KR20190100138A - Cosmetic composition and pharmaceutical composition for improving atopic dermatis, alopetic, wound, or skin wrinkle - Google Patents

Abstract

The present invention relates to a cosmetic composition and a pharmaceutical composition which contain a secretion of natural killer cells so as to have excellent effects of increasing various factors related to atopic dermatitis, hair loss, and wound alleviation or skin wrinkle reduction and improving the expression levels of the factors, thereby having excellent alleviation effects on the symptoms.

Description

Cosmetic compositions and pharmaceutical compositions for improving atopic dermatitis, hair loss, wounds or skin wrinkles {COSMETIC COMPOSITION AND PHARMACEUTICAL COMPOSITION FOR IMPROVING ATOPIC DERMATIS, ALOPETIC, WOUND, OR SKIN WRINKLE}

The present invention relates to cosmetic compositions and pharmaceutical compositions for the improvement of atopic dermatitis, hair loss, wounds or skin wrinkles.

Among the cells constituting the immune system, natural killer cells (NK cells), in particular, function to remove host cells infected with tumor cells, bacteria, intracellular parasites or viruses without prior sensitization by antigen. It is a behavioral cell that acts in the first line of immune system defense mechanisms in vivo such as rejecting inappropriate bone marrow transplantation and regulating T cell immune response.

Among the various functions of these NK cells, in particular, the ability to selectively kill cancer cells has been discovered, many studies have been conducted, in the case of cancer patients, peripheral blood NK cell number or activity decreased than normal people, NK cell activity in animal experiments This decline is known to increase the risk of cancer development and cancer metastasis.

In addition, NK cells play an important role in innate immune responses against host-infected pathogens or cancer cells and in acquired immune responses through cytokine secretion. The main mechanism that NK cells use to kill target cells is perforin. Cell soluble granules, such as granzyme B) and granzyme B, are secreted into the target cell via an immune synapse, and the secreted perforin creates a hole in the target cell wall, and granzyme enters the target cell through the hole. Induces apoptosis.

As described above, NK cells have been found to play a decisive role in the carcinogenic process in which cells are transformed into malignant cells, or in the early defenses of viral infection, but not in the NK cells themselves, but in their secretory substances, and their new uses. Is inadequate.

KR 1344201 KR 1719274

An object of the present invention is to provide a cosmetic composition for improving atopic dermatitis, hair loss, wound or skin wrinkles.

An object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of atopic dermatitis, hair loss, wound or skin wrinkles.

1. Cosmetic composition for improving atopic dermatitis, hair loss, wound or skin wrinkles, including secretion of natural killer cells.

2. The composition of 1 above, wherein the secretion of natural killer cells comprises at least one selected from the group consisting of IL-2, TIMP2, VEGF, PDGF-AA and IL-10.

3. The composition according to the above 1, wherein the natural killer cell secretion is obtained from the culture medium which cultures the natural killer cells, isolates and purifies them.

4. The composition of 3 above, wherein the culture is cultured in a medium containing the feeder cells, supporter cell stimulating factor and growth factor.

5. The composition of 4 above, wherein the raw material cells are human-derived cells that can be differentiated into natural killer cells.

6. In the above 5, wherein the human-derived cells are peripheral blood, peripheral blood leukocytes, PBMC (peripheral blood mononuclear cells), enriched natural killer cells, isolated natural killer cells, cord blood, hematopoietic stem cells and At least one composition selected from the group consisting of pluripotent stem cells.

7. The composition of 4 above, wherein the growth factor is interleukin.

8. A pharmaceutical composition for improving atopic dermatitis, hair loss, wound or skin wrinkles, including secretion of natural killer cells.

9. The composition of the above 8, wherein the secretion of natural killer cells comprises at least one selected from the group consisting of IL-2, TIMP2, VEGF, PDGF-AA and IL-10.

10. The composition according to the above 8, wherein the natural killer cell secretion is obtained from the culture medium which cultures the natural killer cells, isolates and purifies them.

11. The composition of the above 10, wherein the culture is cultured in a medium containing the feeder cells, support cell stimulating factor and growth factor.

12. The composition according to the above 11, wherein the source cells are human-derived cells capable of differentiating into natural killer cells.

13. In the above 12, the human-derived cells are peripheral blood, peripheral blood leukocyte cells, PBMC (peripheral blood mononuclear cells), enriched natural killer cells, isolated natural killer cells, cord blood, hematopoietic stem cells and At least one composition selected from the group consisting of pluripotent stem cells.

14. The composition of 11 above, wherein the growth factor is interleukin.

Secretion according to the invention is excellent in the effect of increasing the various factors, the expression level associated with the improvement of atopic dermatitis, hair loss, wound or skin wrinkles. Thus, the cosmetic composition of the present invention comprising the same is excellent in the improvement effect of the symptoms, the pharmaceutical composition is excellent in the prevention or treatment of the disease.

1 is a result of evaluating the fibroblast proliferation ability difference between NK-CM and NK medium.
2 shows the results of evaluation of cell mobility and cell proliferation of HDF-N cells of NK-CM.
3 is a collagen production evaluation results of NK-CM.
4 is a cell viability confirmation result of keratinocytes by NK-CM treatment.
5 is a cell viability confirmation result of the external root sheath cells (ORS) by NK-CM treatment.
Figure 6 shows the relationship between the expression of inflammatory cytokines and NK-CM in HaCaT cells.
Figure 7 shows the relationship between the expression of inflammatory cytokine and NK-CM in HMC-1 cells.
8 is a result of component analysis of NK conditioned-medium (NK-CM)

Hereinafter, the present invention will be described in detail.

The cosmetic composition for atopic dermatitis, hair loss, wound or skin wrinkle improvement of the present invention includes secretions of natural killer cells.

Natural killer cells (NK cells) are lymphocyte-derived cells that play a role in the immune response. They are present in about 10-15% of normal blood and have high killing capacity when reacted with nonself. . In cells infected with various viruses, invading bacteria, or generating abnormal cells, NK cells react nonspecifically and immediately remove foreign substances. Thus, natural killer cells have been studied for their application as immune anticancer agents.

However, the present inventors have found that the secretion secreted by natural killer cells, rather than the natural killer cells itself, has an effect on atopic dermatitis, hair loss, wound and skin wrinkle improvement, which is different from the above uses. Devised.

Secretion of natural killer cells according to the present invention is IL-2 (interleukin-2), TIMP2 (metallopeptidase inhibitor 2), VEGF (vascular endothelial growth factor), PDGF-AA (platelet-derived growth factor-AA) and IL-10 It may include at least one selected from the group consisting of (interleukin-10).

Among the components included in the secretion of natural killer cells according to the present invention, IL-2, TIMP2 is a factor associated with wrinkle improvement, VEGF, PDGF-AA is a factor associated with hair loss improvement, IL-2, IL- 10 is a factor associated with atopy improvement.

The secretion of natural killer cells according to the present invention may be obtained from, for example, culturing natural killer cells, separating and purifying them, from the remaining culture solution, and using the remaining culture solution itself, but are not limited thereto.

The following animal cell culture medium and the following components are usually components that help the growth of the cells, components that stabilize the cells, which affects the growth of the cells, the degree of cell growth may vary because these components are different, The secretory components of the cells are not affected by them, and the following examples are not limited thereto.

Culture of natural killer cells can be used without limitation conventional animal cell culture medium known in the art, for example, CellGro medium (Cellgenix), AIM-V medium, RPMI1640 medium, XVIVO 20, RPMI1640, etc. .

Cultivation of natural killer cells may include culturing source cells to obtain natural killer cells or culturing natural killer cells.

Seed cell means a cell that can be propagated into natural killer cells through appropriate culture, and specifically, may be a human-derived cell that can be differentiated into natural killer cells. For example, in a group consisting of peripheral blood, peripheral blood leukocytes, PBMCs (peripheral blood mononuclear cells), enriched natural killer cells, isolated natural killer cells, umbilical cord blood, hematopoietic stem cells, and pluripotent induced stem cells One or more selected may be used, but is not limited thereto.

The culturing of the source cells may be carried out in a medium further comprising components for promoting the culture of natural killer cells.

Components for promoting the culture may be, for example, support cells, support cell stimulating factors, growth factors and the like.

Supporting cells prevent division and proliferation, but because they have metabolic activity, they produce a variety of metabolites to help propagate natural killer cells.

The feeder cells to be used may be applied without limitation as long as they help propagation of natural killer cells. For example, animal cell lines into which genes are introduced, or peripheral blood leukocyte cells treated with various cytokines or compounds ( PBL), magnetic or other peripheral blood leukocytes (PBL), T-cells, B-cells, monocytes, CD4 (+) T cells, and the like. PBL) and CD4 (+) T cells can be used, but are not limited thereto.

Support cells can be inactivated and used to ensure safety. As the method of inactivation, a conventional method known in the art may be used, and for example, a method of irradiating gamma-rays may be used. Such inactivated feeder cells include isolated T-cells.

As the support cell stimulating factor, for example, anti-CD3 antibodies can be used.

An anti-CD3 antibody is an antibody that specifically binds to a CD3 antigen, a group of molecules that binds to a T cell receptor (TCR) to form an antigen recognition complex. The CD3 molecule binds to TCR and delivers an antigen recognition signal into a cell. In charge of.

The anti-CD3 antibody usable in the present invention can be used without limitation as long as the antibody has a property of binding to CD3, and is preferably selected from the group consisting of OKT3, UCHT1 and HIT3a, but is not limited thereto. The anti-CD3 antibody may be included, for example, in the range of 0.1-100 ng / ml, but is not limited thereto.

Examples of growth factors include cytokines.

The cytokine is a growth factor, preferably one or more selected from the group of interleukins, but is not limited thereto.

Interleukin is a generic term for proteinaceous bioactive substances produced by immune cells such as lymphocytes, monocytes, and macrophages. The available interleukins include interleukin-2 (IL-2), interleukin-12 (IL-12), and interleukin-15. (IL-15), interleukin-18 (IL-18), and interleukin-21 (IL-21), preferably at least one selected from the group consisting of, and particularly preferably using IL-2, but not limited thereto. However, it will be apparent to those skilled in the art that other cytokines may be used without limitation as long as they meet the object of the present invention. Cytokines may be included, for example, in the range of 10-2000 U / ml, but are not limited thereto.

In addition, it may be cultured by adding an additional growth factor that supports the growth of serum or plasma and lymphocytes. The kind of serum or plasma added to the medium is not particularly limited, and commercially available ones derived from various animals can be used, but those derived from humans are more preferable as human origin. For example, a method known to those skilled in the art can be used, such as a combination of cytokines for propagating lymphocytes from PBMCs, lectins for stimulating lymphocyte proliferation, and the like.

In addition, the medium may further comprise additional growth factors supporting the proliferation of serum or plasma and lymphocytes, and may comprise serum or plasma itself. The kind of serum or plasma to be added to the medium is not particularly limited, and commercially available ones derived from various animals can be used, but those derived from humans are preferred as human origin, and may be, for example, human AB serum or auto plasma.

In the case of a cosmetic composition comprising a secretion according to the present invention, in addition to the active ingredient, it may include components conventionally used in cosmetic compositions, for example, such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings Adjuvants, and carriers.

Cosmetic compositions of the present invention may be prepared in any formulation conventionally prepared in the art and include, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations, packs, massage creams and sprays and the like, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, spray or powder.

When the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethyleneglycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, and microcrystals are used as carrier components. Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

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

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

If the cosmetic composition of the present invention is a soap, a surfactant-containing cleansing formulation or a surfactant-free cleansing formulation, it may be wiped off, peeled off or washed with water after application to the skin. As a specific example, the soap is liquid soap, powdered soap, solid soap and oil soap, the surfactant-containing cleansing formulation is a cleansing foam, cleansing water, cleansing towel and cleansing pack, the surfactant-free cleansing formulation is a cleansing cream , Cleansing lotion, cleansing water and cleansing gel, but are not limited thereto.

The present invention also provides a pharmaceutical composition for improving atopic dermatitis, hair loss, wound or skin wrinkles, including secretion of natural killer cells.

Secretion of natural killer cells may be within the range described above.

The pharmaceutical composition of the present invention may be formulated into a pharmaceutical preparation for the prevention and treatment of degenerative neurological diseases, including a pharmaceutically acceptable carrier, diluent or excipient.

Such carriers, excipients and diluents are, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose , Microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical compositions of the present invention may be formulated into oral dosage forms, external preparations, suppositories, or sterile injectable solutions, such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., according to conventional methods, to prepare pharmaceutical preparations. In general, when formulated, it may be formulated using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose, lactose, gelatin, and the like, in the pharmaceutical composition. Can be prepared by mixing. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition of the present invention may vary depending on the age, sex, and weight of the patient, but generally in an amount of 0.1 to 100 mg / kg, preferably 1 to 30 mg / kg, once to several times a day. It can be administered in divided doses. The dosage may also be increased or decreased depending on the route of administration, degree of disease, sex, weight, age, health condition, diet, time of administration, method of administration, rate of excretion, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be envisaged, for example, by skin application, oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

Hereinafter, the present invention will be described in detail with reference to Examples.

Example. Obtaining natural killer cell secretions

1. Acquisition of natural killer cell secretion

1-1. Preparation of CD3 (-) PBMC Raw Cells

PBS (phosphate buffered saline, LONZA, 17-516Q) was added at a 1: 1 ratio to PBMCs collected from healthy donors and centrifuged at 1500 rpm for 4 minutes. PBMC pellets were suspended in MACS running buffer (PBS containing 2% FBS and 2 mM EDTA) at 10 × 10 ⁶ cells / mL and cell number was measured using an automatic cell counter.

In order to obtain source cells from which CD3 (+) cells were removed (depletion), 5 ~ 10 × 10 ⁷ cells were transferred to a 50 mL tube and centrifuged at 1200 rpm and 4 ° C. for 10 minutes. 400-800 μL of MACS running buffer and 100-200 μL of CD3 magnetic beads (Miltenyi biotech, 130050101) were placed in 5-10 × 10 ⁷ cells of PBMC cells and reacted at 4 ° C. for 20 minutes. After washing with more than 10 times MACS running buffer and centrifuged for 7 minutes at 1350 rpm, 4 ℃ and suspended in the final 1 ~ 2 mL MACS running buffer. Cells were separated by mounting a CS column (column, Miltenyi Biotech, 130-041-305) on VarioMACS (Miltenyi Biotech), and the cells were recovered by washing the column three times. Cell counts were measured using an automatic cell counter. The cells were suspended in a freeze medium and frozen in 1 ~ 2 × 10 ⁷ cells per vial and stored in liquid nitrogen.

On the day of incubation, frozen CD3 (-) PBMC was thawed in a water bath at 37 ° C and transferred to a 50 mL tube, followed by 0.6% ACD (Citrate-dextrose solution, Sigma-Aldrich, C3821) and 0.2% Fetal serum bovine (FBS). It was suspended 10 times with PBS (ACD-A PBS buffer) containing 2 mM EDTA and centrifuged for 10 minutes at 1200 rpm, 4 ℃. CD3 (-) PBMC was suspended in CellGro SCGM medium (Cellgenix, 20802-0500) and cell number was measured. CD3 (-) PBMC feeder cells were suspended in CellGro SCGM medium at 1 × 10 ⁶ cells / mL.

1-2. Preparation of Support Cells

Peripheral blood PBMC support cells were centrifuged at 1500 rpm and 4 ° C. for 10 minutes by adding PBS (phosphate buffered saline, LONZA, 17-516Q) in a 1: 1 ratio. PBMC pellets were suspended in MACS running buffer (PBS containing 2% FBS and 2 mM EDTA) at 10 × 10 ⁶ cells / mL and cell number was measured using an automatic cell counter. PBMC support cells were suspended in freezing medium and frozen 1 x 10 ⁸ cells per vial and stored in liquid nitrogen.

PBMC support cells frozen on the start of culture were thawed in a 37-degree water bath, transferred to a 50 mL tube, suspended in 10-fold ACD-A PBS buffer, and centrifuged at 1200 rpm and 4 ° C for 10 minutes. After measuring the cell number, suspended at 5x10 ⁶ cells / mL with CellGro SCGM medium and inactivated by irradiation at 2000cGy with gamma irradiation.

1-3. Culture of natural killer cells and recovery of culture solution

In NK cell culture, 500 IU / mL IL-2 (Proleukin, Novartis, Korea), 10 ng / mL OKT-3 (eBioscience, 16-0037-85) and 1-2% donor plasma Add culture medium. On day 0 of the cultivation, mix CD3 (-) PBMC source cells and gamma-irradiated support cells at a ratio of 1: 5 and adjust the cell concentration to 2x10 ⁶ cells / mL based on the total cell number for 4 to 5 days in an incubator at 37 ° C. The culture was stationary.

On day 4-5, the same amount of culture medium (Cellgro SCGM + 500 IU IL-2 + 1% by volume donor plasma) was added, followed by stationary culture for 2-3 days. On the 7th day of culture, the cells in cultivation were added in the same manner as on the day of culture, with a 1: 5 ratio of support cell line, 500 IU / mL IL-2, 10 ng / mL OKT-3, and 1 volume% donor plasma. Adjust the cell concentration to the standard 1x10 ⁶ cells / mL and re-stimulate the cells for 3 days. After that, the number of cells was measured at intervals of 2 to 3 days, and cultured to 20 to 21 days with the addition of a culture medium to 0.5 to 1x10 ⁶ cells / mL.

After 20-21 days of culture, the cells in culture were transferred to a 1L centrifuge tube and centrifuged at 1500 rpm and 4 ° C. for 10 minutes. The supernatant was recovered after centrifugation and named NK conditioned-media.

In addition, 500 IU / mL IL-2 (Proleukin, Novartis, Korea), 10 ng / mL OKT-3 (eBioscience, 16-0037-85) and 1-2% donor plasma were added to the Cellgro SCGM medium. One culture medium was named NK cell medium.

Experimental Example 1. Evaluation of Cell Proliferation Capacity of NK-CM

In order to evaluate the proliferative difference between NK cell medium and NK-CM in human fibroblasts, cell proliferation was measured using Cell Counting Kit-8 (CCK-8).

Human fibroblasts (HDF-N) cells suspended in single cells were cultured at 37 ° C by dispensing 5x10 ³ cells / well in 96 well plates. Thereafter, starvation was maintained for 24 hours, followed by further incubation for 24 and 48 hours by treating NK cell medium and NK-CM. To measure the proliferation of the cells, the Cell Counting Kit-8 reaction solution was diluted 1/10 in supplement-free medium and treated with 100 μl of each well for 1 hour, followed by ELISA microplate reader (Model 680, BIO). -RAD) was used to measure the absorbance at 450 nm. The medium to which 10% FBS was added was used as a positive control group, and the cell proliferation rate of each group was calculated as a percentage (%) compared with NK cell medium (M). The results are shown in FIG.

As shown in FIG. 1, NK-CM showed a relatively high proliferative capacity at all concentrations and increased in a concentration-dependent manner when compared with NK cell medium at 24 hours. In NK-CM at 48 hours, there was no difference in the concentration of 2.5% compared to NK cell medium, but it was confirmed that the proliferative capacity increased by 7% at 5% concentration and 37% at 10% concentration. -CM has a higher proliferative effect on the cells.

Experimental Example 2 Evaluation of Cell Proliferation of NK-CM

In order to evaluate the cell migration and wound repair ability of NK-CM in human fibroblasts, the cell scratch assay was used.

HDF-N cells were aliquoted at 1.0 × 10 ⁴ cells / inserts into ibidi culture inserts (μ-Dish35mm, high Culture-Inserts; Thistle Scientific Ltd, UK) at the bottom of a 60 mm dish and incubated for 24 hours in cell culture conditions. Thereafter, the chamber was removed, the sample was diluted in medium at an appropriate concentration, and then treated with cells, and further cultured for 48 hours under cell culture conditions. After 24 and 48 hours, cell mobility was photographed by electron microscopy (Eclipse TS100, Nikon Instruments Inc., NY, USA).

As shown in Figure 2 (A), as compared to the control group at 24 and 48 hours, as the concentration increases in the NK-CM treatment group, it was confirmed that the cell mobility and wound recovery ability increased significantly.

  In order to evaluate the proliferative capacity of NK-CM, HDF-N cells were treated with 2.5, 5, and 10% and measured using Cell Counting Kit-8 (CCK-8) at 24 and 48 hours.

As shown in FIG. 2 (B, C), it was confirmed that cell proliferation was increased in a concentration-dependent manner when comparing the NK-CM treatment group and the control group at 24 and 48 hours. In particular, the highest concentration of NK-CM 10% results in a maximum increase of 1.6 times at 24 hours and 2 times at 48 hours. This means that NK-CM has the effect of improving wrinkles by increasing the mobility and wound repair ability of HDF-N cells, and enhancing the proliferative capacity of the cells.

Experimental Example 3 Evaluation of Collagen Production Capacity of NK-CM

To evaluate the procollagen synthesis ability of NK-CM, procollagen synthesis ability was evaluated at 24 hours after treatment with non-toxic concentrations of 2.5, 5, and 10%.

HDF-N cells were dispensed at 2.0 × 10 ⁴ cells / well in 24 well plates and incubated for 24 hours. Since the medium was removed and the cells were starved for 24 hours, the cells were treated with NK-CM diluted in a medium without media supplement. After 24 hours, the cultured cells were harvested and the amount of procollagen was measured using procollagen type-1 c-peptide (PIP) EIA kit (TAKARA, MK101). Cells attached to the bottom were lysed with 1 N NaOH to measure the total protein amount and procollagen synthesis amount per protein. Each group was calculated as a percentage (%) compared to NK cell medium (M), the results are shown in Figure 3 (A).

As shown in Figure 3 (A), when treated with TGF-β (10 nM) used as a positive control procollagen synthesis was increased 1.7 times compared to the control. When NK culture was treated, procollagen synthesis increased by 3.4 times at all concentrations compared to the control, and statistically significant results were obtained.

Expression changes of collagen protein were measured by Western blot assay using NK cell culture in human fibroblasts.

HDF-N cells were dispensed into 2.0 × 10 ⁶ cells in a 100 mm plate and incubated for 24 hours. Thereafter, after removing the medium and maintaining the starvation of the cells for 24 hours, the test material was diluted in the medium without the media supplement and treated with the cells. After incubation for 24 hours, the medium was removed, washed with DPBS, lysed cells using lysis buffer, and proteins were quantified by the supernatant obtained by centrifugation at 4 ° C. for 20 minutes at 12,000 rpm. The quantified protein was electrophoresed on SDS-PAGE and blotting the protein of the gel with PVDF membrane. After blocking with 5% BSA, the primary antibody was reacted at 4 ° C. for 24 hours and the secondary antibody was reacted for 1 hour. Then, washed three times with TBS-T buffer for 10 minutes and the expression level of the protein was confirmed using the ECL detection solution. The results are shown in FIG. 3 (B).

As shown in Figure 3 (B), it was confirmed that the expression of Collagen I, III effectively increased in the NK-CM treatment group compared to the control group. This means that NK-CM induces collagen synthesis and expression of human fibroblasts and has the effect of improving wrinkles.

Experimental Example 4. Confirmation of cell viability of keratinocytes by NK-CM treatment

Cell Counting Kit-8 (CCK-8), one of the methods for measuring cell viability, was used to confirm the effect of NK-CM on keratinocytes under normal circumstances.

HaCaT cells suspended in single cells were seeded 1 × 10 ⁴ cells per well in 96-well plates, and then cultured in a CO ² incubator at 37 ° C. for 24 hours. After incubation, each was treated as follows: Based on 100 uL of cell culture, 100% NK-CM addition amount was set to the highest concentration, and treated with HaCaT cells for 24 hours until the final 128-fold dilution at 1/2 dilution. Then, the CCK-8 solution was diluted in a 1:10 ratio with NK-CM-treated HaCaT medium at each concentration and then incubated in a CO ² incubator at 37 ° C. for 1 hour.

Thereafter, the absorbance was measured at 450 nm using a spectrophotometer. At this time, the reference was 450 nm. Cell viability of each group was calculated as a percentage (%) compared to the control (Con). The results are shown in FIG.

As a result of measuring the death of HaCaT cells by NK-CM, it was confirmed that cell viability increased with increasing concentration, which indicates that NK-CM is not toxic to cells up to 100% and has a cell growth effect. it means.

Experimental Example 5. Confirmation of cell viability of external root sheath cells (ORS) by NK-CM treatment

Cell Counting Kit-8 (CCK-8), one of the methods for measuring cell viability, was used to confirm the effect of NK-CM on keratinocytes under normal circumstances.

ORS cells suspended in single cells were seeded 1 × 10 ⁴ cells per well in 96-well plates, and then cultured in a CO ² incubator at 37 ° C. for 24 hours. After incubation, each was treated as follows: 100% NK-CM addition amount was set at the highest concentration based on 100 uL of the cell culture, and the ORS cells were treated for 24 hours until the final 4-fold dilution at 1/2 dilution. Then, the CCK-8 solution was diluted in a ratio of 1:10 with NK-CM-treated ORS medium at each concentration and then incubated in a CO ² incubator at 37 ° C. for 1 hour.

Thereafter, the absorbance was measured at 450 nm using a spectrophotometer. At this time, the reference was 450 nm. Cell viability of each group was calculated as a percentage (%) compared to the control (Con). The results are shown in FIG.

As a result of measuring the killing of ORS cells by NK-CM, it was confirmed that cell viability increased with increasing concentration, which means that NK-CM is not toxic to cells up to 100%, and consequently the major follicular tissue It helps to grow one of the constituent cells

Experimental Example 6. Relationship between expression of pro-inflammatory cytokines in HaCaT cells and NK-CM treatment

Real-time-Polymerase Chain Reaction (RT-PCR) was used to measure the expression of messenger RNA (messenger RNA, mRNA) involved in proinflammatory cytokine secretion in cells. .

HaCaT cells were seeded in 12-well plates at 1 × 10 ⁵ cells per well, followed by treatment with NK-CM 50% or 100% for 2 hours and increased expression of proinflammatory cytokines to induce an inflammatory situation with recombinant protein TNF- alpha (50 ng / mL) was treated for 24 hours. Afterwards, all media containing reagents were washed out and then mixed with 1 mL TRIzol® RNA isolation reagent (Invitrogen, USA). The mixed solution was incubated for 30 minutes on ice, and then 400 μL chloroform1 was added thereto for 5 minutes. Centrifuged for 20 minutes at 16,000 rpm. After separating and containing the supernatant, the same amount of isopropyl alcohol was added to the supernatant and reacted for 5 minutes, followed by centrifugation at 16,000 rpm for 10 minutes. The precipitated separated palette was washed three times with the addition of 70% alcohol. Finally, mRNA samples were obtained by diluting in 50 uL of purified water after drying for 4 hours.

The obtained samples consisted of double stranded DNA (cDNA) synthesized using PrimeScript ™ RT master mix (Takara, Japan). 5 uL of 5X PrimeScript ™ RT master mix was added to 20 uL of mRNA sample and heated at 37 ° C. for 20 minutes and at 85 ° C. for 10 seconds to obtain a synthesized DNA double helix structure.

Amplification of specific genes was done using CFX-96 (Bio-Rad, USA). PCR conditions used to amplify all the genes were carried out in 40 cycles of 10 minutes at 95 ℃, 30 seconds at 95 ℃, 15 seconds at 60 ℃. Expression data were calculated from cycle threshold (Ct) values using quantification method ΔCt. Normalization was performed using GAPDH, respectively. Relative values compared to control. The results are shown in FIG.

As shown in FIG. 6, mRNA levels of TSLP, IL-6, TNF-alpha, IL-8, IL-17a and IL-31, which are types of pro-inflammatory cytokines, were significantly increased in the TNF-alpha-treated population. Could confirm. This means that the treatment of TNF-alpha increases the expression of inflammatory cytokines compared to the normal population. However, the results showed that NK-CM significantly reduced the expression level of pro-inflammatory cytokines in the 50% or 100% treated population. This suggests that NK-CM inhibits elevated expression of pro-inflammatory cytokines due to TNF-alpha, and consequently NK-CM has anti-inflammatory effect in HaCaT cells.

Experimental Example 7. Relationship between expression of inflammatory cytokines in HMC-1 cells and NK-CM treatment

Real-time-Polymerase Chain Reaction (RT-PCR) was used to determine the role of NK-CM in inflammatory conditions by measuring the expression level of messenger RNA (messenger RNA, mRNA) involved in intracellular proinflammatory cytokine secretion.

HMC-1 cells, which are human-derived mast cells, were seeded with 2 × 10 ⁶ cells per well in 6-well plates, and then treated with NK-CM 50% or 100% for 2 hours, inducing cellular degranulation to induce an inflammatory condition. PMAC [PMA (25 nM) + A23187 (1 uM)] was treated and incubated for 12 hours.

All media containing reagents were washed out and then mixed with 1 mL TRIzol® RNA isolation reagent (Invitrogen, USA). The mixed solution was incubated for 30 minutes on ice, and then 400 μL chloroform1 was added thereto for 5 minutes. Centrifuged for 20 minutes at 16,000 rpm. After separating and containing the upper transparent liquid, isopropyl alcohol was added to the supernatant in the same amount and reacted for 5 minutes, and then centrifuged at 16,000 rpm for 10 minutes. The precipitated separated palette was washed three times with the addition of 70% alcohol. Finally, mRNA samples were obtained by diluting in 50 uL of purified water after drying for 4 hours.

The obtained samples consisted of double stranded DNA (cDNA) synthesized using PrimeScript ™ RT master mix (Takara, Japan). 5 uL of 5X PrimeScript ™ RT master mix was added to 20 uL of mRNA sample and heated at 37 ° C. for 20 minutes at 85 ° C. for 10 seconds to obtain a synthesized DNA double helix structure.

Amplification of specific genes was done using CFX-96 (Bio-Rad, USA). PCR conditions used to amplify all the genes were carried out in 40 cycles of 10 minutes at 95 ℃, 30 seconds at 95 ℃, 15 seconds at 60 ℃. Expression data were calculated from cycle threshold (Ct) values using quantification method ΔCt. Normalization was performed using GAPDH, respectively. Relative values compared to control. The results are shown in FIG.

As shown in FIG. 7, mRNA levels of TNF-alpha, IL-8, GM-CSF and MCP3, which are proinflammatory inflammatory cytokines, were significantly increased in the PMAC-treated population. This means that cell treatment of PMACs induces degranulation compared to the normal population to increase expression of inflammatory cytokines. However, NK-CM significantly reduced the expression levels of pro-inflammatory cytokines of TNF-alpha, IL-8 and GM-CSF 3 in 50% or 100% treated populations. In addition, the expression level of MCP3 was not reduced at 50% treatment, but it was confirmed that the significantly reduced at a high concentration of 100%. This inhibits the expression of proinflammatory cytokines due to PMAC treatment by NK-CM treatment, which in conclusion means that NK-CM has anti-inflammatory effect by inhibiting the inflammatory response due to degranulation of HMC-1. .

Experimental Example 8. Component Analysis of NK-CM

To analyze the components of NK conditioned-media (NK-CM), analysis was performed using Human Growth Factor Antibody Array C1, human cytokine array C5, and human chemokine array C1 kit (Raybiotech, USA).

The NK cell medium and NK-CM were collected and analyzed according to the protocol description method of each product provided by Raybiotech, and are graphically shown in FIG. 1 based on the microarray results.

As shown in FIG. 8, it was confirmed that IL-2 and IL-10, which are factors related to atopic dermatitis, were increased in NK-CM compared to NK culture medium. In addition, PDGF-AA and VEGF, which are factors related to hair growth, were increased, and the expression level of TIMP-2, which is a factor related to wound healing, was improved. NK-CM improves anti-atopic dermatitis, hair loss, and wrinkles because the number of items related to atopic dermatitis, hair growth, and skin wrinkles has increased in NK-CM, which contains several factors that NK cells release into the medium through growth and proliferation. That means you can help.

Claims (14)

A cosmetic composition for improving atopic dermatitis or wounds comprising secretions of natural killer cells and not containing natural killer cells.
The composition of claim 1, wherein the secretion of natural killer cells comprises at least one selected from the group consisting of IL-2, TIMP2, VEGF, PDGF-AA, and IL-10.
The composition of claim 1, wherein the natural killer cell secretion is obtained from a culture medium in which the natural killer cells are cultured, separated, purified, and left.
The composition of claim 3, wherein the culturing is carried out in a medium containing feeder cells, supporter cell stimulating factors, and growth factors.
The composition of claim 4, wherein the feeder cells are human derived cells capable of differentiating into natural killer cells.
The method of claim 5, wherein the human-derived cells are peripheral blood, peripheral blood leukocytes, PBMC (peripheral blood mononuclear cells), enriched natural killer cells, isolated natural killer cells, umbilical cord blood, hematopoietic stem cells and pluripotent induction At least one composition selected from the group consisting of stem cells.
The composition of claim 4, wherein the growth factor is interleukin.
A cosmetic composition for improving atopic dermatitis or wounds comprising secretions of natural killer cells and not containing natural killer cells.
The composition of claim 8, wherein the secretion of natural killer cells comprises at least one selected from the group consisting of IL-2, TIMP2, VEGF, PDGF-AA, and IL-10.
The composition of claim 8, wherein the natural killer cell secretion is obtained from the culture medium which cultures the natural killer cells, isolates, purifies, and remains.
The composition of claim 10, wherein the culturing is carried out in a medium containing feeder cells, supporter cell stimulating factor and growth factor.
The composition of claim 11, wherein the feeder cells are human derived cells capable of differentiating into natural killer cells.
The method of claim 12, wherein the human-derived cells are peripheral blood, peripheral blood leukocytes, PBMC (peripheral blood mononuclear cells), enriched natural killer cells, isolated natural killer cells, cord blood, hematopoietic stem cells and pluripotent induction At least one composition selected from the group consisting of stem cells.
The composition of claim 11, wherein the growth factor is interleukin.

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