition to the natural moisturizing factors. HA is synthesized mainly by hyaluronate synthase (Hyaluronic acid synthase, HAs) of keratinocytes and fibroblasts and accumulates in the extracellular matrix.
The composition may be prepared into cosmetic dosage forms commonly used in the art, as needed.
The cosmetic composition may be prepared into, for example, a solution, suspension, emulsion, paste, gel, cream, emulsion, powder, soap, surfactant-containing cleanser, oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. In particular, the composition can be prepared into the dosage forms of skin softening lotion, nourishing cream, massage cream, essence, eye cream, face cleansing foam, face cleansing lotion, mask, spray or powder. In addition, when the formulation of the cosmetic composition is a paste, cream or gel, a carrier component selected from the group consisting of an animal oil, a vegetable oil, a wax, paraffin, starch, tragacanth (tragacanth), a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc, zinc oxide, and mixtures thereof may be included.
In addition, when the formulation of the cosmetic composition is a solution or emulsion, a carrier component selected from the group consisting of solvents, solvating agents, emulsifiers, and mixtures thereof may be included. Examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butanediol oil, glycerin fatty acid ester, polyethylene glycol, sorbitan fatty acid ester, a mixture thereof, and the like.
In addition, when the formulation of the cosmetic composition is a suspension, a carrier component selected from the group consisting of a liquid diluent such as water, ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, tragacanth, and mixtures thereof may be included.
The carrier component may be present in an amount of about 1 wt% to about 99.99 wt%, preferably about 80 wt% to about 90 wt%, based on the total weight of the cosmetic composition.
In another aspect, the present invention relates to a pharmaceutical external composition for improving skin, comprising the neural stem cell culture broth as an active ingredient, wherein the improvement of skin is a relief of wounds, an improvement of wrinkles, a skin regeneration, an increase of skin elasticity, skin whitening, anti-inflammatory or anti-oxidative.
The neural stem cell culture fluid is as described above.
In the present invention, the "medical external product" refers to a fiber, a rubber product or a product similar thereto which is used for the purpose of treating, alleviating, disposing or preventing a disease of a human or an animal, a product which is not an appliance or a machine and a product similar thereto which has little or no direct effect on a human body, a product which is not an appliance, a machine or a device among products which are used for the purpose of diagnosing, treating, alleviating, disposing or preventing a disease of a human or an animal, products which are not an appliance, a machine or a device among products used for the purpose of diagnosing, alleviating, disposing or preventing a disease of a human or an animal, and products which are not an appliance, a machine or a device among products used for the purpose of pharmacologically affecting the structure and function of a human or an animal, including skin external preparations and personal hygienic products.
When the composition according to the present invention is contained in a pharmaceutical external product for the purpose of improving skin or the like, the composition may be used by directly containing the composition, or may be used together with other pharmaceutical external product components, and may be used appropriately according to a usual method. The amount of the active ingredient to be mixed can be appropriately determined according to the purpose of use.
In still another aspect, the present invention relates to a pharmaceutical composition for treating skin wounds, skin regeneration, skin anti-inflammatory, or treating or preventing autoimmune diseases, comprising the neural stem cell culture broth as an active ingredient.
The neural stem cell culture fluid is as described above.
The neural stem cell culture fluid as an active ingredient may be contained in an amount of about 0.1 to about 90 wt%, specifically about 0.5 to about 75 wt%, and more specifically about 1 to about 50 wt%, based on the total weight of the pharmaceutical composition.
The pharmaceutical composition may comprise conventional and non-toxic pharmaceutically acceptable additives formulated into a formulation according to conventional methods. For example, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, diluent or excipient.
The pharmaceutical composition may be applied to the skin. The dosage form of the pharmaceutical composition can be a skin external preparation dosage form. The external preparation for skin is not particularly limited, and for example, can be prepared into the form of an ointment, a emulsion, a spray, a patch, a cream, a powder, a suspension, a patch or a gel.
In yet another aspect, the present invention provides a use of a neural stem cell culture fluid in the preparation of a cosmetic for improving skin.
In yet another aspect, the present invention provides a use of a neural stem cell culture fluid in the preparation of a pharmaceutical external product for improving skin.
In yet another aspect, the invention provides the use of a neural stem cell culture broth in the manufacture of a medicament for treating a skin wound, skin regeneration, skin anti-inflammatory, treating or preventing atopic dermatitis or autoimmune disease.
Examples
The above will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of describing the present invention, and the scope of the present invention is not limited thereto.
Preparation example 1 preparation of adipose-derived stem cell culture solution
After 3 or 4 times subculture of adipose-derived stem cells purchased from Promocell (Cat#C-12978), DMEM/Low (Hyclone Co.), 10% FBS, 10ng/mL bFGF (basic Fibroblast Growth Factors, basic fibroblast growth factor) were added and further cultured. When the confluence (confluency) reached 70% to 80%, the medium was replaced with low-sugar DMEM (phenol-red free Low DMEM) free of phenol red, and a adipose stem cell culture broth was obtained during the 48 hours of culture.
Preparation example 2 preparation of neural Stem cells and neural Stem cell culture solution
The L-Myc expression vector [ pMXs-hu-L-Myc ] was transduced (transferred) into Neural Stem Cells (NSC) (LM-NSC 008, city of Hope institute) extracted from fetal brain tissue for 10 to 14 weeks, and immortalized neural stem cells (immortalized neural stem cell, imNSC) were prepared by a selection process (Okita et al, 2013).
Preparation of a composition comprising phenol-free DMEM/F12 (Gibco Co.), 2% supplement (reduced glutathione (Glutathione reduced), superoxide dismutase (Superoxide Dismutase), human Holo-transferrin, L-Carnitine, D + Galactose (D) + -galactose), putrescine (Putrescine), linoleic acid (Linoleic acid), linolenic acid (Lin)The culture medium was used to culture immortalized neural stem cells after the culture of olenic acid), vitamin A acetate (Retinol acetate), DL-alpha tocopherol (vitamin E), DL-alpha tocopherol acetate (DL-alpha tocopherol acetate)), 20ng/mL bFGF and 20ng/mL EGF (Epidermal Growth Factor ). The culture solution is obtained during the process of subjecting it to subculture, and the non-adherent cells are removed after the culture to obtain neural stem cells. Further, the obtained culture broth was subjected to centrifugal separation, and the supernatant was filtered with a 0.22 μm filter to obtain a neural stem cell culture broth.
Experimental example 1 characterization of neural Stem cells
Experimental example 1.1. Observation of growth rate of neural Stem cells
The neural stem cells obtained in preparation example 2 were observed with a microscope at X40 magnification and X100 magnification. Then, the cell growth rate of the neural stem cells and the timing of diploid formation under different subcultures were measured and represented graphically. FIG. 1a is a photomicrograph, FIG. 1b is a graph of cell growth rate, and FIG. 1c is a graph showing the time at which cells become diploid.
Experimental example 1.2. Confirmation of expression of neural stem cell-specific Gene markers
The gene expression pattern of the neural stem cells obtained in preparation example 2 was confirmed by a flow cytometry method and a real-time polymerase chain reaction method (qRT-PCR).
Experimental example 1.2.1 confirmation of Gene expression Using flow cytometry
When the confluency of the neural stem cells obtained in preparation example 2 reached 80% to 90%, the medium was removed and washed with phosphate buffer (phosphate buffered saline, PBS). Then, using
Cells were isolated and washed with PBS. The cell number was counted to make 1×10 6 After cells/mL, the cells were fixed with 4% p-formaldehyde at 4℃for 20 minutes. The cells after fixation were washed with PBS The antibody was then permeabilized by treatment with PBS diluted with 0.01% Triton X-100 (surfactant), followed by a permeabilization (permeization) procedure for 10 minutes at room temperature, followed by washing with PBS.
Then, FACS buffer was added to the cells to react NESTIN-PE, PAX6-PE, SOX2-PE antibodies, and then the expression of NESTIN (NESTIN), PAX6, and SOX2 was confirmed by fluorescence flow cytometry (FACS). FIG. 2a is a graph showing the expression amounts of nestin, PAX6 and SOX2, 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 preparation example 2 significantly 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 preparation example 2 by using phenol/chloroform. The extracted RNA was subjected to reverse transcription to synthesize cDNA. qRT-PCR was performed on the Applide Biosystems 700 sequence system (foster, calif.) to analyze the gene expression level of cDNA. At this time, the synthesized cDNA was performed using each specific primer set shown in Table 1 below. qPCR was repeated 30 times in cycles of 10 minutes at 95℃for 15 seconds at 95℃and 1 minute at 56 ℃. mRNA levels were normalized by beta-actin values and compared. The results are shown in FIG. 3.
[ Table 1 ]
As a result, it was confirmed that SOX1, SOX2, PAX6 and nestin were significantly expressed in the neural stem cells.
From this, it was found that the cells obtained in preparation example 2 had characteristics of neural stem cells since SOX1, SOX2, PAX6 and nestin, which were specific gene markers of neural stem cells, were expressed.
Experimental example 2 confirmation of differentiation ability of neural Stem cells
After the neural stem cells obtained in preparation example 2 were differentiated into neural cells (neuron), astrocytes (astrocytes) and oligodendrocytes (oligodendrocyte), respectively, the following immunofluorescent staining method was performed to confirm the expression of each intracellular differentiation-related protein.
First, neural stem cells maintained in 4-well chamber slides were fixed with 4% p-formaldehyde at 37 ℃ for 20 minutes, and then washed 2 times with PBS containing calcium and magnesium ions. Thereafter, treatment was performed with PBS diluted with 0.1% Triton X-100 (surfactant) for 10 minutes, followed by washing again with PBS. To prevent detection of non-specific antibodies due to their attachment, bovine serum albumin (Bovine Serum Albumin, BSA) was diluted to 5% in 0.1% Triton X-100/PBS, then added to the sample and reacted for 1 hour.
The target antibody and the dilution amount according to the protein are shown in Table 2 below, depending on the type of the primary antibody added to the cells. After the addition of the primary antibody, the reaction was carried out in a shaker at 4℃for 16 hours. The secondary antibody was selected and used according to the host (host) and wavelength of the primary antibody, and specifically, the coat Anti-Mouse IgG-conjugated Alexa 488 (abcam Co., cat.no. ab150113, 1000-fold dilution), and Anti-Mouse FITC (eBioscience Co., cat.no. ab150113, 1000-fold dilution) were used.
In addition, nuclei were stained with DAPI (1,000 fold dilution). The stained samples were photographed using a fluorescence microscope. The nuclei of the nerve cells are represented by DAPI (blue), MAP2 (green), GFAP (green), O1 (green). The results are shown in FIG. 4.
[ Table 2 ]
| Differentiated cells | First antibody | Second antibody |
| Nerve cell (neuron) | MAP2 resistance | Goat anti-mouse IgG Alexa 488 |
| Astrocytes (astrocyte) | anti-GFAP | Goat anti-mouse IgG Alexa 488 |
| Oligodendrocytes (oligocodendrocyte) | anti-O1 | Anti-mouse FITC |
As a result, it was confirmed that MAP2, GFAP and O1 were expressed in neural cells, astrocytes and oligodendrocytes differentiated from neural stem cells, respectively.
From this, it was found that the neural stem cells obtained in preparation example 2 had the ability to differentiate into neural cells, astrocytes and oligodendrocytes.
Experimental example 3 evaluation of cytotoxicity and cell proliferation Effect of neural Stem cell culture fluid
The following experiments were performed using human epidermal cells (HaCaT) and human dermal fibroblasts (HS 68) to confirm cytotoxicity and cell proliferation effects of the neural stem cell culture solution obtained in preparation example 2.
HaCaT and HS68 were each run at 1 kW 10 3 
Is divided into 96-well plates and carried out for 24 hoursAfter the culture of (a) was performed, a negative control group (N.C; no treatment group), a fat stem cell culture solution having a concentration of 10%, 25%, 50%, 100% as a comparative control group, and a neural stem cell culture solution having a concentration of 10%, 25%, 50%, 100% as an experimental group were used.
After treatment with the culture solution, the absorbance at 450nm was measured at the same time on days 1, 2 and 3 by using CCK8 (Dojindo, CK 04-13) reagent, and the change in cell activity was confirmed. The viability of human epidermal cells is shown in FIG. 5a, and the viability of human dermal fibroblasts is shown in FIG. 5 b.
As a result, it was confirmed that when human epidermal cells and human dermal fibroblasts were treated with the neural stem cell culture solution, most of the cells exhibited excellent survival rates.
Experimental example 4 confirmation of the effect of neural Stem cell culture solution on alleviating skin wounds
Human epidermal cells (HaCaT) were processed to hIg 10 per well 3 5 cells, human dermal fibroblasts (HS 68) 2 to 10 per well 5 cells were aliquoted in 24 well plates and cultured until confluence reached 100%. After scraping the well in the middle of the well with a 1000P white tip (white tip) to form a wound (wound), treatment was performed with a negative control group (N.C), a adipose stem cell culture solution (comparative control group; concentrations 10%, 25%, 50%, 100%) and a neural stem cell culture solution (experimental group; concentrations 10%, 25%, 50%, 100%).
Wound areas of human epidermal cells and human dermal fibroblasts were measured at the time of culture treatment and after 24 hours, respectively, to confirm recovery rates. At this time, a microscopic photograph of cells stained with crystal violet (crystal violet) reagent after 24 hours is shown together. Fig. 6a and 6b are photomicrographs showing the wound recovery rate of human epidermal cells (HaCaT), and fig. 6c and 6d are photomicrographs showing the wound recovery rate of human dermal fibroblasts (HS 68).
As a result, it was confirmed that, when the human epidermal cells and human dermal fibroblasts were treated with the neural stem cell culture solution, the wound was recovered rapidly as compared with the negative control group and the adipose stem cell culture solution.
Experimental example 5 confirmation of collagen Synthesis Effect of neural Stem cell culture fluid
Experimental example 5.1 analysis of collagen Gene expression Using RT-PCR
Human dermal fibroblasts (HS 68) were seeded at 1.0 mW 10 per well 5 cell amounts were plated in 6-well plates and cultured for 24 hours. After addition of a negative control group (N.C), an adipose-derived stem cell culture solution (comparative control group; concentration: 10%, 25%, 50%, 100%) and a neural stem cell culture solution (experimental group; concentration: 10%, 25%, 50%, 100%) and cultivation for 24 hours, qRT-PCR was performed by the method described in experimental example 1.2.2 using each of the specific primer sets described in Table 3 below to measure the expression or absence and the expression amount of collagen genes (COL 1A1 and COL3A 1).
[ Table 3 ]
Fig. 7a and 7b are diagrams showing an electrophoresis photograph of whether COL1A1 is expressed or not and measuring the expression level thereof, and fig. 7c and 7d are diagrams showing an electrophoresis photograph of whether COL3A1 is expressed or not and measuring the expression level thereof.
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 used for treatment, as compared with the negative control group and the adipose stem cell treatment.
Experimental example 5.2 evaluation of ability to promote collagen Synthesis Using ELISA
Human dermal fibroblasts (HS 68) were seeded at 1.0 mW 10 per well 5 cell amounts were plated in 6-well plates and cultured for 24 hours. The culture medium was subjected to 48 hours of culture after addition of a negative control group (N.C), a positive control group (TGF-. Beta.), a fat stem cell culture solution (comparative control group; concentration: 10%, 25%, 50%, 100%) and a neural stem cell culture solution (experimental group: concentration: 10%, 25%, 50%, 100%)Centrifugal separation to obtain supernatant. The degree of synthesis of procollagen was analyzed by using type I procollagen carboxy terminal peptide (Procollagen Type I C-peptide, PICP) EIA kit (Takara Corp., cat. #MK101) to confirm the ability to promote collagen synthesis. The results are shown in FIG. 8.
As a result, compared with the negative control group and the adipose-derived stem cell culture fluid (comparative control group; 10%, 25%, 50%, 100%), the expression level of PICP was higher in the case of treatment with neural stem cells and also higher than in the case of the conventional known positive control group.
From these results, the neural stem cell culture fluid has an effect of improving skin wrinkles and increasing skin elasticity.
Experimental example 6 confirmation of the effect of neural Stem cell culture fluid on inhibition of elastase Activity
The extent of inhibition of elastase activity according to the neural stem cell broth treatment was measured by elastase activity inhibition assay (Elastase inhibition assay).
Specifically, human dermal fibroblasts (HS 68) were seeded at 1.0 hx 10 per well 5 cell amounts were plated in 6-well plates and cultured for 24 hours. Thereafter, 50. Mu.l of 0.6unit/mL porcine pancreatic elastase (Porcine Pancreatic elastase, PPE; in 0.2M Tris buffer, pH 8.0), 50. Mu.l of each sample, 100. Mu.l of 1mg/mL N-Succ- (Ala) 3-nitroanilide (SANA; DMSO) were added and absorbance at 410nm was measured over 10 minutes. In this case, the untreated group was used as a negative control group (N.C), and sodium phosphonodipeptide (Phosphoramidon disodium salt, PPDS;2.5 mM) was used as a positive control group, adipose stem cells (at concentrations of 10%, 25%, 50%, 100%) were used as a comparative control group, and neural stem cell culture solutions (at concentrations of 10%, 25%, 50%, 100%) were used as experimental groups, respectively. The results are shown in FIG. 9.
As shown in fig. 9, the inhibition activity of elastase increased in a concentration-dependent manner in the case of treatment with the neural stem cell culture solution, as compared with the negative control group and the comparative control group treated with the adipose stem cell culture solution.
From this, it is clear that the neural stem cell culture fluid inhibits degradation of elastin, and thus has an effect of increasing skin elasticity.
Experimental example 7 confirmation of skin whitening Effect of neural Stem cell culture solution
Experimental example 7.1 effects of neural Stem cell culture solution on inhibiting melanin synthesis
Rodent B16F10 melanoma cells were at 1.0 to 10 per well 5 cell amounts were plated in 6-well plates, washed with PBS for a second time to remove non-adherent cells, and then treated with negative control (N.C), 10Mm arbutin (Albutin) having an effect of inhibiting melanogenesis in cells as a positive control, adipose stem cell culture fluid (ASC; concentration of 10%, 25%, 50%, 100%) as a comparative control, and neural stem cell culture fluid (NSC; concentration of 10%, 25%, 50%, 100%) as an experimental group. After 48 hours, cells were washed with PBS and obtained with Trypsin (Trypsin-EDTA).
After that, 1ml of a 1N NaOH+50% DMSO solution was added to each well, and after the cells were lysed at 95 ℃, the absorbance of melanin was measured at 405nm by using a microplate reader (micro plate reader) to measure the secretion amount and intracellular content of melanin. Fig. 10a shows a photograph of whether melanin is synthesized in melanoma cells when treated with an adipose stem cell culture solution or a neural stem cell culture solution, and fig. 10b shows a graph representing the content of melanin.
Further, after treatment with 100nM melanocyte stimulating hormone (alpha-MSH), the amount of melanin secretion and intracellular content were measured in the same manner as described above. Fig. 10c shows a photograph of whether melanin is synthesized in melanoma cells when treated with an alpha-MSH and then treated with an adipose stem cell culture solution or a neural stem cell culture solution, and fig. 10d shows a graph showing the melanin content.
As a result, it was confirmed that the effect of inhibiting melanin synthesis was exhibited in proportion to the concentration in the case of treatment with the neural stem cell culture solution, and the effect of inhibiting melanin synthesis was more superior to the case of treatment with the negative control group and the adipose stem cell culture solution.
Experimental example 7.2 effects of neural Stem cell culture fluid on inhibiting tyrosinase Activity
To a 96-well plate, 220. Mu.l of 0.1M phosphate buffer (pH 6.8,0.1% PMSF) and 20. Mu.l of neural stem cell culture broth were added, followed by 50. Mu.l of 1.5mM Tyrosine (Tyrosine) and 20. Mu.l of Tyrosinase (Tyrosinase) and the reaction was performed, and then absorbance at 492nm was measured by an enzyme-labeled instrument. At this time, the untreated group was treated as a negative control group (N.C), 1mM arbutin as a positive control group, adipose stem cell culture solutions (at concentrations of 10%, 25%, 50%, 100%) as a comparative control group, and neural stem cell culture solutions (at concentrations of 10%, 25%, 50%, 100%) as experimental groups. The results are shown in FIG. 11.
As a result, it was confirmed that the effect of inhibiting tyrosinase activity was more excellent in the case of the treatment with the neural stem cell culture solution than in the negative control group and the comparative control group treated with the adipose stem cell culture solution.
From this, it was found that the neural stem cell culture solution has a skin whitening effect by inhibiting melanin synthesis and tyrosinase activity.
Experimental example 8 confirmation of the anti-inflammatory Effect of neural Stem cell culture fluid
Human dermal fibroblasts (HS 68) were seeded at 2.5 to 10 rounds per well 5 cell amounts were plated in 6-well plates and cultured until confluence reached 80% and then replaced with serum-free medium. After 24 hours, the cells were treated with lipopolysaccharide (LPS; 20. Mu.g/mL), negative control (N.C), adipose stem cell cultures (10%, 25%, 50%, 100%) as comparative control, and neural stem cell cultures (10%, 25%, 50%, 100%) as experimental groups.
After 24 hours, RNA was isolated from the cells to synthesize cDNA, and qRT-PCR was performed in accordance with the method described in Experimental example 1.2.2 to confirm the expression levels of COX-2, IL-1. Beta. And IL-6. FIG. 12a is an electrophoretically photograph showing whether or not IL-6, IL-1β and COX-2 are expressed, and FIGS. 12b to 12d are graphs showing the expression amounts of COX-2, IL-1β and IL-6.
When treated with the neural stem cell culture solution, the expression levels of COX-2, IL-1β and IL-6 were lower than those in the negative control group and the adipose stem cell treatment.
From these results, it was found that the neural stem cell culture fluid 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 eliminating intracellular Reactive Oxygen Species (ROS)
The following experiment was performed using an ROS assay kit (Abcam Inc.) containing carboxy-H2 DCFDA to confirm the effect of neural stem cell culture broth on the production of intracellular reactive oxygen species (reactive oxygen species; ROS).
DCFH-DA readily penetrates the cell membrane and diffuses into the cell, is hydrolyzed to DCFH, which loses fluorescence by intracellular esterases, and then rapidly oxidizes to DCF, which exhibits high fluorescence, in the presence of ROS. Thus, the fluorescence intensity of DCF is proportional to the amount of ROS in the cell.
Human dermal fibroblasts (HS 68) were seeded at 2.5 to 10 rounds per well 4 cell mass was plated in 24-well microplates and cultured in a medium containing 10% FBS at 37℃and 5% CO 2 The culture was performed in a conditioned incubator for 24 hours. Thereafter, the untreated group was used as a negative control group, 250. Mu.M ascorbic acid (Vitamin C) was added as a positive control group, hydrogen peroxide was added as a comparative control group, and neural stem cell culture solution (at concentrations of 10%, 25%, 50%, 100%) was added as an experimental group, and culture was performed for 24 hours. Subsequently, 25. Mu.M DCFDA was added simultaneously, and after a reaction was carried out at 37℃for 45 minutes, 50. Mu.M TBHP (Tert-Butyl Hydrogen Peroxide Solution, tert-butyl hydroperoxide solution) was used for treatment, and a reaction was carried out at 37℃for 1 to 5 minutes. After 1 washing with 1 Xphosphate buffered saline, 100. Mu.l of each well of 1 Xphosphate buffered saline was added, respectively, and fluorescence values were measured at excitation wavelength (excitation) 485nm and emission wavelength (emission) 528nm using a fluorescence microplate reader. The results are shown in FIG. 13.
As a result, it was confirmed that ROS levels were significantly lower in the case of the addition of the neural stem cell culture medium, compared with the oxidative damage-induced group in which ROS levels were increased by hydrogen peroxide. This means that by pre-adding the neural stem cell culture broth, even if exposed to the same concentration of hydrogen peroxide due to the increase in the activity of the antioxidant system in human dermal fibroblasts, a lower level of ROS can be maintained.
Experimental example 9.2 measurement of Total antioxidant Effect
In experimental example 9.1, it was confirmed that apoptosis by peroxide was reduced and intracellular ROS levels were also reduced in human dermal fibroblasts (HS 68) to which the neural stem cell culture solution was added. Thus, TAC was measured according to the Trolox equivalent antioxidant capacity (Trolox equivalent antioxidant capacity) method for the negative control group (N.C), the adipose stem cell culture broth (concentration of 10%, 25%, 50%, 100%) as the comparative control group, and the neural stem cell culture broth (concentration of 10%, 25%, 50%, 100%) as the experimental group to confirm the total antioxidant capacity of the neural stem cell culture broth (Total antioxidant status).
Antioxidants are of three types: enzyme systems (GSH reductase, catalase, peroxidase, etc.), small molecules (ascorbic acid, uric acid, GSH, vitamin E, etc.), and proteins (albumin, transferrin, etc.). Trolox was used to normalize antioxidants, all other antioxidants were measured as Trolox equivalents. The measurement was performed using a total oxidation capability assay kit (Total Antioxidant Capacity Assay Kit) capable of measuring a combination of a small molecule antioxidant and a protein or a small molecule, both of which are Cu 2+ Ion over-conversion to Cu + . Protein Mask (Protein Mask) can prevent Cu caused by Protein 2+ And thus only small molecule antioxidants were analyzed. Reduced Cu + Ions are sequestered by the colorimetric probe (probe) and a broad absorption peak occurs at about 570nm in proportion to the total antioxidant capacity. Colorless reduced 2,2 '-diazabis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (2, 2' -azinobis (3-ethyllb)Enzothiazo-thiazoline-6-sulfonate); ABTS) is oxidized by hydrogen peroxide to a bluish green ABTS. If an antioxidant substance is present in the sample, ABTS is decolorized in proportion to these concentrations, and the result of this color change reaction is determined by absorbance at 570 nm. A standard curve was made using Trolox as a standard reagent to determine TAC of the sample substance. Trolox was used as a representative standard reagent widely used to determine total antioxidant capacity, and TAC activity was expressed in mM Trolox equivalent (mM Trolox equivalent).
Cu is added with 2+ Reagents and samples were mixed with the protein mask and added to 200 μl in a 96-well plate, and the reaction was performed in an orbital shaker for 90 minutes under dark conditions, followed by measurement with absorbance at 570 nm. The results are shown in FIG. 14.
As a result, it was confirmed that the ABTS radical scavenging activity was increased in a concentration-dependent manner when the neural stem cell culture medium was treated, compared with the negative control group and the adipose stem cell culture medium.
From this, it was found that the neural stem cell culture solution had an antioxidant effect.
Experimental example 10 confirming the effects of neural Stem cell culture fluid on skin moisturization and Barrier enhancement
Human epidermal cells (HaCaT) were at 1.0 to 10 panel per well 6 cell/well aliquots were plated in 6-well plates and cultured and then replaced with serum-free medium. After 24 hours, a negative control group (N.C), retinoic acid (R.A, sigma-aldrich company, R2625,1 uM) was treated as a positive control group, and neural stem cell culture solution (at a concentration of 10%, 25%, 50%, 100%) was treated as an experimental group.
After 24 hours, RNA was isolated from the cells to synthesize cDNA, and qRT-PCR was performed according to the method described in Experimental example 1.2.2 to confirm the expression amounts of paphiopediin (Loricrin), endocapelin (Involurin), hyaluronate synthase-2 (Hyaluronic acid synthase, HAS-2) and HAS-3. At this time, the synthesized cDNA was performed using each specific primer set shown in Table 4 below.
[ Table 4 ]
FIG. 15a is an electrophoresis photograph showing whether or not pocket A protein, endo-coat protein, HAS-2 and HAS-3 are expressed, and FIG. 15b is a graph showing the expression amounts of pocket A protein, endo-coat protein, HAS-2 and HAS-3.
As a result, it was found that the expression levels of paphiopediin, endo-chaperonin, HAS-2 and HAS-3 were higher in the case of treatment with the neural stem cell culture solution than in the case of treatment with the negative control group.
From this, it is clear that the neural stem cell culture solution has an effect of moisturizing skin and enhancing skin barrier.
Experimental example 11 secretome (secretome) analysis of neural Stem cell culture fluid
Human cytokine/growth factor antibodies from RayBio Inc. (Human Cyokine/Growth Factor Antibody; from RayBiotech, noncross, georgia, U.S.) were used to confirm neural stem cell culture broth composition in a serum-free state.
After incubating the Array membrane (Array membrane) in blocking buffer for 30 minutes at room temperature, it was treated with 2ml of neural stem cell culture solution for 1 hour. After 5 washes of the membrane, biotin-conjugated antibodies were treated at room temperature for 1 to 2 hours, and 2ml of HRP-streptavidin conjugate as a substrate was added. After 2 hours, the neural stem cell culture broth was treated with detection buffer (detection buffer) for 2 minutes, and the composition of the neural stem cell culture broth was confirmed using iBright (CL 1000 imaging system, sammer feichi technologies). In addition, the signal intensity of the secreted proteome analysis of the neural stem cell culture broth is graphically represented. The results are shown in table 4, fig. 16 and fig. 17.
[ Table 5 ]
As a result, it was found that the neural stem cell culture medium contains many growth factors, cytokines, and the like.
In particular, it is known that it comprises proteins involved in skin regeneration and anti-aging, namely, activin RIA/ALK-2, adiponectin 1, tenascin-like protein 1 (chord-like 1), csk, EDA-A2, EGF, bFGF, GDF3, GDF5, GDF15, GLO-1, IGFBP-2, IGFBP-3, IGFBP-7, latent TGF-beta binding protein 1, MFG-E8, MMP-20, PDGF-AA, PECAM-1, smad4, sonic hedgehog (Shh N-terminus), TGF-beta 5, thrombospondin 1, tie-2, VEGF and VEGF-C. In addition, it specifically comprises proteins involved in promoting collagen synthesis and improving wrinkles, i.e., comprises MCP-1, TIMP-1, bFGF, insulin, latent TGF-beta binding-protein 1, smad4, TGF-beta 5, thrombospondin 1. In addition, it comprises the protein TGF-beta 5 known to be involved in whitening and the protein GLO-1 involved in antioxidant effects. In addition, it is known that it contains proteins required for anti-inflammatory effects and prevention of autoimmune diseases, that is, axl, GRO, thrombospondin 1, tgf- β5.
<110> Kagaku Kogyo Han Shifa ma
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<151> 2020-05-22
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Claims (14)
1. A cosmetic composition for improving skin comprises a neural stem cell culture solution as an active ingredient, the neural stem cell culture fluid comprises activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 Isoarm GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1, S100A10, sFRP-4, sgp130, DPP homolog 1 (d 1), d4, N-factor, hedgehog receptor 1, vascular kinase 1, vascular endothelial cell receptor 1, vascular kinase 1, vascular pathway 1-beta-5, and vascular receptor 1; and the improvement of skin is a relief of wounds, improvement of wrinkles, skin regeneration, increase of skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturization, or enhancement of skin barrier.
2. The cosmetic composition of claim 1, wherein the neural stem cells are derived from dead-born fetal brain tissue for 10 weeks to 14 weeks.
3. The cosmetic composition of claim 1, wherein the neural stem cells are cells transduced with L-MYC genes.
4. Cosmetic composition according to claim 1, characterized in that it is intended to promote collagen synthesis by skin cells.
5. Cosmetic composition according to claim 1, characterized in that it is for inhibiting the elastase activity of skin cells.
6. Cosmetic composition according to claim 1, characterized in that it is for inhibiting melanin synthesis of skin cells.
7. Cosmetic composition according to claim 1, characterized in that it is used for inhibiting the tyrosinase activity of skin cells.
8. Cosmetic composition according to claim 1, characterized in that it is intended to inhibit the production of reactive oxygen species of skin cells.
9. Cosmetic composition according to claim 1, characterized in that it is intended to promote the differentiation of keratinocytes and the synthesis of hyaluronic acid.
10. A pharmaceutical composition for external use for improving skin, which comprises a neural stem cell culture solution as an active ingredient, the neural stem cell culture fluid comprises activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1, S100A10, sFRP-4, sgp130, DPP homolog 1, d4, tone factor N-terminus, hemiechian 1, vascular kinase 1, vascular pathway 1-5, vascular kinase 1, vascular inhibitor; and the improvement of skin is a relief of wounds, improvement of wrinkles, skin regeneration, increase of skin elasticity, skin whitening, anti-inflammatory, antioxidant, skin moisturization, or enhancement of skin barrier.
11. A pharmaceutical composition for treating skin wounds, skin regeneration, skin anti-inflammatory, treating or preventing atopic dermatitis or autoimmune diseases, comprising a neural stem cell culture broth as an active ingredient, the neural stem cell culture fluid comprises activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1, S100A10, sFRP-4, sgp130, DPP homolog 1, d4, tone factor N-terminus, hemiechian-1, vascular kinase 1, vascular pathway 1, vascular kinase 1, vascular endothelial factor 1, vascular kinase 1, and vascular pathway 1-5 inhibitors.
12. Use of a neural stem cell culture broth comprising activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 isotom GGF2, PDGF-AA, platelet adhesion molecule 1, S100a10, sgp-4, sgp1, d-130, VEGF-1, vascular endothelial factor receptor 1, VEGF-5, vascular pathway inhibitors, vascular endothelial growth factor 1, vascular endothelial growth factor inhibitors, VEGF-5, and tissue factor inhibitors for the preparation of cosmetics for improving skin.
13. Use of a neural stem cell culture broth comprising activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 isotom GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1, S100a10, sgp-4, gp1, snf-130, VEGF-1, VEGF-5, vascular pathway receptor inhibitors, VEGF-1, vascular endothelial growth factor inhibitors, VEGF-5, vascular pathway inhibitors, and the like.
14. Use of a neural stem cell culture fluid in the manufacture of a medicament for treating skin wounds, skin regeneration, skin anti-inflammatory, treating or preventing atopic dermatitis or autoimmune diseases, the neural stem cell culture fluid comprises activin RIA/ALK-2, adiponectin 1, axl, BIK, tenascin-like protein 1, csk, EDA-A2, epidermal growth factor, endostatin, basic fibroblast growth factor, galectin 3, growth differentiation factor-3, GDF5, GDF-15, glypican 3, GRO, GLO-1, intercellular adhesion molecule 2, insulin-like growth factor binding protein 2, IGFBP-3, IGFBP-7, IL-1 F6, IL-1 F8, IL-7, IL-15R Alpha, IL-20R beta, insulin, latent TGF-beta binding protein 1, monocyte chemotactic protein 1, MFG-E8, MMP-20, NRG1 Isoform GGF2, PDGF-AA, platelet endothelial cell adhesion molecule 1, S100A10, sFRP-4, sgp130, DPP homolog 1, d4, tone factor N-terminus, hemiechian-1, vascular kinase 1, vascular pathway 1, vascular kinase 1, vascular endothelial factor 1, vascular kinase 1, and vascular pathway 1-5 inhibitors.
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