Method for producing aging model skin cell line by using house dust mites, and aging model skin cell line produced using same

Abstract

The present invention relates to a method for producing an aging model skin cell line by using house dust mites and, more specifically, to a method for producing an aging model skin cell line, and an aging model skin cell line produced using same, the method comprises the steps of: culturing isolated skin cells in a medium comprising fetal bovine serum; and adding a purified house dust mite extract at a concentration of 10 to 300 μg / ml to the cultured skin cells so as to induce aging of the cultured skin cells. The present invention provides the aging model skin cell line, which can be used as an alternative to animal experiments and has the effect of not causing a decrease in copy number even upon subculture.

Description

Method for preparing an aging model skin cell line using house dust mites and an aging model skin cell line prepared thereby

[0001] The present invention provides a method to replace animal testing in the study of aging phenomena or anti-aging substances by preparing an aging model skin cell line induced by a specific concentration of house dust mites (HDM). By preparing an aging model skin cell line similar to actual skin aging conditions, the present invention can serve as an optimal test model for verifying the anti-aging effects of cosmetics or pharmaceuticals in vitro and for use in screening for the development of cosmetic compositions or drugs for anti-aging.

[0002] The skin, which protects the body from the external environment, undergoes aging primarily through natural aging (or intrinsic aging) and extrinsic aging. Natural aging is difficult to control artificially because it is influenced by metabolic processes and genetic factors within the human body, whereas extrinsic aging is relatively easy to control artificially because it is influenced by environmental factors. Representative causes of aging include ultraviolet radiation, reactive oxygen species (ROS), and stress. Under normal conditions, the activity of receptor tyrosine kinase (RTK) on the surface of skin cells is inhibited by receptor protein tyrosine kinase (RPTP). However, when exposed to ultraviolet radiation, the skin generates reactive oxygen species that bind to RPTP. As a result, RTK is phosphorylated, inducing the transcription factors NF-kB (nuclear factor kappa-light-chain-enhancer of activated B cells) and AP-1 (activator protein-1). These transcription factors inhibit collagen production by reducing TGF-β receptors and decrease collagen content by increasing the transcription of MMP (matrix metalloproteinase) genes. Research on cosmetics and drugs aimed at preventing or reversing such skin aging is active, and studies on skin aging and treatment are continuously being conducted under extracellular laboratory conditions.

[0003] Recently, methods to improve extrinsic aging are being actively researched, and in particular, efforts to identify substances for anti-aging or improvement are continuing. Conventional technologies for manufacturing skin cell aging models include replicative senescence models, which take a long time to produce, or models that induce excessive stress (such as oxidizing agent treatment, UV treatment, or radiation treatment). Skin cell lines produced using these aging models have been utilized in studies to verify and discover anti-aging substances. However, these models have the disadvantage of not being applicable to in vivo aging models due to the long production time required or the excessive stress that does not occur in vivo.

[0004] Therefore, there is a need for research on aging model skin cell lines that can be used as an alternative to animal testing for the verification and discovery of anti-aging substances to inhibit or prevent skin cell aging, thereby shortening production time, and can be usefully applied to conducting physiological, molecular biological, and biochemical studies related to the skin of animals, including humans.

[0005] [Prior Art Literature]

[0006] [Patent Literature]

[0007] Korean Patent Publication No. 10-2023-0011059

[0008] The problem that the present invention aims to solve is to establish an aging model skin cell line that can be produced in a short period of time and allows for repeated experiments in vitro using a purified house dust mite extract. In addition, the invention aims to confirm the anti-aging effects of cosmetics or pharmaceuticals, etc., without relying on animal testing by using the aging model skin cell line produced thereby, and to provide a method for screening anti-aging substances for the development of cosmetic compositions or drugs for preventing aging.

[0009] This patent is based on research conducted with support from Gyeongsangbuk-do and Gyeongsan-si in 2024 (No. 20245130000000167302, Anti-aging Research and Technology Development Project).

[0010] To solve the above problem, the present invention provides a method for preparing an aging model skin cell line comprising the steps of: culturing isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml; and measuring the p21 protein expressed in the cultured skin cells to induce aging of the cultured skin cells, and an aging model skin cell line prepared therefrom.

[0011] According to the method for producing an aging model skin cell line according to the present invention, by culturing isolated skin keratinocytes or skin epithelial cells in a medium containing fetal bovine serum and house dust mite extract, an aging model skin cell line that can be produced quickly without a decrease in copy number even by subculture can be provided.

[0012] In addition, according to the method for preparing an aging model skin cell line of the present invention, the aging model skin cell line established by the above method can be used to replace animal experiments in the study of aging phenomena or anti-aging substances by establishing an environment in vitro that is similar to the aging phenomenon occurring in vivo.

[0013] FIG. 1 is a schematic diagram of a method for preparing an aging model skin cell line using house dust mites according to one embodiment of the present invention.

[0014] FIG. 2 shows the expression levels of p21 protein expressed on the 1st and 2nd days after culture of skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF) cultured in a medium composition containing house dust mite purified extract (HDM) according to one embodiment of the present invention, and the p21 protein expressed after culture of cells cultured in a medium composition containing doxorubicin as a control.

[0015] Figure 3 shows p21 protein and β-actin expressed after culturing skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF) in a culture medium composition comprising 10% fetal bovine serum and a purified house dust mite extract at a concentration of 100 μg / ml according to one embodiment of the present invention.

[0016] Figure 4 shows the p21 protein expression levels according to the day of culture of skin epithelial cells (NS-HDF) cultured in a medium composition containing 50 nM doxorubicin according to one embodiment of the present invention and skin epithelial cells cultured in a medium composition containing 3% fetal bovine serum and 100 μg / ml of purified house dust mite extract.

[0017] Figure 5 shows the results of screening anti-aging substances using an aging model skin cell line according to one embodiment of the present invention, and it can be confirmed that when an anti-aging candidate substance is administered, the expression level of p21 protein in aging skin cells decreases.

[0018] Hereinafter, various embodiments of the present invention are described with reference to the accompanying drawings. The present invention is not limited to specific embodiments and should be understood to include various modifications, equivalents, and / or alternatives of the embodiments of the present invention. In relation to the description of the drawings, similar reference numerals may be used for similar components.

[0019] In this document, expressions such as "have," "can have," "include," or "can include" refer to the existence of the relevant feature (e.g., numerical values, functions, actions, or components, etc.) and do not exclude the existence of additional features.

[0020] In this document, expressions such as “A or B,” “at least one of A or / and B,” or “one or more of A or / and B” may include all possible combinations of items listed together. For example, “A or B,” “at least one of A and B,” or “at least one of A or B” may refer to cases including (1) at least one A, (2) at least one B, or (3) both at least one A and at least one B.

[0021] As used in this document, the expression "configured to" may be replaced, depending on the context, with, for example, "suitable for," "having the capacity to," "designed to," "adapted to," "made to," or "capable of." The term "configured to" does not necessarily mean "specifically designed to."

[0022] The terms used in this document are used merely to describe specific embodiments and are not intended to limit the scope of other embodiments. Singular expressions may include plural expressions unless the context clearly indicates otherwise. Terms used herein, including technical or scientific terms, may have the same meaning as generally understood by those skilled in the art described in this document. Terms used in this document that are defined in general dictionaries may be interpreted as having the same or similar meaning as they have in the context of the relevant technology, and are not to be interpreted in an ideal or overly formal sense unless explicitly defined in this document. In some cases, even terms defined in this document may not be interpreted to exclude the embodiments of this document.

[0023] The embodiments disclosed in this document are presented for the purpose of explaining and understanding the disclosed technical content and are not intended to limit the scope of the invention. Accordingly, the scope of this document should be interpreted to include all modifications or various other embodiments based on the technical concept of the invention.

[0024] Hereinafter, preferred embodiments of the present invention will be described in detail. Prior to this, terms and words used in this specification and claims should not be interpreted as being limited to their ordinary or dictionary meanings, and should be interpreted in a meaning and concept consistent with the technical spirit of the present invention, based on the principle that the inventor can appropriately define the concept of the terms to best describe his invention.

[0025] Therefore, it should be understood that the configurations of the embodiments described in this specification are merely some of the most preferred embodiments of the present invention and do not represent all of the technical ideas of the present invention, and that various equivalents and modifications that can replace them may exist at the time of filing this application.

[0026] Throughout the specification, when a part is described as "including" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0027] The present invention will be described in detail below.

[0028]

[0029] A method for preparing an aging model skin cell line according to one embodiment of the present invention may include the steps of: culturing isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml; and measuring the p21 protein expressed in the cultured skin cells to induce aging of the cultured skin cells. Each step will be described in detail below.

[0030] The step of culturing the above-mentioned isolated skin cells in a medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml is to cultivate them in a medium containing fetal bovine serum to improve sensitivity and infectivity to purified house dust mite extract that causes skin cell aging.

[0031] The aforementioned purified house dust mite extract refers to a product obtained by purifying house dust mites. House dust mites are very small mites, typically measuring about 0.4 mm in length and 0.3 mm in width, and are one of the causes of allergic reactions. House dust mites feed on dead skin cells and dandruff shed from the human body. Since house dust mites prefer these dead skin cells and dandruff to be partially decomposed by mold, they break down enzymes to decompose their prey; the main cause of allergies is that the effects of these components persist even after the mites have excreted them. These house dust mites or their excrement can cause dermatitis and skin aging upon contact with the skin, and can damage the lungs if inhaled.

[0032] The isolated skin cells mentioned above may be skin cells capable of detecting aging indicators or measuring cell proliferation rates to measure the degree of skin aging. Specifically, the isolated skin cells may be one or more selected from the group consisting of skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF), but are not limited thereto.

[0033] In the step of culturing the isolated skin cells in a culture medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, the fetal bovine serum may be 1-20% fetal bovine serum. Preferably, the fetal bovine serum may be 3-15% fetal bovine serum. More preferably, the fetal bovine serum may be one or more of 3% fetal bovine serum or 10% fetal bovine serum. Fetal bovine serum is a substance containing various growth factors, and when included in the culture medium during cell culture, it has an excellent cell proliferation effect. When cells are cultured using a medium containing fetal bovine serum at a concentration below the above concentration range, cell proliferation occurs insufficiently and the expression of p21, a cell aging marker, cannot be confirmed; conversely, when cells are cultured using a medium containing fetal bovine serum at a concentration exceeding the above concentration range, there may be mutability of the cell tissue and the generation of toxins that may cause deformation of the cell surface.

[0034] In the step of culturing isolated skin cells in a medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, the medium composition refers to a medium capable of supporting skin cell growth and survival in vitro, and includes all conventional media used in the field suitable for cell culture. The medium composition and culture conditions may be selected according to the type of isolated skin cells. Examples of the medium composition include MR-IVF TMThere are DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, aMEM (a Minimal Essential Medium), GMEM (Glasgow's Minimal Essential Medium), Iscove's Modified Dulbecco's Medium, etc., but preferably DMEM culture medium can be used, but is not limited thereto.

[0035] In the step of culturing the isolated skin cells in a culture medium composition containing fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, aging of the skin cells can be artificially induced by the purified house dust mite extract. Preferably, the concentration of the purified house dust mite extract may be 50 to 200 μg / ml. Most preferably, the concentration of the purified house dust mite extract may be 100 to 200 μg / ml. If purified house dust mite extract is administered at a concentration below the above numerical range, it is difficult to confirm the expression of one or more of p16, p21, p53, and β-actin, which are aging-related biomarkers capable of confirming skin cell aging; and if purified house dust mite extract is administered at a concentration above the above numerical range, this results in the injection of excessive stress, which is very different from aging that actually occurs in the skin or in vivo.

[0036] In the step of culturing the isolated skin cells in a culture medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, the weight ratio of the fetal bovine serum to the purified house dust mite extract at a concentration of 10 to 300 μg / ml may be 1:0.001 to 0.5. Preferably, the weight ratio may be 1:0.003 to 0.3. Most preferably, the weight ratio may be 1:0.01 to 0.3. If the purified house dust mite extract is included in an amount less than the above numerical range, it is difficult to create an environment similar to biological aging conditions in the cultured skin cells, and if it is included in an amount exceeding the above numerical range, the isolated skin cells may die from excessive stress.

[0037] The step of inducing aging in cultured skin cells by measuring the p21 protein expressed in the cultured skin cells is intended to create an aging environment similar to the aging phenomenon occurring in vivo by measuring the expression amount of the p21 protein expressed in the cells on day 1 to day 7 after culture.

[0038] The p16, p21, and p53 genes and β-actin, which promote human cell aging, can cause aging-related diseases such as skin aging, chronic skin damage, and rheumatoid arthritis.

[0039] Referring to Figure 2, the p21 protein expressed on the 1st and 2nd day after culture of skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF) cultured in a medium composition treated with doxorubicin (doxo) as a control instead of house dust mite purified extract can be confirmed. Additionally, the p21 protein expressed on the 1st and 2nd day after culture of skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF) cultured in medium compositions containing house dust mite purified extract (HDM) at concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively, can be confirmed.

[0040] Doxorubicin (doxo) was used as a control group instead of the house dust mite purified extract. Doxorubicin is an effervescent substance that causes severe tissue damage or blister formation if it exits the vein and is classified as a chemotherapy drug used in cancer treatment. It is used to treat breast cancer, bladder cancer, Kaposi's sarcoma, lymphoma, and acute lymphoblastic leukemia, and can be used in combination with other chemotherapy agents. Skin keratinocytes (HaCaT cells) cultured in culture compositions treated with Doxorubicin (doxo) at concentrations of 50 nM, 100 nM, and 200 nM, respectively, did not express p21 protein on day 1 after culture. Skin keratinocytes (HaCaT cells) cultured in a culture composition treated with Doxorubicin (doxo) at a concentration of 200 nM did not express p21 protein even on day 2 after culture. Skin epithelial cells (NS-HDF) cultured in a culture composition treated with 200 nM of doxorubicin did not express p21 protein on day 1 after culture. Skin epithelial cells (NS-HDF) cultured in a culture composition treated with 50 nM of doxorubicin (doxo) showed a decrease in p21 protein expression on day 2 after culture. Presumably, when doxorubicin is administered, it may inhibit cell proliferation by blocking the cell cycle of skin cells, thereby causing p21 protein not to be expressed or its expression to decrease.

[0041] Skin keratinocytes (HaCaT cells) cultured in culture media compositions containing purified house dust mite extract (HDM) at concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively, showed significantly higher expression of p21 protein on day 1 after culture compared to skin keratinocytes (HaCaT cells) cultured in culture compositions treated with doxorubicin (doxo) at concentrations of 50 nM, 100 nM, and 200 nM, respectively. Skin keratinocytes (HaCaT cells) cultured in culture media compositions containing purified house dust mite extract (HDM) at concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively, showed a further increase in p21 protein expression on day 2 after culture.

[0042] In addition, skin epithelial cells (NS-HDF) cultured in culture media compositions containing purified house dust mite extract (HDM) at concentrations of 50 μg / ml, 100 μg / ml, and 200 μg / ml, respectively, showed higher expression of p21 protein on day 1 after culture compared to skin epithelial cells (NS-HDF) cultured in culture compositions treated with doxorubicin (doxo) at concentrations of 50 nM, 100 nM, and 200 nM, respectively. In particular, skin epithelial cells (NS-HDF) cultured in a culture media composition containing purified house dust mite extract (HDM) at a concentration of 200 μg / ml showed significantly higher expression of p21 protein on day 1 after culture compared to skin epithelial cells (NS-HDF) cultured in a culture composition treated with doxorubicin (doxo) at a concentration of 200 nM.

[0043] Presumably, when isolated skin keratinocytes (HaCaT cells) or skin epithelial cells (NS-HDF) are cultured in a medium composition containing fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, the purified house dust mite extract acts on the skin keratinocytes or skin epithelial cells as a stress that causes aging, thereby creating an environment similar to the aging environment in vivo, and it is presumed that the expression level of p21, an aging-related biomarker, is high on the 1st and 2nd days after culture. Preferably, the concentration of the purified house dust mite extract may be 50 to 200 μg / ml.

[0044] In the step of culturing the isolated skin cells in a medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, the cells may be cultured in a medium containing fetal bovine serum for one day of culture, and then cultured in a medium not containing fetal bovine serum from the second day of culture. The culture in a medium containing fetal bovine serum for one day of culture and then culture in a medium not containing fetal bovine serum from the second day of culture is intended to promote cell growth and increase in cell number during the first day of culture, and to confirm the expression of p21, a cell aging marker, while minimizing cell proliferation and cell tissue variability by culturing in a medium not containing fetal bovine serum on the second day of culture.

[0045] In the step of culturing the above-described isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, the cells are cultured in a medium containing fetal bovine serum for the first day of culture, and from the second day of culture, they are cultured in a medium not containing fetal bovine serum, and the concentration of the purified house dust mite extract may be 100 μg / ml.

[0046] In the step of culturing the isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, the cells are cultured in a medium containing fetal bovine serum for one day of culture, cultured in a medium not containing fetal bovine serum from the second day of culture, and cultured in a medium containing fetal bovine serum for one day of culture, the fetal bovine serum may be 10% fetal bovine serum.

[0047] Referring to Figure 3, the p21 protein and β-actin expressed on the first day of culture of skin keratinocytes (HaCaT cells) and skin epithelial cells (NS-HDF) cultured in a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract can be observed. In skin keratinocytes (HaCaT cells) cultured in a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract, it was confirmed that the expression level of p21 was very high on the first day of culture and decreased on the second day. Additionally, in skin keratinocytes (HaCaT cells) cultured in a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract, the expression level began to decrease on the second day of culture, and almost no p21 expression was observed on the third day of culture. Presumably, by replacing the medium with one that does not contain fetal bovine serum from the second day of culture and culturing skin keratinocytes (HaCaT cells), cell proliferation was minimized, which is why the expression level of p21 protein, an aging-related biomarker, was also rapidly reduced.

[0048] Skin epithelial cells (NS-HDF) cultured in a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract showed very high p21 expression on day 1 of culture and decreased on day 2. Additionally, skin epithelial cells (NS-HDF) cultured in a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract showed an increase in p21 expression on day 3 of culture and then decreased again. It is presumed that by replacing the medium with one that does not contain fetal bovine serum from day 2 of culture to culture the skin epithelial cells (NS-HDF), cell proliferation is minimized, which in turn causes the expression of the aging-related biomarker p21 protein to gradually decrease.

[0049] In the step of culturing the isolated skin cells in a culture medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, the fetal bovine serum may be 3% fetal bovine serum and the concentration of the purified house dust mite extract may be 100 μg / ml. By culturing in a culture medium composition comprising the 3% fetal bovine serum and the purified house dust mite extract at a concentration of 100 μg / ml, and measuring the p21 protein expressed in the cultured skin cells, the cultured skin cells on the 2nd to 3rd day after culture may be used for screening antioxidant substances in vitro.

[0050] Referring to Figure 4, the p21 protein expression levels according to the day of culture can be observed in skin epithelial cells (NS-HDF) cultured in a medium composition containing 50 nM doxorubicin and skin epithelial cells cultured in a medium composition containing 3% fetal bovine serum and 100 μg / ml of purified house dust mite extract. Presumably, the p21 protein expression levels of skin epithelial cells cultured in a medium composition containing 3% fetal bovine serum and 100 μg / ml of purified house dust mite extract on the 2nd or 3rd day after culture can be seen as establishing an in vitro aging model skin cell line that can be used as a substitute for animal experiments in the study of aging phenomena or anti-aging substances, as this establishes an environment in vitro similar to the aging phenomenon occurring in vivo.

[0051] In the step of culturing the isolated skin cells in a culture medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, when the fetal bovine serum is 3% fetal bovine serum and the concentration of the purified house dust mite extract is 100 μg / ml, the culture composition may not be replaced for 6 days after culture.

[0052] According to another embodiment of the present invention, an aging model skin cell line obtained by any one of the above manufacturing methods is provided.

[0053] The above aging model skin cell line is characterized by a 10 to 50% increase in p21 protein expression compared to a skin cell line cultured in a medium composition containing doxorubicin at a concentration of 50 to 200 nM. The p21 protein expression may be measured on the 2nd or 3rd day after culture.

[0054] According to another embodiment of the present invention, a method for screening anti-aging substances using an aging model skin cell line is provided, comprising the steps of: culturing isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml; measuring p21 protein expressed in the cultured skin cells to induce aging of the cultured skin cells; and administering an anti-aging candidate substance to the aging skin cells after the step of inducing aging of the cultured skin cells.

[0055] In a method for screening anti-aging substances using the above-mentioned aging model skin cell line, the step of culturing the isolated skin cells in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, wherein the fetal bovine serum may be 3% fetal bovine serum.

[0056] In a method for screening anti-aging substances using the above-mentioned aging model skin cell line, the isolated skin cells are cultured in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, wherein the concentration of the purified house dust mite extract may be 100 μg / ml.

[0057] After the step of inducing aging of the cultured skin cells, in the step of administering the anti-aging candidate substance to the aging skin cells, the anti-aging candidate substance may be administered on the 2nd to 4th day after cell culture. Preferably, it may be administered on the 4th day after culture.

[0058] In the step of administering an anti-aging candidate substance to the skin cells that have undergone aging after the step of causing aging of the cultured skin cells, the anti-aging candidate substance may be one or more selected from the group consisting of fisetin and liposomal fisetin.

[0059] In the step of administering an anti-aging candidate substance to the skin cells that have undergone aging after the step of inducing aging of the cultured skin cells, the anti-aging candidate substance is fisetin and can be administered at a concentration of 40 to 100 μM. Preferably, the fisetin can be administered at a concentration of 40 μM.

[0060] In the step of administering an anti-aging candidate substance to the skin cells that have undergone aging after the step of inducing aging of the cultured skin cells, the anti-aging candidate substance is liposomal fisetin and can be administered at a concentration of 50 to 200 μg / ml. Preferably, the liposomal fisetin can be administered at a concentration of 100 μg / ml.

[0061] Referring to Figure 5, it can be seen that the expression level of p21 protein decreases in aged skin cells administered with the anti-aging candidate substances fisetin and liposomal fisetin, respectively.

[0062] In the step of culturing the isolated skin cells in a culture medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml, when the fetal bovine serum is 3% fetal bovine serum and the concentration of the purified house dust mite extract is 100 μg / ml, the culture composition may not be replaced for 6 days after culture.

[0063] The present invention will be described in more detail below through examples. These examples are intended solely to explain the present invention more specifically, and it will be obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

[0064]

[0065] Examples and Comparative Examples

[0066] Example 1

[0067] The basic medium was DMEM, and isolated skin keratinocytes (HaCaT cells) were inoculated into a medium composition containing 10% fetal bovine serum and 50 μg / ml of purified house dust mite extract (100,000 cells / well, 12-well for Western blot (p21)) and cultured (S1).

[0068] The cultured skin keratinocytes (HaCaT cells) were collected on days 1 to 6 of culture, respectively, and the expression level of p21 protein was measured (S2).

[0069] Example 2

[0070] The above Example 1 was carried out in the same manner as Example 1, except that in step S1 of Example 1, the culture medium composition contained a house dust mite purified extract at a concentration of 100 μg / ml.

[0071] Example 3

[0072] The above Example 1 was carried out in the same manner as Example 1, except that in step S1 of Example 1, the culture medium composition contained a purified house dust mite extract at a concentration of 200 μg / ml.

[0073] Examples 4 to 6

[0074] The procedure was carried out in the same manner as Examples 1 to 3, except that isolated skin epithelial cells (NS-HDF) were cultured instead of the isolated skin keratinocytes (HaCaT cells) isolated in Step S1 of Examples 1 to 3.

[0075] Example 7

[0076] Isolated skin keratinocytes (HaCaT cells) were inoculated into a medium composition containing 10% fetal bovine serum and 100 μg / ml of purified house dust mite extract (50,000 cells / well, 12-well for Western blot (p21)) and cultured for 1 day (S1').

[0077] From the second day of culture, the medium was replaced with one that did not contain fetal bovine serum, and the skin keratinocytes cultured for the first day were cultured (S2').

[0078] The cultured skin keratinocytes were collected on days 1 to 6 of culture, respectively, and the p21 protein expression level was measured (S3).

[0079] Example 8

[0080] The procedure was carried out in the same manner as Example 7, except that isolated skin epithelial cells (NS-HDF) were cultured instead of the isolated skin keratinocytes (HaCaT cells) isolated in step S1' of Example 7.

[0081] Example 9

[0082] Isolated skin epithelial cells (NS-HDF) were inoculated into a medium composition containing 3% fetal bovine serum and 100 μg / ml of purified house dust mite extract (50,000 cells / well, 12-well for Western blot (p21)) and cultured for 1 day (S1).

[0083] The cultured skin epithelial cells (NS-HDF) were collected on days 1 to 6 of culture, respectively, and the p21 protein expression level was measured (S2).

[0084] Example 10

[0085] An aging model skin cell line was established by carrying out the same procedure as in Example 9, except that skin keratinocytes (HaCaT cells) were cultured for 1 day instead of skin epithelial cells (NS-HDF) in the S1" step of Example 9.

[0086] Anti-aging substances were screened by administering 40 μM fisetin and 100 μg / ml liposomal fisetin, respectively, to the above-prepared aging model skin cell line (skin keratinocyte cell line (HaCaT)).

[0087]

[0088] Experimental Example 1

[0089] Skin keratinocytes and skin epithelial cells cultured according to Examples 1 to 6 above were collected, and the amount of p21 protein was quantified by the intensity of the gel bands using the Western blotting method and observed visually (see Fig. 2).

[0090] Experimental Example 2

[0091] Skin keratinocytes and skin epithelial cells cultured according to Examples 7 and 8 above were collected, and the amount of p21 protein was quantified by the intensity of the gel bands using the Western blotting method and observed visually (see Fig. 3).

[0092] Experimental Example 3

[0093] Skin epithelial cells cultured according to Example 9 above were collected, and the amount of p21 protein was quantified by the intensity of the gel bands using the Western blotting method and observed visually (see Fig. 4).

[0094] Experimental Example 4

[0095] Skin keratinocytes were collected according to Example 10 above, and the amount of p21 protein was quantified by the intensity of the gel bands using the Western blotting method and observed visually (see Fig. 5).

[0096]

[0097] Example 11

[0098] Isolated skin keratinocytes (HaCaT cells) were inoculated into a medium composition containing 1 μg of 10% fetal bovine serum and 0.001 μg of purified house dust mite extract at a concentration of 100 μg / ml (100,000 cells / well, 12-well for Western blot (p21)) and cultured (S4).

[0099] The above-mentioned cultured skin keratinocytes (HaCaT cells) were collected on the second day of culture, and the expression level of p21 protein was measured to establish an aging model skin cell line (S5).

[0100] Example 12

[0101] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.003 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0102] Example 13

[0103] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.01 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0104] Example 14

[0105] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.3 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0106] Example 15

[0107] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.5 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0108] Example 16

[0109] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.7 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0110] Example 17

[0111] The above Example 11 was carried out in the same manner as Example 11, except that in step S4 of Example 11, 0.05 μg of purified house dust mite extract at a concentration of 100 μg / ml was included.

[0112] Comparative example

[0113] The above Example 11 was carried out in the same manner as Example 11, except that the S4 step of Example 11 did not include fetal bovine serum.

[0114]

[0115] Experimental Example 5

[0116] The number of cells in the aging model skin keratinocyte (HaCaT cell) lines obtained according to Examples 11 to 17 above was measured and is shown in [Table 1].

[0117] Number of cells on day 1 Number of cells on day 2 Example 110.9 x 10 6 0.7x 10 6 Example 1 20.8 x 10 6 0.6x 10 6 Example 132.0x 10 6 2.2x 10 6 Example 142.2x 10 6 2.4x 10 6 Example 151.0x 10 6 0.8x 10 6 Example 160.5x 10 6 0.4x 10 6 Example 172.6x 10 6 2.8x 10 6 Comparative Example 0.3x 10 6 0.2x 10 6

Claims

1. A step of culturing isolated skin cells in a medium composition comprising fetal bovine serum and purified house dust mite extract at a concentration of 10 to 300 μg / ml; and A step of inducing aging of cultured skin cells by measuring the p21 protein expressed in the cultured skin cells; A method for preparing an aging model skin cell line including 2. In Claim 1, A method for preparing an aging model skin cell line, wherein the isolated skin cells are cultured in a medium composition comprising fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, wherein the fetal bovine serum is 1-20% fetal bovine serum.

3. In Claim 1, A method for preparing an aging model skin cell line in which the isolated skin cells are one or more selected from the group consisting of skin keratinocytes and skin epithelial cells.

4. In Claim 1, A method for preparing an aging model skin cell line, wherein the isolated skin cells are cultured in a medium composition containing fetal bovine serum and a purified house dust mite extract at a concentration of 10 to 300 μg / ml, wherein the cells are cultured in a medium containing fetal bovine serum for the first day of culture and then cultured in a medium not containing fetal bovine serum from the second day of culture onwards.

5. An aging model skin cell line obtained by any one of the methods of claims 1 to 4.

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