KR102175470B1 - Method for preparing senescent model skin cell lines, skin cell lines therefrom and method for screening anti-aging material using the same - Google Patents

Abstract

The present invention is a method for preparing an aging model skin cell line that can be used for the study of aging phenomena, cultured by treatment with pyruvic acid in a specific range, and an anti-aging model skin cell line prepared by the method and the cells. It relates to a method for screening aging substances, and the aging model skin cell line prepared by the manufacturing method of the present invention can alleviate cytotoxicity generated when culturing skin cells in a state of deficiency of pyruvate, and in vivo ( in In vivo ) model-like environment has an excellent effect of screening anti-aging substances.

Description

Method for preparing senescent model skin cell lines, skin cell lines therefrom and method for screening anti-aging material using the skin cell line and the skin cell line produced by the method using the same}

In the present specification, a method for preparing an aging model skin cell line that can be used for the study of aging phenomena cultured by treating a specific range of concentration of pyruvic acid, an aging model skin cell line prepared by the method, and anti-aging using the cells A method of screening a substance is disclosed.

With the recent improvement in living standards, in addition to maintaining a healthy body, modern people are paying much attention to maintaining healthy skin. Therefore, interest in skin care and skin aging improvement is increasing.

The skin is the largest organ of the human body, accounting for about 16% of the total volume of the human body, and while in direct contact with the external environment, it is important to protect the human body from many deadly harmful factors such as temperature, humidity, and ultraviolet rays. It acts as a protective film. However, skin cells are damaged due to external environments such as various contaminants and strong ultraviolet rays, and cell proliferation is not performed properly, resulting in wrinkles, loss of elasticity, keratinization, and irregular pigmentation in the skin.

Skin aging is largely divided into natural aging (or endogenous aging) and exogenous aging, and natural aging is influenced by genetic factors, so artificial control is difficult, whereas exogenous aging is affected by environmental factors, so artificial control is relatively difficult. It's easy. As representative exogenous aging factors, ultraviolet rays, reactive oxygen species, and stress are known.

Accordingly, recently, methods for improving exogenous aging are being actively studied, and in particular, efforts to identify substances for preventing or improving aging are being continued. Conventional skin cell aging model manufacturing technology includes a replica senescence model that takes a long production period or an excessive stress-inducing model (oxidant treatment, UV, radiation treatment, etc.) manufacturing technology, and an aging model skin cell line manufactured using this Was used for verification and discovery of anti-aging substances. However, these models take a long time to manufacture or are not applied to in vivo aging models due to excessive stress that does not occur in vivo .

Accordingly, the present inventor has completed the present invention by studying a method of manufacturing an aging model for manufacturing in a shorter time without excessive stress and manufacturing in an environmental state similar to a living body.

JP 2015-051654 A KR 10-2015-0085546 A

Na Liu et al., Pyruvate prevents aging of mouse oocytes, Reproduction. 2009 Aug;138(2):223-34.

In one aspect, it is an object of the present invention to provide a method for producing an aging model skin cell line, comprising culturing the isolated skin cells in a medium containing pyruvate in a specific concentration range.

In another aspect, an object of the present invention is to provide an isolated, aging model skin cell line prepared by the method for producing an aging model skin cell line.

In another aspect, an object of the present invention is to provide a composition for preparing an aging model skin cell line, comprising as an active ingredient pyruvate in a specific concentration range.

In another aspect, an object of the present invention, the aging model skin cell line produced by the aging model skin cell line manufacturing method, the step of treating an anti-aging candidate material; And it provides an anti-aging substance screening method comprising the step of detecting an aging index before and after treatment with the candidate substance or measuring a cell proliferation rate.

In another aspect, an object of the present invention is to provide an in vitro model for screening anti-aging substances, including an aging model skin cell line prepared by the method for producing an aging model skin cell line.

In another aspect, an object of the present invention, the aging model skin cell line produced by the aging model skin cell line manufacturing method; And it is to provide a kit for screening anti-aging substances, including instructions.

In one aspect, the present invention provides a method for producing an aging model skin cell line, comprising culturing the isolated skin cells in a medium containing 0.01 to 0.9 mM pyruvate.

In another aspect, the present invention provides an isolated, aging model skin cell line prepared by the method for producing an aging model skin cell line.

In another aspect, the present invention provides a composition for preparing an aging model skin cell line comprising 0.01 to 0.9 mM or less of pyruvate as an active ingredient.

In another aspect, the present invention, the aging model skin cell line produced by the aging model skin cell line manufacturing method, the steps of treating an anti-aging candidate material; And it provides an anti-aging substance screening method comprising the step of detecting an aging index before and after treatment with the candidate substance or measuring a cell proliferation rate.

In another aspect, the present invention provides an in vitro model for screening anti-aging substances, including an aging model skin cell line prepared by the method for producing an aging model skin cell line.

In another aspect, the present invention, the aging model skin cell line produced by the aging model skin cell line manufacturing method; And it provides a kit for screening anti-aging substances, including instructions.

The present invention shows that when culturing skin cells in a medium containing pyruvate of 0.01 to 0.9 mM or less similar to the concentration of pyruvate in a living body, an aging model skin cell line can be prepared in an environmental state similar to a living body within about 2 weeks without excessive stress. Confirmed. The aging model skin cell line prepared by the manufacturing method of the present invention can alleviate the cytotoxicity that occurs when culturing skin cells in a state of deficiency of pyruvate, and is an anti-aging substance in an environment similar to an in vivo model. There is an excellent effect that can be screened.

1 is a diagram comparing the degree of aging of an aging model skin cell line according to the concentration of pyruvate contained in the culture medium. 1A is a graph showing the cell proliferation rate of three aging model skin cell lines prepared in a culture medium containing 0 mM, 0.1 mM, and 1 mM pyruvate, respectively, and FIG. 1B is a graph showing the cell proliferation rate of the three aging model skin cell lines. It is a graph showing the aging-related beta-galactosidase (SA-βgal) activity.
Figure 2 is a graph showing the cell survival rate when anti-aging candidate substances are treated in an aging model skin cell line prepared in a pyruvate deficient environment (0 mM pyruvate).
Figure 3 is a graph showing the cell viability when treated with anti-aging candidate substances in each of five aging model skin cell lines prepared in a culture medium containing 0 mM, 0.1 mM, 0.2 mM, 0.5 mM and 1 mM pyruvate, respectively.
4 is a diagram showing the results of anti-aging evaluation of each anti-aging candidate substance when anti-aging candidate substances are treated in an aging model skin cell line prepared in a pyruvate deficient environment (0 mM pyruvate). Figure 4a is a graph showing the cell proliferation rate evaluation results, Figure 4b is a graph showing the aging-related beta-galactosidase (SA-βgal) activity.
5 is a diagram showing the results of anti-aging evaluation of each anti-aging candidate substance when anti-aging candidate substances are treated in an aging model skin cell line prepared in a culture medium containing 0.1 mM pyruvate. Figure 5a is a graph showing the cell proliferation rate evaluation results, Figure 5b is a graph showing the aging-related beta-galactosidase (SA-βgal) activity.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a method for producing an aging model skin cell line, comprising culturing the isolated skin cells in a medium containing 0.01 to 0.9 mM pyruvate.

In the aging model skin cell line production method of the present invention, the concentration of pyruvic acid contained in the medium may be 0.01 to 0.9 mM, specifically 0.01 to 0.5 mM, and more specifically 0.01 mM or more, 0.02 mM or more, 0.03 mM Or more, 0.04 mM or more, 0.05 mM or more, 0.06 mM or more, 0.07 mM or more, 0.08 mM or more, 0.09 mM or more, 0.1 mM or more, 0.11 mM or more, 0.12 mM or more, 0.13 mM or more, 0.14 mM or more, 0.15 mM or more, 0.16 mM or more, 0.17 mM or more, 0.18 mM or more, 0.19 mM or more, 0.2 mM or more, 0.21 mM or more, 0.22 mM or more, 0.23 mM or more, 0.24 mM or more, 0.25 mM or more, 0.26 mM or more, 0.27 mM or more, 0.28 mM Or more, 0.29 mM or more, 0.3 mM or more, 0.31 mM or more, 0.32 mM or more, 0.33 mM or more, 0.34 mM or more, 0.35 mM or more, 0.36 mM or more, 0.37 mM or more, 0.38 mM or more, 0.39 mM or more, 0.4 mM or more, 0.41 mM or more, 0.42 mM or more, 0.43 mM or more, 0.44 mM or more, 0.45 mM or more, 0.46 mM or more, 0.47 mM or more, 0.48 mM or more, 0.49 mM or more, or 0.5 mM or more, and 0.9 mM or less, 0.8 mM or less, 0.7 mM or less, 0.6 mM or less, 0.5 mM or less, 0.49 mM or less, 0.48 mM or less, 0.47 mM or less, 0.46 mM or less, 0.45 mM or less, 0.44 mM or less, 0.43 mM or less, 0.42 mM or less, 0.41 mM or less, 0.4 mM Or less, 0.39 mM or less, 0.38 mM or less, 0.37 mM or less, 0.36 mM or less, 0.35 mM or less, 0.34 mM or less, 0.33 mM or less, 0.32 mM or less, 0.31 mM or less, 0.3 mM or less, 0.29 mM or less, 0.28 mM or less, 0.27 mM or less, 0.26 mM or less, 0.25 mM or less, 0.24 mM or less, 0.23 mM or less, 0.22 mM or less, 0.21 mM or less, 0.2 mM or less, 0.19 mM or less, 0.18 mM or less, 0.17 mM Hereinafter, it may be 0.16 mM or less, 0.15 mM or less, 0.14 mM or less, 0.13 mM or less, 0.12 mM or less, 0.11 mM or less, or 0.1 mM or less, but is not limited thereto.

In the method of manufacturing an aging model skin cell line of the present invention, the skin cells are not limited as long as they are skin cells capable of detecting an aging index or measuring a cell proliferation rate in order to measure the degree of skin aging, and specifically, used for screening anti-aging substances. It may be a skin cell that can be, more specifically, may be a fibroblast, but is not limited thereto.

In the aging model skin cell line manufacturing method of the present invention, the culturing step may be to cultivate the isolated skin cells in a medium containing 0.01 to 0.9 mM pyruvate for 7 to 30 days, and specifically culture for 10 to 20 days More specifically, 7 days or more, 8 days or more, 9 days or more, 10 days or more, 11 days or more, 12 days or more, 13 days or more, 14 days or more, 15 days or more, 16 days or more, 17 days Culture for more than, 18 days or more, 19 days or more, 20 days or more, 21 days or more, 22 days or more, 23 days or more, 24 days or more, 25 days or more, 26 days or more, 27 days or more, 28 days or more, or 29 days or more May be 30 days or less, 29 days or less, 28 days or less, 27 days or less, 26 days or less, 25 days or less, 24 days or less, 23 days or less, 22 days or less, 21 days or less, 20 days or less, 19 Days or less, 18 days or less, 17 days or less, 16 days or less, 15 days or less, 14 days or less, 13 days or less, 12 days or less, 11 days or less, 10 days or less, 9 days or less, or 8 days or less However, it is not limited thereto.

According to an embodiment of the present invention, when the concentration of pyruvate contained in the culture medium is 1 mM, when the concentration of pyruvate is 0 mM or 0.1 mM, cell senescence of skin cells is promoted to prepare an aging model skin cell line for a shorter time. It was confirmed that it can be (Experimental Example 1). In addition, according to an embodiment of the present invention, when the concentration of pyruvate contained in the culture medium is 0.1 to 0.5 mM, cytotoxicity caused by anti-aging substances is alleviated, and aging model skin in the state most optimized for anti-aging substance screening. It was confirmed that a cell line could be obtained (Experimental Examples 2 and 3).

The senescence model skin cell line optimized in the present specification, for example, is a cell whose metabolic activity is degraded, excluding normal cells that have not undergone senescence and cells that are not alive due to cell death. It may be a cell in which the senescence index to be measured increases or decreases, the cell proliferation rate is decreased, and when treated with an anti-aging substance, it may mean a cell in which aging may be delayed or may not occur. That is, it may mean cells in a state that does not induce cell proliferation any more and exhibits an acceptable survival rate.

In another aspect, the present invention provides an isolated, aging model skin cell line prepared by the method for producing an aging model skin cell line. The aging model skin cell line may be for screening for anti-aging substances.

According to an embodiment of the present invention, an aging model skin cell line prepared in a pyruvate-deficient environment (0 mM pyruvate) has cytotoxicity due to antioxidants (Experimental Example 2-1), and an anti-aging substance (candidate). There is little change in the aging index or cell proliferation rate before and after treatment (Experimental Example 3-1), and it is not suitable for use in screening for anti-aging substances. On the other hand, the aging model skin cell line cultured in a culture medium containing 0.1 to 0.5 mM pyruvate alleviates or inhibits cytotoxicity caused by anti-aging substances (Experimental Example 2-2), and in particular, a culture containing 0.1 mM pyruvate The aging model skin cell line prepared in the medium was able to detect a distinct change in the aging index before and after treatment with the anti-aging candidate substance, and the cell proliferation rate was different, so it was confirmed that it is suitable for use in screening for anti-aging substances (Experimental Example 3-2).

In another aspect, the present invention provides a composition for preparing an aging model skin cell line comprising 0.01 to 0.9 mM or less of pyruvate as an active ingredient. The composition may be a culture medium for preparing an aging model skin cell line, and the description of the pyruvate concentration, the aging model skin cell line, and the aging model skin cell line production are as described above.

In another aspect, the present invention comprises the steps of treating an anti-aging candidate substance on an isolated aging model skin cell line prepared by the above manufacturing method; And it provides an anti-aging substance screening method comprising the step of detecting an aging index before and after treatment with the candidate substance or measuring a cell proliferation rate. The manufacturing method and the description of the aging model skin cell line are as described above.

The anti-aging substance screening method may further include determining an anti-aging substance when the expression level of the aging indicator after treatment with the candidate substance decreases by 10% or more compared to a control group not treated with the candidate substance. In the present specification, the senescence indicator may refer to a gene capable of confirming the degree of senescence of the cell by increasing or decreasing the amount of expression specifically in an aged cell or cell line, and an expression product of the gene. The aging indicator is, for example, beta-galactosidase (β-galactosidase) or aging-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase) activity, p16, p21 or p53 gene or The protein may be, but is not limited thereto, and includes known indicators of aging known in the art. The step of determining the anti-aging substance may be a step of determining an anti-aging substance when the expression level of the aging indicator after treatment with the candidate substance decreases by 10% or more compared to the control group not treated with the candidate substance, and specifically 10% or more, 12 % Or more, 14% or more, 16% or more, 18% or more, 20% or more, 30% or more, 32% or more, 34% or more, 36% or more, 38% or more, 40% or more, 42% or more, 44% or more , 46% or more, 48% or more, 50% or more, 52% or more, 54% or more, 56% or more, 58% or more, 60% or more, 62% or more, 64% or more, 66% or more, 68% or more, 70 % Or more, 72% or more, 74% or more, 76% or more, 78% or more, 80% or more, 82% or more, 84% or more, 86% or more, 88% or more, 90% or more, 92% or more, 94% or more If it is reduced by more than 96% or more than 98%, it may be a step of determining as an anti-aging substance.

The anti-aging substance screening method may further include determining an anti-aging substance when the cell proliferation rate after treatment with the candidate substance increases by 1.1 times or more compared to the control group not treated with the candidate substance. In the present specification, the cell proliferation rate means that the rate at which cells proliferate over time is quantified, and may be a cell proliferation rate measured using the following Equation 1, but is not limited thereto, and known in the art It includes those measured by the method of measuring the cell proliferation rate of.

[Equation 1]

The step of determining the anti-aging substance may be a step of determining an anti-aging substance when the cell proliferation rate after treatment with the candidate substance increases by 1.1 times or more compared to the control group not treated with the candidate substance, and specifically 1.1 times or more, 1.2 times or more, 1.3 times or more, 1.4 times or more, 1.5 times or more, 1.6 times or more, 1.7 times or more, 1.8 times or more, 1.9 times or more, 2 times or more, 2.1 times or more, 2.2 times or more, 2.3 times or more, 2.4 times An increase of more than, 2.5 times or more, 2.6 times or more, 2.7 times or more, 2.8 times or more, 2.9 times or more, or 3 times or more may be a step of determining as an anti-aging substance.

In another aspect, the present invention provides an in vitro model for screening anti-aging substances, including an isolated aging model skin cell line, prepared by the above manufacturing method. The manufacturing method, the aging model skin cell line, and the description of the anti-aging substance screening are as described above.

In another aspect, the present invention is an isolated aging model skin cell line prepared by the above manufacturing method; And an instruction sheet, wherein, after detecting an aging index before and after treatment with the candidate substance or measuring a cell proliferation rate, the expression level of the aging index after treatment with the candidate substance is 10% compared to the control group not treated with the candidate substance. Provides a kit for screening an anti-aging substance, including the content determined to be an anti-aging substance, when the cell proliferation rate after the treatment of the candidate substance decreases or increases by 1.1 times or more compared to the control group not treated with the candidate substance. The manufacturing method, aging model skin cell line, anti-aging substance screening, aging index, and description of the cell proliferation rate are as described above.

The skin cell line may be cryopreserved or carrier-preserved. The aging model skin cell line included in the kit may be included in the kit with the optimal aging state preserved by techniques well known to those skilled in the art in addition to the above-listed techniques in order to minimize additional cell proliferation or aging.

The senescence indicator is, for example, beta-galactosidase (β-galactosidase) or senescence-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase) activity, p16, p21 or p53 gene or The protein may be, but is not limited thereto, and includes known indicators of aging known in the art.

The cell proliferation rate may be a cell proliferation rate measured using Equation 1 below, but is not limited thereto, and includes those measured by a known cell proliferation rate measurement method known in the art.

[Equation 1]

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to experimental examples. However, the experimental examples below are provided for illustrative purposes only to aid understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[ Reference example ] Manufacture of anti-aging candidate substances

Each of the four anti-aging candidates was prepared by the following method.

1) Anti-aging candidate material 1 (Sprouted seed and fruit complex) preparation

Anti-aging candidate 1 (Sprouted seed and fruit complex) are Prunus mume fruit, Pinus koraiensis seed, Chaenomeles sinensis fruit, germinated Rosa multiflora fruit, and It is a mixture of five substances including germinated black Sesamum indicum seeds. The five substances of the same volume were extracted for 6 hours at 60 to 70°C using an aqueous ethanol solution (50% (v/v) ethanol-water) of 4 or 5 times the volume (v/w). At this time, the dissolved substances were filtered, dried, and powdered to prepare anti-aging candidate material 1.

2) Anti-aging candidate substance 2 (Tangja fruit extract ( Poncirus trifoliata fruit extract)) manufacturing

After mixing Poncirus trifoliata fruit with saline (salt solution, 0.5%), dissolve at 1 to 4°C for 7 days, mix 5 times the volume (v/w) of 70% ethanol aqueous solution, and then filter to melt. Only the solution was obtained. At this time, the separated material was dried and powdered to prepare an anti-aging candidate material 2.

3) Anti-aging candidate substance 3 (Jaum balancing complex) preparation

Anti-aging candidate 3 (Jaum balancing complex) is the roots of Paeonia lactiflora , flowers of Nelumbo nucifera , roots of Rehmannia glutinosa , bulbs of Lilium candidum , and rhizomes. (rhizomes of Polygonatum officinale ) is a mixture of five substances. After dissolving the mixture in a 10% ethanol aqueous solution of the same volume as the mixture for 18 hours or more, the dissolved mixture was filtered, dried, and powdered to prepare anti-aging candidate material 3.

4) Anti-aging candidate substance 4 (Plum tree extract ( Prunus mume extract)) manufacturing

The dried plum tree ( Prunus mume ) flowers were dissolved in a 10-fold volume (v/w) 70% ethanol aqueous solution at room temperature for 2 hours, filtered, dried, and powdered to prepare anti-aging candidate material 4.

[ Experimental example 1] Aging model according to pyruvate concentration Skin cell line Comparison of the degree of aging

Normal human dermal fibroblasts (Lonza) were seeded in a 12-well plate at 1-5 X 10 ⁴ and then culture medium containing 0 mM, 0.1 mM, and 1 mM pyruvate ( DMEM, WelGene) using a CO ₂ incubator under 37 ℃, 5% CO ₂ conditions for 2 weeks, three kinds of aging model skin cell lines were prepared.

Then, in order to compare the degree of senescence according to the pyruvate concentration of the three senescence model cell lines prepared above, the cell proliferation rate and senescence-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase) activity were evaluated. Measured.

Specifically, the cell proliferation rate was quantified for each of the three senescence model cell lines using the following formula 1, and the results are shown in FIG. 1A.

[Equation 1]

In addition, after staining each of the three aging model cell lines using the SA-βgal kit (Abcam), more than 200 cells were counted using MetaMorph (Molecular Devices), and the results were It is shown in Figure 1b.

As shown in FIG. 1A, the proliferation rate of a senescent model cell line cultured in a culture medium containing 1 mM pyruvate is about three times that of a aging model cell line cultured in a culture medium containing 0 mM and 0.1 mM pyruvate, respectively. It was confirmed to the extent. In addition, as shown in Figure 1b, the senescence model cell line cultured in a culture medium containing 0 mM and 0.1 mM pyruvate, respectively, compared to the aging model cell line cultured in a culture medium containing 1 mM pyruvate, SA-βgal activity It was confirmed that this increased significantly by more than about 12 times. At this time, it was confirmed that there was no significant difference in the degree of cellular senescence of the senescent model cell line when the concentration of pyruvate was 0 mM and 0.1 mM.

Through this, it was found that when the concentration of pyruvate contained in the fibroblast culture medium is less than 1 mM, for example, 0 to 0.1 mM, cell senescence is promoted, and thus an aging model skin cell line can be prepared for a shorter time. .

[Experimental Example 2] Evaluation of cytotoxicity during screening for anti-aging substances using an aging model skin cell line

The following experiment was performed to confirm whether cytotoxicity occurred by anti-aging candidate substances in aging model skin cell lines.

Experimental Example 2-1. Cytotoxicity evaluation when screening for anti-aging substances using an aging model skin cell line manufactured in a pyruvate deficient environment

The following experiment was performed to confirm whether cytotoxicity was caused by anti-aging candidate substances in an aging model skin cell line prepared in a pyruvate deficient environment (0 mM pyruvate).

Specifically, an aging model skin cell line was prepared in the same manner as in Experimental Example 1, but fibroblasts were cultured for 2 weeks in a culture medium containing 1 mM pyruvate without treatment with an anti-aging candidate substance to prepare an aging model skin cell line. (1 mM pyruvate treatment group), and the culture medium containing 0 mM pyruvate contains each of the anti-aging candidate substances 1 to 4 prepared in the reference example at the concentrations shown in FIG. The blast cells were cultured to prepare an aging model skin cell line (0 mM pyruvate treated group). Then, the prepared aging model skin cell line was stained with trypan blue and then the proportion of the stained cells was compared, and the cell viability was calculated using Equation 2 below. Cytotoxicity was measured through the derived cell viability, and the results are shown in FIG. 2.

[Equation 2]

As shown in FIG. 2, in the case of an aging model skin cell line not treated with an anti-aging substance, it was confirmed that the cell survival rate of the 0 mM pyruvate-treated group was lower than that of the 1 mM pyruvate-treated group. In addition, in the 0 mM pyruvate-treated group, there was no difference in cell viability even when anti-aging candidate substances 1, 3 and 4 were treated together, whereas when anti-aging candidate substance 2 was treated together, the cell viability was not treated with anti-aging substances. It was confirmed that the cell viability decreased in a concentration-dependent manner compared to the control group.

Through this, it was found that the aging model skin cell line prepared in a pyruvate-deficient environment (0 mM pyruvate) has an excellent effect of promoting cellular senescence, but is not suitable for use in screening anti-aging substances due to cytotoxicity.

Experimental Example 2-2. Cytotoxicity evaluation when screening for anti-aging substances using an aging model skin cell line manufactured with different concentrations of pyruvic acid

In order to confirm the concentration of pyruvate that can alleviate cytotoxicity caused by an anti-aging substance (candidate), the anti-aging candidate substance 2 was added to a culture medium containing pyruvate at a concentration of 0, 0.1 0.2, 0.5, and 1 mM, respectively. Treated together to prepare an aging model skin cell line in the same manner as in Experimental Example 1, and the cell viability of the aging model skin cell line was measured in the same manner as in Experimental Example 2-1, and the results are shown in FIG. 3.

As shown in Figure 3, the aging model skin cell line prepared in a pyruvate-deficient environment (0 mM pyruvate) has a very low cell viability, resulting in cytotoxicity due to anti-aging substances, whereas a concentration of less than 1 mM, specifically 0.1 to It was found that the aging model skin cell line cultured in a culture medium containing 0.5 mM pyruvate inhibited cytotoxicity by anti-aging substances.

[Experimental Example 3] Anti-aging evaluation of anti-aging candidate substances when screening for anti-aging substances using an aging model skin cell line

In order to confirm whether the aging model skin cell line was used for screening for anti-aging substances, an anti-aging evaluation experiment was performed on the anti-aging candidate substances in the following manner.

Experimental Example 3-1. Anti-aging evaluation of anti-aging candidate substances using an aging model skin cell line prepared in a pyruvate deficient environment

For the anti-aging evaluation of an anti-aging candidate substance using an aging model skin cell line prepared in a pyruvate-deficient environment (0 mM pyruvate), 1 mM pyruvate treatment group (1 mM pyruvate was used in the same manner as in Experimental Example 2-1). An aging model skin cell line prepared by culturing fibroblasts for 2 weeks without treatment with an anti-aging candidate substance in the included culture medium) and a 0 mM pyruvate treatment group (0 mM pyruvate, and the anti-aging candidate substances 1, 3 and 4) An aging model skin cell line) was prepared by culturing fibroblast cells for 2 weeks in a culture medium containing each of the concentrations in FIG. 4A.

Then, each of the prepared aging model skin cell lines was measured for cell proliferation rate and aging-related beta-galactosidase (SA-βgal) activity in the same manner as in Experimental Example 1, and the results are shown in FIGS. 4A and 4B. It is shown in 4b. At this time, the group without anti-aging candidate substance treatment group among the 0 mM pyruvic acid treatment group was used as a control group.

As shown in Figure 4a, the cell proliferation rate of the group not treated with the anti-aging candidate material (control group) of the 0 mM pyruvate-treated group was about 0.2 days, and the cells treated with the anti-aging candidate materials 1, 3 and 4, respectively It was confirmed that the growth rate was similar to that of the control group. In addition, as shown in FIG. 4B, it was confirmed that there was little difference in SA-βgal activity before and after treatment with the anti-aging candidate substance in the 0 mM pyruvate-treated group.

As a result, the aging model skin cell line prepared in a pyruvate-deficient environment (0 mM pyruvate) has little change in aging indicators or cell proliferation rate before and after treatment with anti-aging candidate substances, which is not suitable for use in screening anti-aging substances. And it was found.

Experimental Example 3-2. Anti-aging evaluation of anti-aging candidate substances using an aging model skin cell line prepared in a culture medium containing 0.1 mM pyruvate

Through the Experimental Example 2-2, it was confirmed that the aging model skin cell line cultured in a culture medium containing 0.1 to 0.5 mM pyruvate inhibits cytotoxicity by anti-aging substances, and culture containing 0.1 mM pyruvate Anti-aging evaluation of anti-aging candidate substances was performed using an aging model skin cell line prepared in a medium.

Specifically, in the same manner as in Experimental Example 2-1, 1 mM pyruvate-treated group (an aging model skin cell line prepared by culturing fibroblasts for 2 weeks without treatment with anti-aging candidate substances in a culture medium containing 1 mM pyruvate ) And 0.1 mM pyruvate-treated group (an aging model skin cell line prepared by culturing fibroblasts for 2 weeks in a culture medium containing 0.1 mM pyruvate and each of the anti-aging candidate substances 1 to 4 at the concentration of FIG. 5A) Was prepared.

Then, each of the prepared aging model skin cell lines was measured for cell proliferation rate and aging-related beta-galactosidase (SA-βgal) activity in the same manner as in Experimental Example 1, and the results are shown in FIGS. 5A and 5B. It is shown in 5b. At this time, the group not treated with anti-aging candidate substances among the 0.1 mM pyruvic acid treatment group was used as a control group.

As shown in Figure 5a, the cell proliferation rate of the group not treated with the anti-aging candidate material (control group) among the 0.1 mM pyruvate-treated group was about 0.2 days, and the cell proliferation rate when the anti-aging candidate material 1 was treated was compared with the control group. Similar, it was confirmed that the cell proliferation rate when treated with anti-aging candidate material 2 decreased compared to the control, whereas the cell proliferation rate when treated with anti-aging candidate material 3 and 4, respectively, increased compared to the control. In addition, as shown in FIG. 5B, there is little difference in SA-βgal activity before and after treatment with each of anti-aging candidate substances 1 and 2 in the 0.1 mM pyruvate-treated group, whereas anti-aging candidate substances 3 and 4 were each treated. It was confirmed that the SA-βgal activity decreased to about 1/3 to 1/5 of the control group.

Through this, it can be seen that an anti-aging effect occurs when anti-aging candidate substances 3 and 4 are treated among anti-aging candidate substances 1 to 4, and an aging model skin cell line prepared in a culture medium containing 0.1 mM pyruvate is an anti-aging candidate. It was found that a clear change in the aging index before and after treatment with the substance could be detected, and the cell proliferation rate was different, so it was suitable for use in screening for anti-aging substances.

Therefore, the aging model skin cell line prepared according to the aging model skin cell line manufacturing method comprising the step of treating the isolated skin cells according to an aspect of the present invention with a pyruvate of 0.01 to 0.9 mM or less is used to screen for anti-aging substances. It was confirmed that there is an excellent effect on.

Claims (17)

A method for producing an aging model skin cell line comprising culturing the isolated skin cells in a medium containing 0.1 to 0.5 mM pyruvate. The method of claim 1,
The skin cells are fibroblasts, aging model skin cell line manufacturing method. The method of claim 1,
The culture is to culture the isolated skin cells for 7 to 30 days, aging model skin cell line manufacturing method. The method of claim 3,
The culture is to culture the separated skin cells for 10 to 20 days, aging model skin cell line manufacturing method. An isolated, aging model skin cell line prepared by the method of any one of claims 1 to 4. The method of claim 5,
The aging model skin cell line is for screening for anti-aging substances, aging model skin cell line. A composition for preparing an aging model skin cell line comprising 0.1 to 0.5 mM of pyruvate as an active ingredient. Treating an anti-aging candidate substance on the aging model skin cell line of claim 5; And
An anti-aging substance screening method comprising the step of detecting an aging index before and after treatment with the candidate substance or measuring a cell proliferation rate. The method of claim 8,
When the expression amount of the aging indicator after the treatment of the candidate substance decreases by 10% or more compared to the control group not treated with the candidate substance, the method further comprises determining an anti-aging substance. The method of claim 8,
The aging indicator is beta-galactosidase (β-galactosidase), anti-aging substance screening method. The method of claim 8,
If the cell proliferation rate after treatment with the candidate substance increases by 1.1 times or more compared to the control group not treated with the candidate substance, the method further comprises determining an anti-aging substance. The method of claim 8,
The cell proliferation rate is measured using the following formula 1, anti-aging substance screening method.
[Equation 1]

An in vitro model for screening anti-aging substances, comprising the aging model skin cell line of claim 5. The aging model skin cell line of claim 5; And
Contains instructions,
In the above directive, after detecting the aging index before and after treatment with the candidate substance or measuring the cell proliferation rate, the expression level of the aging index after treatment with the candidate substance decreases by 10% or more compared to the control group not treated with the candidate substance. A kit for screening anti-aging substances, which includes the content determined as anti-aging substances when the cell proliferation rate after treatment increases by 1.1 times or more compared to the control group not treated with the candidate substance. The method of claim 14,
The skin cell line is cryopreserved or carrier-preserved, anti-aging material screening kit. The method of claim 14,
The aging indicator is beta-galactosidase (β-galactosidase), anti-aging material screening kit. The method of claim 14,
The cell proliferation rate is measured using the following formula 1, anti-aging material screening kit.
[Equation 1]

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