KR20200055979A - Method for making skin senescent models, skin senescent models therefrom and method for screening anti-aging material using the same - Google Patents

Abstract

The present invention relates to a method for manufacturing a skin aging model which can be used for the study of aging phenomena manufactured by treating dichloroacetate on isolated adipose-derived stem cells, the skin aging model manufactured by the method, and a method for screening anti-aging matter using the skin aging model. By treating dichloroacetate on isolated adipose-derived stem cells, there is an excellent effect of manufacturing the skin aging model in a time and cost-effective manner.

Description

Method for making skin senescent models, skin senescent models therefrom and method for screening anti-aging material using the same}

In the present specification, a method for producing a skin aging model that can be used for the study of aging phenomena produced by treating isolated adipose-derived stem cells with dichloroacetate, a skin aging model produced by the method, and anti-aging using the model A method for screening a material is disclosed.

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

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

Skin aging is largely divided into natural aging (or endogenous aging) and exogenous aging. 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 It is easy. Representative exogenous aging factors include ultraviolet light, reactive oxygen species, and stress.

Therefore, recently, methods for improving exogenous aging have been actively researched, and in particular, efforts are being made to identify substances for preventing or improving aging. There is a method of subculture several times in order to manufacture a conventional skin aging model, and the skin aging model produced using it was used for verification and discovery of anti-aging substances. However, this method has a disadvantage in that it takes a cell culture for at least 1 month, so that it takes time and money.

Accordingly, the present inventor has completed the present invention by studying a method capable of manufacturing a skin aging model at a lower cost in a shorter time.

KR 10-2018-0019613 A KR 1,040,486 B JP 平 11-0501653 A

Protective effects of adipose-derived stem cells secretome on human dermal fibroblasts from ageing damages, Int J Clin Exp Pathol, 2015; 8 (12): 15739-15748. Characterization of Senescence of Culture-expanded Human Adipose-derived Mesenchymal Stem Cells, Int J Stem Cells. 2016 May; 9 (1): 124-136. P.W.Stacpoole, The pyruvate dehydrogenase complex as a therapeutic target for age-related diseases, Aging Cell, 2012, vol. 11, pp371-377.

In one aspect, an object of the present invention is to provide a method for preparing a skin aging model comprising the step of treating dichloroacetate (DCA) on isolated fat-derived stem cells (ADSCs).

In another aspect, an object of the present invention is to provide an isolated, skin aging model, produced by the method for preparing a skin aging model.

In another aspect, an object of the present invention is to provide a composition for preparing a skin aging model comprising dichloroacetate (DCA) as an active ingredient.

In another aspect, an object of the present invention, the skin aging model produced by the skin aging model manufacturing method, treating the anti-aging candidate material; And detecting an aging index before and after treatment with the candidate material, to provide an anti-aging screening method.

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

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

In one aspect, the present invention provides a method for producing a skin aging model comprising the step of treating dichloroacetate (DCA) on isolated fat-derived stem cells (ADSC).

In another aspect, the present invention provides a separated, skin aging model prepared by the method for preparing a skin aging model.

In another aspect, the present invention provides a composition for preparing a skin aging model comprising dichloroacetate (DCA) as an active ingredient.

In another aspect, the present invention, the skin aging model produced by the skin aging model manufacturing method, treating an anti-aging candidate material; And detecting an aging index before and after the candidate material treatment.

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

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

In the present invention, unlike the conventional method for preparing a skin aging model through passage culture that takes more than one month, skin aging is time and cost-effective by treating dichloroacetate derived fat-derived stem cells for a period of about 2 weeks or less. It was confirmed that a model could be prepared. The skin aging model produced by the manufacturing method of the present invention has an excellent effect of quickly and accurately screening anti-aging substances.

1 is a graph showing the aging-related beta-galactosidase (SA-βgal) activity of a skin aging model (Example) and a control prepared by treating dichloroacetate (DCA) on adipose-derived stem cells for 2 weeks. .
FIG. 2 is a diagram showing the results of Western blot of pPDH S232, PDH, p21 and HMGB1 of skin aging model (Example) and control prepared by treating dichloroacetate (DCA) on adipose-derived stem cells for 2 weeks. .

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention provides a method for preparing a skin aging model, comprising the step of treating dichloroacetate (DCA) on isolated fat-derived stem cells (ADSC).

The isolated adipose-derived stem cells may be passaged 3 to 20 times, specifically 3 or more times, 4 or more times, 5 or more times, 6 or more times, 7 or more times, 8 or more times, 9 or more times, 10 More than 11 times, more than 11 times, more than 12 times, more than 13 times, more than 14 times, more than 15 times, more than 16 times, more than 17 times, more than 18 times, or more than 19 times Below, 18 or below, 17 or below, 16 or below, 15 or below, 14 or below, 13 or below, 12 or below, 11 or below, 10 or below, 9 or below, 8 or below, 7 or below , 6 or less, 5 or less or 4 or less may be passaged, but the number of passages is not limited if fat-derived stem cells capable of producing a skin aging model by dichloroacetate treatment.

In the method for preparing a skin aging model of the present invention, the dichloroacetate increases the expression or activity of pyruvate dehydrogenase (PDH) present in the mitochondria of adipose-derived stem cells, and pyruvate dehydrogenase, which is an activity inhibitory enzyme of the PDH. By inhibiting the expression or activity of phosphatase (pyruvate dehydrogenase kinase, PDK), it may be to induce aging in adipose-derived stem cells. The pyruvate dehydrogenase phosphatase (PDK) is an enzyme (kinase) that phosphorylates the pyruvate dehydrogenase (PDH), and is inactive when the pyruvate dehydrogenase (PDH) is phosphorylated. The dichloroacetate is an inhibitor of the expression or activity of pyruvate dehydrogenase phosphatase (PDK), and the expression or activity of pyruvate dehydrogenase phosphatase (PDK) is inhibited by the dichloroacetate treatment, thereby inhibiting the phosphorylation of pyruvate dehydrogenase (PDH). As it is, the expression or activity of the pyruvate dehydrogenase (PDH) is increased or enhanced, and aging may be induced. According to one embodiment of the present invention, when dichloroacetate is treated for 2 weeks on isolated adipose-derived stem cells passaged 10 or more times (Example), the protein of phosphorylated pyruvic acid dehydrogenase (pPDH S232) compared to the control group As the amount (expression amount) was reduced, it was confirmed that phosphorylation of pyruvate dehydrogenase (PDH) was suppressed by dichloroacetate treatment, thereby making it possible to prepare a skin aging model efficiently and timely by enhancing the pyruvate dehydrogenase activity. (Experimental Example 2).

In the method for preparing a skin aging model of the present invention, the concentration of the dichloroacetate may be 0.01 mM to 1000 mM, specifically, 0.01 mM or more, 0.1 mM or more, 0.5 mM or more, 1 mM or more, 2 mM or more, 3 mM or more , 4 mM or more, 5 mM or more, 6 mM or more, 7 mM or more, 8 mM or more, 9 mM or more, 10 mM or more, 20 mM or more, 30 mM or more, 40 mM or more, 50 mM or more, 60 mM or more, 70 mM or more, 80 mM or more, 90 mM or more, 100 mM or more, 200 mM or more, 300 mM or more, 400 mM or more, 500 mM or more, 600 mM or more, 700 mM or more, 800 mM or more or 900 mM or more, 1000 mM or less, 900 mM or less, 800 mM or less, 700 mM or less, 600 mM or less, 500 mM or less, 400 mM or less, 300 mM or less, 200 mM or less, 100 mM or less, 90 mM or less, 80 mM or less, 70 mM or less , 60 mM or less, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 10 mM or less, 9 mM or less, 8 mM or less, 7 mM or less, 6 mM or less, 5 mM or less, 4 mM or less, 3 It may be mM or less, 2 mM or less, 1 mM or less, 0.5 mM or less, or 0.1 mM or less, but the concentration of dichloroacetate capable of producing a skin aging model from adipose derived stem cells is not limited.

In the skin aging model manufacturing method of the present invention, the treatment of the dichloroacetate may be for 7 to 30 days, specifically for 10 to 20 days, and more specifically for 7 days or more, 8 days or more , 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 Days, 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, 29 days or more, 30 days or less, 29 days or less, 28 days 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, It may be 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, but is not limited thereto.

According to one embodiment of the present invention, when treated with dichloroacetate for 2 weeks on isolated adipose-derived stem cells passaged 10 or more times (Example), SA-βgal activity as an aging index increased and p21 compared to the control group. It was confirmed that the expression level of and the expression level of HMGB1 decreased, so that a skin aging model could be produced efficiently and timely (Experimental Examples 1 and 2).

The skin aging model in an optimized state in the present specification is a cell in which metabolic activity is lowered, except for normal cells that have not undergone aging and cell death that has not already occurred during cell population, and measures the degree of cell aging. The aging indicator may be a cell that increases or decreases, a cell that decreases the rate of cell proliferation, and may mean a cell that may or may not delay aging when treated with an anti-aging agent. That is, it may mean a cell in a state showing an allowable survival rate without inducing cell proliferation.

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

According to an embodiment of the present invention, the skin aging model prepared by treating dichloroacetate for 2 weeks on isolated adipose-derived stem cells passaged 10 or more times (Example) is SA-βgal, an aging index compared to a control group. As the activity increased, the expression level of p21 increased, and the expression level of HMGB1 decreased, it was confirmed that it is suitable for use in screening anti-aging substances (Experimental Examples 1 and 2).

In another aspect, the present invention provides a composition for preparing a skin aging model comprising dichloroacetate (DCA) as an active ingredient. The composition may be a culture medium for preparing a skin aging model, but is not limited thereto.

In the composition of the present invention, dichloroacetate may be included in an amount of 0.001 to 50% by weight based on the total weight of the composition, specifically 0.001% by weight or more, 0.005% by weight or more, 0.01% by weight or more, 0.02% by weight or more, 0.03% by weight or more , 0.04 wt% or more, 0.05 wt% or more, 0.06 wt% or more, 0.07 wt% or more, 0.075 wt% or more, 0.08 wt% or more, 0.09 wt% or more, 0.1 wt% or more, 0.5 wt% or more, 1 wt% or more , 5 wt% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, 30 wt% or more, 35 wt% or more, 40 wt% or more, or 45 wt% or more, and 50 wt% % Or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 1% %, 0.5% or less, 0.1% or less, 0.09% or less, 0.08% or less, 0.075% or less, 0.07% or less, 0.06% or less, or 0.05% or less If the content of dichloroacetate that can be prepared is not limited thereto.

In another aspect, the present invention comprises the steps of treating the anti-aging candidate material to the separated skin aging model, prepared by the above manufacturing method; And detecting an aging index before and after the candidate material treatment. The manufacturing method and description of the skin aging model are as described above.

The anti-aging material screening method may further include determining an anti-aging material when the expression level of the aging index after treatment of the candidate material is increased or decreased by 10% or more compared to a control group not treated with the candidate material. In the present specification, the aging index may mean a gene capable of confirming the degree of aging of a cell by specifically increasing or decreasing the expression level in an aged cell or cell line, and an expression product of the gene. The aging indicator is, for example, activity of beta-galactosidase (β-galactosidase) or aging-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase), p16, p21, p53, High mobility group box 1 (HMGB1) gene, or may be a protein, specifically, beta-galactosidase (β-galactosidase), p21 and high mobility group box 1 (High mobility group box 1) , HMGB1) may be one or more selected from the group consisting of, but is not limited to, it includes known aging indicators known in the industry. The anti-aging substance determination step may be a step of determining as an anti-aging substance when the expression level of HMGB1 after treatment of the candidate substance after treatment of the candidate substance increases by 10% or more compared to a control group not treated with the candidate substance, 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, 96% or more or 98% or more, it may be a step of determining as an anti-aging material. Alternatively, in the step of determining the anti-aging substance, if the expression level of beta-galactosidase or p21 after the treatment of the candidate substance decreases by 10% or more compared to the control group not treated with the candidate substance, it is determined as an anti-aging substance. It may be a step, 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% Above, above 40%, above 42%, above 44%, above 46%, above 48%, above 50%, above 52%, above 54%, above 56%, above 58%, above 60%, above 62%, 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 Above, 90% or more, 92% or more, 94% or more, 96% or more or 98% or more reduction may be a step of determining as an anti-aging material.

In another aspect, the present invention provides an in vitro model for screening anti-aging substances, including a separated skin aging model, prepared by the above method. Descriptions of the manufacturing method, skin aging model, and anti-aging material screening are as described above.

In another aspect, the present invention is a separate skin aging model prepared by the above manufacturing method; And an instruction, wherein after detecting the aging indicator before and after treatment with the candidate substance, the expression level of the aging indicator after treatment of the candidate substance increases or decreases by 10% or more compared to the control group not treated with the candidate substance. Provided is a kit for screening for anti-aging substances, which includes what is judged to be an aging substance. Description of the manufacturing method, skin aging model, anti-aging material screening, and aging indicators are as described above.

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

The aging indicator is, for example, activity of beta-galactosidase (β-galactosidase) or aging-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase), p16, p21, p53, High mobility group box 1 (HMGB1) gene, or may be a protein, specifically, beta-galactosidase (β-galactosidase), p21 and high mobility group box 1 (High mobility group box 1) , HMGB1) may be one or more selected from the group consisting of, but is not limited to, it includes known aging indicators known in the industry.

Hereinafter, the configuration and effects of the present invention will be described in more detail with reference to Examples and Experimental Examples. However, the following examples and experimental examples 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 thereby.

 [ Manufacturing example ] Skin aging model manufacturing

Adipose-derived stem cells (ADSC) (Lonza) in adipose derived stem cell growth media (ADSCGM) (Lonza) supplements and growth factors (ADSC) -GM SingleQuots ^TM Kit, Lonza) was added and cultured in a 5% CO ₂ incubator at 37 ° C. In the following, fat-derived stem cells of passage 10 or less (passage number-10 or less) were used.

In order to examine the effect of dichloroacetate (DCA) on aging, a skin aging model was prepared by treating DCA 5 mM (Sigma) with fat-derived adipose-derived stem cells for 2 weeks (Example). At this time, the control group was treated with distilled water (DW) instead of DCA. During the treatment period, the medium was changed every two days, and each time the medium was changed, distilled water was treated as a control and DCA 5 mM was treated in the above example.

[ Experimental Example  1] Aging-related beta-galactosidase (SA- βgal , senescence-associated β-galactosidase) activity measurement

In order to measure the aging degree of the skin aging model of the above example, aging-related beta-galactosidase (SA-βgal, senescence-associated β-galactosidase) activity was measured.

Specifically, in order to measure aging-related beta-galactosidase activity, which is an aging indicator (SA-βgal activity assay) (Enzo life science), washing the cells of the control and the examples prepared in the preparation example with PBS After that, each cell lysate was obtained using a lysis buffer. After each of the obtained cell lysate was mixed with an assay buffer and reacted at 37 ° C. for 2 hours, the intensity of each fluorescence was measured, and the results are shown in FIG. 1.

As shown in FIG. 1, it was confirmed that SA-βgal activity increased by about 1.4 times or more in cells prepared by treating DCA (Example) compared to a control group not treated with DCA.

Through this, it was found that when DCA was treated with adipose-derived stem cells, a skin aging model could be produced at a lower cost for a shorter time.

[ Experimental Example  2] Phosphorylated PDH , p21 and HMGB1  Expression level measurement

In order to measure the degree of aging of the skin aging model of the above example, the amount of expression of phosphorylated pyruvate dehydrogenase (PDH), p21 and High mobility group box 1 (HMGB1) was Western blot. ).

Specifically, after obtaining the cell lysate of each of the control and the examples prepared in the above preparation example using a RIPA lysis buffer (Millipore), a protein assay kit (Bio Protein was quantified using -rad). Cell lysate containing the same amount of protein was separated using SDS-PAGE, transferred to a PVDF membrane, and then blocked using a blocking solution (thermos scientific). Anti-pPDH S232 (calbiochem), anti-PDH (novex), anti-p21 (cell signaling) or anti-HMGB1 (abcam), tris-buffered saline (TBS) with 0.1% tween 20 (sigma) (thermo scientific ) And the membrane was incubated for at least 12 hours, and then the protein amount was measured using an ECL chemiluminescence system (Amersham Pharmacia Biotech), and the results are shown in FIG. 2. In FIG. 2, there was no difference in the amount of protein in PDH in the cells of the control and the example, and PDH was used as a loading control.

As shown in FIG. 2, the protein amount of pPDH S232 was decreased in cells prepared by treating DCA as compared to a control group not treated with DCA, and phosphorylation of pyruvate dehydrogenase (PDH) was inhibited by DCA treatment. It means that In addition, unlike the control group not treated with DCA, the expression level of p21, which is known to increase during cell aging, is increased in cells (Example) prepared by treating DCA, and the expression level of HMGB1, which is known to decrease when cell aging, decreases. It could be observed.

Through this, it was found that when DCA was treated on adipose-derived stem cells, a skin aging model could be produced for a shorter time and at a lower cost.

Therefore, a method for preparing a skin aging model comprising the step of treating dichloroacetate (DCA) on an isolated adipose-derived stem cell (ADSC) according to an aspect of the present invention is time and cost efficient. It was confirmed that a skin aging model can be prepared.

Claims (15)

A method of manufacturing a skin aging model, comprising the step of treating dichloroacetate (DCA) on isolated fat-derived stem cells (ADSC). According to claim 1,
The isolated fat-derived stem cells are passaged 3 to 20 times, skin aging model manufacturing method. According to claim 1,
The treatment of the dichloroacetate is to be treated for 7 to 30 days, skin aging model manufacturing method. According to claim 3,
The treatment of the dichloroacetate is to be treated for 10 to 20 days, skin aging model manufacturing method. An isolated, skin aging model produced by the method of claim 1. The method of claim 5,
The skin aging model is for screening anti-aging substances, the skin aging model. A composition for preparing a skin aging model comprising dichloroacetate (DCA) as an active ingredient. Treating the anti-aging candidate substance in the skin aging model of claim 5; And
And detecting an aging index before and after the candidate substance treatment. The method of claim 8,
The aging indicator is at least one selected from the group consisting of beta-galactosidase (β-galactosidase), p21 and High mobility group box 1 (HMGB1), anti-aging screening method. The method of claim 8,
When the expression level of the aging indicator after treatment of the candidate substance increases or decreases by 10% or more compared to a control group not treated with the candidate substance, the method further comprises determining as an anti-aging substance, the anti-aging substance screening method. The method of claim 10,
The anti-aging substance determination step,
Determining an anti-aging substance when the expression level of HMGB1 after treatment of the candidate substance increases by 10% or more compared to a control group not treated with the candidate substance; or
When the expression level of beta-galactosidase (p-galactosidase) or p21 after treatment of a candidate substance decreases by 10% or more compared to a control group not treated with the candidate substance, the step of determining an anti-aging substance is an anti-aging substance screening method. An in vitro model for screening for anti-aging substances, including the skin aging model of claim 5. The skin aging model of claim 5; And
Includes instructions,
In the above instructions, after detecting the aging indicator before and after treatment with the candidate substance, if the expression level of the aging indicator after treatment of the candidate substance is increased or decreased by 10% or more compared to the control group not treated with the candidate substance, it is determined to be an anti-aging substance. Containing, anti-aging material screening kit. The method of claim 13,
The skin aging model is a cryopreservation or carrier preservation state, anti-aging material screening kit. The method of claim 13,
The aging indicator is at least one selected from the group consisting of beta-galactosidase (β-galactosidase), p21 and High mobility group box 1 (HMGB1), anti-aging screening kit.

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