Evaluation / screening method for tissue morphology / function control agents

Abstract

The purpose of the present invention is to provide a novel method for evaluating / screening superior healthcare materials and pharmaceutical products, on the basis of a new concept.　To this end, the present invention provides a method for evaluating / screening a tissue structure and a tissue function control agent, the method being characterized by including: a step for applying a substance under test to a tissue or cells that can express mechanical stress signaling molecules; and a step for evaluating the expression, activity and intracellular localization of mechanical stress signaling molecules of the tissue and the cells.

Description

[Technical field]

[0001] The present invention relates to a method for evaluating / screening an agent for controlling tissue morphology / tissue function. [Background technology]

[0002] The decline and change in the cellular functions that make up each tissue have been pointed out as the cause of various diseases and aging. As a pharmacological approach to improve these conditions, low molecular weight compounds, nucleic acids, proteins, etc. are being considered as drug discovery materials. However, depending on the type and symptoms of the disease, there are many cases where sufficient therapeutic and improving effects cannot be obtained, and solutions based on new ideas are required.

[0003] On the other hand, recent research has revealed that mechanical stress controls the functionality of tissues and cells and is involved in aging and pathogenesis, and the possibility of developing healthcare materials and drug discovery focusing on mechanical stress is being considered in areas where sufficient treatment and improvement methods have not been developed in the past. For example, the importance of mechanical stress response in the pathophysiology of heart failure and treatment strategies based on it are also being considered (see Non-Patent Document 1). In addition, in the field of drug discovery, molecules have been discovered that utilize the mechanism of muscle mechanotransduction to exert effects equivalent to those of exercise when administered orally (see Non-Patent Document 2). Furthermore, development of new treatment methods for vascular-related diseases targeting the extracellular matrix involved in the mechanical stress response in blood vessels is also being promoted (see Non-Patent Document 3). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Heart, Vol.47, No.12, 2015 [Non-Patent Document 2] Biochemistry, Vol. 81, No. 6, 2009, pp. 494-501 [Non-Patent Document 3] PNAS May 5, 2020, 117(18)9896-9905 Summary of the Invention [Problem to be solved by the invention]

[0005] The present invention has been made under such circumstances, and aims to provide a novel method for evaluating / screening excellent healthcare materials and pharmaceuticals based on a new concept. [Means for solving the problem]

[0006] As a result of intensive research conducted by the present inventors to solve the above problems, they discovered that the "tension equilibrium state" (tension balance) present in the skin controls the functionality of the skin through a specific mechanical stress signal. Previous research results have shown that a transient tension load caused by an external force controls the functionality of cells, but the present inventors are the first to discover that a continuous "tension balance" similar to the state of a living body directly controls skin function. Based on these findings, the present inventors have developed a new concept for evaluating / screening health care materials, medicines, and devices. Effect of the Invention

[0007] According to the method for evaluating / screening tissue morphology / tissue function control agents of the present invention, it is possible to find health care materials and pharmaceutical materials that can transmit signals to cells that constitute tissues equivalent to signals obtained by mechanical stress due to continuous tension balance under normal conditions of the living body. The materials found by the method of the present invention can appropriately control the tissue morphology and tissue function of the living body, and are therefore expected to be effective against symptoms caused by reduced mechanical stress. For example, they are useful for treating aesthetic deterioration such as wrinkles, sagging, age spots, loss of transparency, and enlarged pores due to aging, as well as skin fibrosis such as scars, stretch marks, and keloids, and epithelial cell cancer. [Brief description of the drawings]

[0008] [Figure 1]Changes in gene expression of mechanical stress signaling molecules by tension balancing stimulation (Dunnett's Test *: P<0.05, **: P<0.01, ***: P<0.001) [Diagram 2] Expression and localization of mechanical stress signaling molecules induced by tension balancing stimulation [Diagram 3] Nuclear translocation rate of MRTFA by tension balance stimulation [Figure 4] Changes in the expression of ECM synthesis-related genes due to tension balancing stimulation (Dunnett's Test *: P<0.001) [Diagram 5] Changes in type 1 collagen fibril formation induced by tension balancing stimulation [Figure 6] Changes in KGF gene expression due to tension balancing stimulation (Dunnett's Test *: P<0.001) [Figure 7] Distribution of Ki67 positive cells by tension balance stimulation [Figure 8] Changes in the rate of Ki67-positive proliferating cells in epidermal keratinocytes due to tension balancing stimulation (Dunnett's Test *:p<0.01, **:p<0.001) [Figure 9] Morphological changes in dermal fibroblasts and collagen fibers induced by tension balancing stimulation [Figure 10] Changes in nuclear orientation in dermal fibroblasts induced by tension balancing stimulation [Figure 11] Evaluation of collagen fiber orientation in dermal fibroblasts by tension balance stimulation using two-dimensional Fourier transform [Figure 12] Induction of MRTFA gene expression in tension-balanced artificial skin model by ATRA administration (TTEST *: P<0.01) [Figure 13] Induction of COL1A1 and HAS3 gene expression by mechanical stress signal promoting materials (Tukey-Kramer Test *:P<0.01, **:P<0.001) [Figure 14] Collagen fiber formation promotion effect by mechanical stress signal promoting material [Figure 15] Changes in expression and localization of mechanical stress signaling molecules by Y27632 [Figure 16] Changes in the nuclear translocation rate of MRTFA by materials that suppress mechanical stress signals [Figure 17] Changes in ECM synthesis-related gene expression by mechanical stress signal suppression materials (Dunnett's Test *: P<0.05, **: P<0.01, ***: P<0.001) [Figure 18] Changes in type 1 collagen fibrillogenesis by materials that suppress mechanical stress signals [Figure 19] Tissue morphological changes induced by mechanical stress signal suppression materials DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present invention will now be described in detail. In addition, the terms used in this specification are to be interpreted as having the meanings usually used in the technical field unless otherwise specified.

[0010] [Evaluation / screening methods for tissue morphology / function control agents] The method for evaluating / screening a tissue morphology / tissue function controlling agent of the present invention is characterized by comprising the steps of: (i) applying a test substance to a tissue or cell capable of expressing a mechanical stress signaling molecule; and (ii) evaluating the expression, activity, or intracellular localization of the mechanical stress signaling molecule in the tissue or cell. The method of the present invention preferably further comprises the step of (iii) evaluating / screening a test substance that can be used as a tissue morphology / tissue function controlling agent based on the results of the step (ii). According to the method of the present invention, it is possible to find health care or pharmaceutical materials that can transmit signals to cells constituting tissues similar to signals obtained by mechanical stress due to continuous tension balance experienced by a living organism in a normal state.

[0011] "Mechanical stress" refers to mechanical stimuli such as stretching and compression applied to tissues and cells. Mechanical stress is an important parameter in various aspects, such as fetal development, aging, metabolism, and maintenance of the circulatory and musculoskeletal systems. Within the body, the musculoskeletal system, such as skeletal muscles and bones, the circulatory system, such as the heart and blood vessels, and the skin are strongly subjected to mechanical stress. It is believed that an increase or decrease in mechanical stress in these tissues causes major changes in their development and structural maintenance.

[0012] The term "mechanical stress signaling molecule" refers to a molecule that exists downstream of an extension ion channel or integrin that is activated when tissues or cells are subjected to mechanical stress, and transmits a signal from these. For example, examples of mechanical stress signaling molecules that are activated when mechanical stress is applied to the skin include YAP / TAZ, glycocalyx, lipid raft / caveolin-1, cell adhesion structures (ITGA2, desmosomes, hemidesmosomes, cadherins), ion channels (CaSR / TRP / Piezo), MRTFA, and Rho / ROCK-related molecules (ACTA2(αSMA)). Among these, MRTFA and ACTA2(αSMA) are preferred, and MRTFA is particularly preferred, from the viewpoint of improving the efficiency of the method of the present invention.

[0013] The term "tissue morphology / tissue function control agent" refers to an agent that controls tissue morphology and / or tissue function. The type of agent that controls tissue morphology and / or tissue function varies depending on the tissue. For example, an agent that controls tissue morphology in the case of skin (skin morphology control agent) can control the firmness and texture of the skin, and can improve or suppress wrinkles, sagging, etc., and can be exemplified. In addition, an agent that controls tissue function in the case of skin (skin function control agent) can be exemplified by an agent that can promote skin turnover and improve moisturizing function and barrier function. More specifically, examples of the tissue morphology / tissue function control agent in the present invention include skin morphology control agents, skin function control agents, agents that prevent or improve wrinkles and sagging of the skin, agents that prevent or improve the decrease in firmness / slackness of the skin, skin barrier function improvers, dermal ECM formation control agents, collagen fiber formation control agents, whitening agents, skin color improvers, texture improvers, hair growth / hair growth inhibitors, acne treatment agents, etc. These can be suitably used in cosmetics for anti-aging and skin care, and therapeutic agents for skin diseases, etc.

[0014] The method for evaluating / screening a tissue morphology / tissue function controlling agent of the present invention will be explained below in detail in each step.

[0015] (i) applying a test substance to a tissue or cell capable of expressing a mechanical stress signaling molecule; In this step, a test substance and tissues or cells capable of expressing a mechanical stress signaling molecule are prepared, and the test substance is applied to these tissues or cells.

[0016] The tissue or cells used in this step are not particularly limited as long as they are capable of expressing the mechanical stress signaling molecule, and include tissue or cells isolated from a mammal, or a culture thereof, capable of expressing the mechanical stress signaling molecule or its gene. Specifically, examples include skin tissue, epidermal tissue, dermal tissue, epidermal cells (such as epidermal keratinocytes), dermal cells (such as dermal fibroblasts), cultures thereof (including established cell lines), and artificial skin models composed of any of these, which are collected from a mammal. Among these, epidermal keratinocytes and dermal fibroblasts are preferred from the viewpoint of simplicity. Furthermore, an artificial skin model is preferred from the viewpoint of obtaining results closer to actual skin, and an artificial skin model composed of epidermal keratinocytes and dermal fibroblasts is more preferred. Such artificial skin may be any commercially available artificial skin known in the art, and is not particularly limited, but a preferred example is an artificial dermis prepared by dispersing and seeding normal human dermal fibroblasts in a collagen gel or the like and solidifying the artificial dermis, on which normal human epidermal keratinocytes are seeded and cultured in an air layer.Furthermore, the state of the artificial skin may be a tension-balanced artificial skin model in which the edge of the artificial dermis is fixed in a culture vessel to reproduce a state in which the tension between cells is balanced as in natural skin, or a bell-shaped artificial skin in which tension is released by culturing the artificial dermis in a floating state, with the tension-balanced artificial skin model being particularly preferred.

[0017] The tissue or cell used in this step may be a mammalian tissue or cell genetically modified to express the mechanical stress signaling molecule or its gene, or a culture thereof. The genetically modified mammalian tissue or cell and its culture may be prepared, for example, by introducing a gene encoding the mechanical stress signaling molecule into any mammalian tissue or cell, and transforming the tissue or cell to express the mechanical stress signaling molecule or to enhance the expression of the mechanical stress signaling molecule. Alternatively, a cell or a culture thereof into which a construct in which a reporter gene such as luciferase is fused downstream of the promoter region of the gene of the mechanical stress signaling molecule is introduced may be used. A method for introducing these genes or constructs into cells may be a conventional method, such as vector introduction by electroporation or lipofection.

[0018] Examples of mammals from which the tissues or cells used in this step are derived include humans, mice, rats, hamsters, rabbits, pigs, monkeys, etc. Of these, humans are preferred.

[0019] The test substance in this step is not particularly limited, and may be a naturally occurring substance or a substance synthesized by a chemical or biological method, etc. The test substance may also be a low molecular weight compound, a peptide, a protein, a nucleic acid, an extract from a natural product such as a plant extract, a mixture of these, or a composition containing these.

[0020] A test substance can be applied to tissues or cells capable of expressing a mechanical stress signaling molecule by, for example, adding the test substance to a medium in advance to a predetermined concentration, and then seeding the tissue or cells in the medium, or adding the test substance to a predetermined concentration to the medium in which the tissue or cells have been seeded. After application of the test substance, the tissue or cells are preferably cultured under normal conditions, for example, in the case of dermal fibroblasts, epidermal keratinocytes, and artificial skin models, at 25 to 37°C for about 8 to 240 hours, preferably about 24 to 120 hours.

[0021] The concentration of the test substance to be added may be set to an appropriate concentration depending on the type of test substance, and may be, for example, in the range of 0.00001 to 10% (w / v).

[0022] The medium for culturing the above-mentioned tissues or cells can be appropriately selected from media suitable for the tissues or cells used, and examples thereof include, but are not limited to, DMEM medium containing or not containing 10% fetal bovine serum (FBS).

[0023] (ii) evaluating the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cells. In this step, in order to evaluate / screen the test substance applied in step (i) above, changes in the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cell caused by the test substance are measured.

[0024] The expression of the mechanical stress signaling molecule may be the expression level of the gene encoding the mechanical stress signaling molecule, or the expression level of the mechanical stress signaling molecule (protein) itself. The expression level of the gene and the expression level of the protein can be measured according to a known method. When the expression level of the gene is detected at the mRNA level, for example, after extracting total RNA from the cell, the mRNA transcribed from the mechanical stress signaling molecule gene can be detected and quantified using real-time RT-PCR, RNase protection assay, DNA array, Northern blot analysis, etc. The expression level of the protein can be measured by a conventional immunoassay method such as RIA, ELISA, bioassay, and Western blot. Western blot is inexpensive and convenient, and is therefore desirable.

[0025] The activity of the mechanical stress signaling molecules can be measured by a method appropriate for each molecule, for example, by quantifying the amount of RNA transcription by RT-PCR, detecting phosphorylation of a specific molecule by Western blotting, or detecting molecular activity by detecting the amount of fluorescence / luminescence after introducing a reporter gene.

[0026] The intracellular localization of mechanical stress signaling molecules can be confirmed by immunohistochemical staining using antibodies that specifically bind to each molecule, and the effects of the test substance (such as nuclear translocation of transcription factors, polymerization levels of cytoskeletal molecules, and changes in integrin subtypes on the cell membrane) can be assessed by image analysis in comparison with controls.

[0027] (iii) A step of evaluating / screening test substances that can be used as tissue morphology / tissue function control agents based on the results of the above step (ii). In this step, based on the results of the measurement of changes in expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cell caused by the test substance in step (ii), the test substance is evaluated and selected as to whether it can be used as a tissue morphology / tissue function control agent.

[0028] In this step, when evaluating and selecting whether the test substance is usable as a tissue morphology / tissue function control agent, the expression, activity, or intracellular localization of mechanical stress signaling molecules in the above tissue or cells to which the test substance has been applied is compared with that of a control group. Examples of the control group include the same tissue or cells as the test group but to which the test substance has not been applied, or a control group to which only the solvent for the test substance has been applied. The expression, activity, or intracellular localization of mechanical stress signaling molecules in the control group can also be measured or detected by the same method as described in step (ii).

[0029] When the expression or activity level of the mechanical stress signaling molecule or its gene in the test group is higher than that in the control group, the test substance is selected as a substance that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. For example, when the expression or activity level in the test group is statistically significantly increased compared to the expression or activity level in the control group, the test substance is selected as a substance that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. Specifically, when the expression or activity level in the control group is taken as 100%, the expression or activity level in the test group is 110% or more, preferably 130% or more, and more preferably 150% or more, and the test substance can be selected as a substance that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. The selected test substance may be selected as a skin morphology regulator, a skin function regulator, a preventive or improving agent for wrinkles and sagging skin, a preventive or improving agent for loss of skin firmness / looseness, a skin barrier function improver, a dermal ECM formation regulator or a collagen fiber formation regulator, a skin whitening agent, a skin color improver, a skin texture improver, a hair growth / hair growth inhibitor, an acne treatment agent, etc., or as a candidate substance thereof. These substances are preferably used, for example, as cosmetics, skin care products, etc. for the purpose of anti-aging.

[0030] When the expression or activity level of the mechanical stress signaling molecule or its gene in the test group is lower than that in the control group, the test substance is selected as a substance that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. For example, when the expression or activity level in the test group is statistically significantly lower than that in the control group, the test substance is selected as a substance that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. In addition, when the expression or activity level in the test group is 90% or less, preferably 70% or less, and more preferably 50% or less, when the expression or activity level in the control group is 100%, the test substance is selected as a substance that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. The selected test substance can be selected as a skin morphology regulator, a skin function regulator, a dermal ECM formation regulator, a collagen fiber formation regulator, or a candidate substance thereof. These substances are useful, for example, in the treatment of scars, skin fibrosis such as stretch marks and keloids, and epithelial cell cancer.

[0031] In the method of the present invention, the substance selected in the above procedure may be subjected to further screening, if necessary. The test substance selected in the above step (iii) as a substance that enhances or reduces the expression or activity level of a mechanical stress signaling molecule or its gene, or a substance that changes its intracellular localization, is obtained as a candidate substance for the agent of the present invention. Next, these candidate substances are administered to tissues or cells capable of expressing mechanical stress signaling molecules (skin tissue, epidermal tissue, dermal tissue, epidermal keratinocytes, dermal fibroblasts, or a culture thereof, or an artificial skin model composed of any of these), and then the ECM synthesis function, type 1 collagen synthesis function, epidermal keratinocyte proliferation promotion function and the turnover promotion function therewith, epidermal keratinocyte proliferation promotion function, function of maintaining the alignment of collagen fibers in the same horizontal direction as the tension direction, etc. are measured and confirmed, and a better test substance can be selected by comparing with a control group as necessary. The ECM synthesis function, type 1 collagen synthesis function, epidermal keratinocyte proliferation-promoting function and the resulting turnover-promoting function, epidermal keratinocyte proliferation-promoting function, and function of maintaining the alignment of collagen fibers in the horizontal direction, which is the same as the tension direction, can be measured by known methods such as RT-PCR, microarray, immunoassay, Western blotting, and immunohistochemical staining.

[0032] [Methods for evaluating devices for controlling tissue morphology and function] The method for evaluating a device for controlling tissue morphology and tissue function of the present invention is characterized by comprising the steps of: (i) applying mechanical stress to a tissue or cell capable of expressing a mechanical stress signaling molecule using a test device; and (ii) evaluating the expression, activity, or intracellular localization of the mechanical stress signaling molecule in the tissue or cell. The method of the present invention preferably further comprises the step of (iii) evaluating whether or not the test device can be used as a device for controlling tissue morphology and tissue function based on the results of the step (ii). According to the method of the present invention, it is possible to appropriately evaluate a healthcare device or a medical device that can transmit a signal to cells constituting a tissue similar to the mechanical stress caused by continuous tension balance experienced by a living organism under normal conditions.

[0033] The term "device for controlling tissue morphology and function" refers to a device, machine, equipment, etc. that applies mechanical stress to control tissue morphology and / or function, specifically, pressure stimulation, ultrasonic stimulation, and other physical stimulation to tissue. The device for appropriately controlling tissue morphology and / or function varies depending on the tissue to which it is applied. For example, in the case of skin, a device that can control the firmness, texture, etc. of the skin and improve / suppress wrinkles, sagging, etc. can be mentioned. In addition, an example of a device for controlling tissue function in the case of skin is a device that can promote turnover of the skin and improve moisturizing function and barrier function. More specifically, the device for controlling tissue morphology and function in the present invention is a device that can be expected to control skin morphology, control skin function, prevent or improve wrinkles and sagging of the skin, prevent or improve the decrease in firmness / slackness of the skin, improve skin barrier function, control dermal ECM formation, control collagen fiber formation, whitening, improve skin color, improve texture, hair growth / hair inhibition, treat acne, etc. These can be suitably used for the purpose of anti-aging, skin care, treatment of skin diseases, etc.

[0034] The method for evaluating the device for controlling tissue morphology and tissue function of the present invention will be explained below by dividing it into each step.

[0035] (i) applying a mechanical stress load by a test device to a tissue or cell capable of expressing a mechanical stress signaling molecule; In this step, a test device and tissues or cells capable of expressing a mechanical stress signaling molecule are prepared, and mechanical stress is applied to these tissues or cells by the test device.

[0036] The explanation of the tissues or cells used in this step can be applied directly to the explanation in the section on the evaluation / screening method of tissue morphology / tissue function controlling agents.

[0037] The test device in this step is not particularly limited, but refers to any device capable of applying pressure stimulation, ultrasonic stimulation, or other physical stimulation to the skin.

[0038] The application of mechanical stress load by the test device to tissues or cells capable of expressing mechanical stress signaling molecules is performed according to the specifications of each device.

[0039] (ii) evaluating the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cells. In this step, in order to evaluate the test device (applied with mechanical stress) applied in step (i) above, changes in the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cell caused by mechanical stress application with the test device are measured. Note that the explanation of the expression, activity, and intracellular localization of the mechanical stress signaling molecules can be directly applied to the explanation in the section on the evaluation / screening method for tissue morphology / tissue function controlling agents above.

[0040] (iii) A step of evaluating whether the test device can be used as a device for controlling tissue morphology and tissue function. In this step, based on the results of step (ii) of measuring changes in the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cells due to mechanical stress applied by the device, the test device is evaluated as to whether it is effective as a device for controlling tissue morphology and tissue function.

[0041] In this step, when evaluating whether the test substance can be used as a device for controlling tissue morphology and tissue function, the expression, activity, or intracellular localization of mechanical stress signaling molecules in the tissue or cells to which mechanical stress has been applied by the device is compared with that of a control group. Examples of the control group include tissue or cells that are the same as the test group but to which mechanical stress has not been applied. The expression, activity, or intracellular localization of mechanical stress signaling molecules in the control group can also be measured or detected by the same method as described in step (ii).

[0042] If the expression or activity level of the mechanical stress signaling molecule or its gene in the test group is higher than that in the control group, the test device (or the condition of mechanical stress loading by it) is selected as a device (or the above-mentioned condition) that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. For example, if the expression or activity level in the test group is statistically significantly increased compared to the expression or activity level in the control group, the test device (or the above-mentioned condition) is selected as a device (or the above-mentioned condition) that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. Specifically, when the expression or activity level in the control group is taken as 100%, if the expression or activity level in the test group is 110% or more, preferably 130% or more, and more preferably 150% or more, the test device (or the above-mentioned condition) can be evaluated as a device (or the above-mentioned condition) that enhances the expression or activity level of the mechanical stress signaling molecule or its gene. The evaluated test devices can be suitably used for controlling skin morphology, controlling skin function, preventing or improving wrinkles and sagging skin, preventing or improving loss of skin firmness / looseness, improving skin barrier function, controlling dermal ECM formation or collagen fiber formation, whitening, improving skin color, improving skin texture, hair growth / inhibition, treating acne, etc. These devices (or the above conditions) can be used for cosmetic purposes such as anti-aging and skin care, and for medical purposes such as treating acne.

[0043] If the expression or activity level of the mechanical stress signaling molecule or its gene in the test group is lower than that in the control group, the test device (or the condition of mechanical stress application thereto) is selected as a device (or the above-mentioned condition) that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. For example, if the expression or activity level in the test group is statistically significantly lower than the expression or activity level in the control group, the test device (or the above-mentioned condition) is selected as a device (or the above-mentioned condition) that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. In addition, if the expression or activity level in the test group is 90% or less, preferably 70% or less, and more preferably 50% or less, when the expression or activity level in the control group is 100%, the device (or the above-mentioned condition) is selected as a device (or the above-mentioned condition) that reduces the expression or activity level of the mechanical stress signaling molecule or its gene. The evaluated devices (or the above conditions) can be suitably used for cosmetic purposes such as skin morphology control, skin function control, dermal ECM formation control, collagen fiber formation control, etc. Furthermore, these devices (or the above conditions) can also be suitably used for medical purposes such as treatment of scars, skin fibrosis such as stretch marks and keloids, and epithelial cell cancer.

[0044] In the method of the present invention, the device evaluated by the above procedure may be further evaluated as necessary. The device (or the above condition) evaluated in the above step (iii) as a device (or the above condition) that enhances or reduces the expression or activity level of a mechanical stress signaling molecule or its gene, or a device (or the above condition) that changes its intracellular localization, is applied to tissue or cells capable of expressing a mechanical stress signaling molecule (skin tissue, epidermal tissue, dermal tissue, epidermal keratinocytes, dermal fibroblasts, or a culture thereof, or an artificial skin model composed of any of these), and then the ECM synthesis function, type 1 collagen synthesis function, epidermal keratinocyte proliferation promotion function and the turnover promotion function therewith, epidermal keratinocyte proliferation promotion function, function of maintaining the alignment of collagen fibers in the same horizontal direction as the tension direction, etc. are measured and confirmed, and compared with a control group as necessary, to evaluate and select a superior device (or the above condition). The ECM synthesis function, type 1 collagen synthesis function, epidermal keratinocyte proliferation-promoting function and the resulting turnover-promoting function, epidermal keratinocyte proliferation-promoting function, and function of maintaining the alignment of collagen fibers in the horizontal direction, which is the same as the tension direction, can be measured by known methods such as RT-PCR, microarray, immunoassay, Western blotting, and immunohistochemical staining.

[0045] [Application of the present invention] As described above, the skin morphology regulator, skin function regulator, wrinkle / sagging prevention or improvement agent, skin firmness / looseness reduction prevention or improvement agent, skin barrier function improvement agent, dermal ECM formation regulator, collagen fiber formation regulator, skin whitening agent, skin color improvement agent, texture improvement agent, and hair growth / hair growth inhibitor selected by the evaluation / screening method of the present invention can be suitably used in cosmetics, quasi-drugs, and oral preparations that promote skin care such as anti-aging (prevention / improvement of wrinkles, sagging, age spots, loss of transparency, enlarged pores, etc.), moisturizing, whitening, and promoting skin turnover. They can also be suitably used in pharmaceuticals such as scars, stretch marks, keloids, and other skin fibrosis, epithelial cell cancer treatment agents, and acne treatment agents.

[0046] As described above, the devices for controlling skin morphology, controlling skin function, preventing or improving skin wrinkles and sagging, preventing or improving loss of skin firmness / slackness, improving skin barrier function, controlling dermal ECM formation, controlling collagen fiber formation, whitening, improving skin color, improving skin texture, and hair growth / inhibition, which are evaluated and selected by the evaluation method of the present invention, can be suitably used as health care devices such as beauty devices for the purpose of skin care such as anti-aging (preventing and improving wrinkles, sagging, age spots, loss of transparency, enlarged pores, etc.), moisturizing, whitening, promoting skin turnover, etc. In addition, they can be suitably used as medical devices for treating scars, stretch marks, keloids, and other skin fibrosis, epithelial cell cancer, and acne.

[0047] The above-mentioned agents selected by the evaluation / screening method of the present invention can be used in combination with health care devices such as beauty treatment equipment, or treatment methods such as massage and relaxation, to enhance the effect. The ideas in the screening method and evaluation method of the present invention can also be applied to the development of treatment methods. That is, in the above-mentioned treatment methods, it is possible to develop agents that enhance or reduce the expression or activity level of mechanical stress signaling molecules or their genes in target cells, or change the intracellular localization in a favorable direction. EXAMPLES

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0049] [Example 1] Expression and localization changes of mechanical stress signal molecules by tension balance stimulation Normal human dermal fibroblasts (Kurashiki Boseki Co., Ltd.) were cultured in DMEM medium (Thermo Fisher Scientific Co., Ltd.) containing 10 ng / ml bFGF (Peprotech), 10% fetal bovine serum, and 1% penicillin / streptomycin in tissue culture-coated petri dishes. Normal human epidermal keratinocytes (Kurashiki Boseki Co., Ltd.) were cultured in Humedia-KG2 medium (Kurashiki Boseki Co., Ltd.) in tissue culture-coated petri dishes. Normal human epidermal keratinocytes were seeded on an artificial dermis made by dispersing and solidifying normal human dermal fibroblasts in collagen gel (Koken Co., Ltd.), and cultured in an air layer using an artificial skin maintenance medium containing 10% fetal bovine serum, 1% penicillin / streptomycin, 5μg / ml insulin (Fujifilm Wako Pure Chemical Co., Ltd.), 1mM magnesium ascorbyl phosphate (Fujifilm Wako Pure Chemical Co., Ltd.), 10ng / ml bFGF (Peprotech), and 1μM hydrocortisone (Fujifilm Wako Pure Chemical Co., Ltd.). In constructing the artificial skin, a bell-shaped artificial skin in which tension is released by culture in a floating state, which is a conventional method for constructing artificial skin, and a tension-balanced artificial skin model in which the edge of the artificial dermis is fixed in a culture vessel to reproduce a state in which tension between cells is balanced like in natural skin, were constructed. After five days of air layer culture, the tension-balanced artificial skin model was detached from the culture vessel to create a tension-released artificial skin model.

[0050] Tissues were collected from the three models, the bell-shaped artificial skin, the tension-balanced artificial skin model, and the tension-released artificial skin model, on the seventh day of air layer culture. Total RNA was immediately collected from a portion of the collected tissue using RNeasy Plus mini kit (Qiagen, Inc.) according to the attached protocol. cDNA was reverse transcribed from the obtained total RNA using SuperScript VILO (Thermo Fisher Scientific, Inc.) according to the attached protocol. Real-time PCR of mechanical stress signaling genes, namely ITGA2, MRTFA, and ACTA2 (αSMA), was performed using SYBR Premix Ex Taq II or TaqMan (registered trademark) Gene Expression Assay system (Thermo Fisher Scientific, Inc.) on an Applied Biosystems QuantStudio 12K Flex (Thermo Fisher Scientific, Inc.). The measured expression values ​​of each gene were corrected by the ΔΔCT method, with GAPDH set as an endogenous gene expression control.

[0051] Furthermore, a portion of the collected tissue was immediately fixed in 4% paraformaldehyde phosphate buffer, paraffin sections were prepared using standard methods, and immunohistochemical staining for each of the mechanical stress signaling molecules was performed using the following procedures.

[0052] ITGA2 Immunohistochemical staining was performed using rabbit anti-human ITGA2 antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength: 470 nm) and ITGA2 expression (excitation wavelength: 594 nm, observation wavelength: 620 nm) were observed using a confocal microscope.

[0053] ·MRTFA Immunohistochemical staining was performed using rabbit anti-human MRTFA antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength 470 nm) and MRTFA expression (excitation wavelength 594 nm, observation wavelength 620 nm) were observed using a confocal microscope. Furthermore, the amount of MRTFA translocation into the nucleus was quantitatively evaluated by calculating the average value of the Alexa594 fluorescent signal intensity in the Hoechst-positive nuclear region using image analysis using ImageJ.

[0054] αSMA Immunohistochemical staining was performed using rabbit anti-human αSMA antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength 470 nm) and αSMA expression (excitation wavelength 594 nm, observation wavelength 620 nm) were observed using a confocal microscope.

[0055] Figure 1 shows the expression levels of mechanical stress signaling genes in each artificial skin model. The tension-balancing artificial skin model showed statistically significantly higher gene expression levels of ITGA2, MRTFA, and ACTA2 (αSMA) compared with the other two models without tension-balancing stimulation.

[0056] Immunohistochemical staining images of mechanical stress signaling molecules in each artificial skin model are shown in Figure 2. The tension-balancing artificial skin model showed increased expression of ITGA2, MRTFA, and ACTA2 (αSMA) compared with the other two models without tension-balancing stimulation.

[0057] Figure 3 shows the results of quantitative evaluation of the amount of MRTFA translocation into the nucleus of each cell by image analysis of immunohistochemical staining images. The tension-balanced artificial skin model showed statistically significantly higher nuclear translocation of ITGA2, MRTFA, and ACTA2 (αSMA) than the other two models without tension-balanced stimulation.

[0058] From the above, it was found that the tension-balanced artificial skin model, which reflects actual good skin condition, promoted the expression of mechanical stress signaling molecules (ITGA2, MRTFA, ACTA2 (αSMA)) compared to the other two models without tension-balanced stimulation.

[0059] [Example 2] Control of the functionality of skin cells by adjusting tension balance stimuli A bell-shaped artificial skin model, a tension-balanced artificial skin model, and a tension-release artificial skin model were prepared in the same manner as in Example 1.

[0060] For the bell-shaped artificial skin, tension-balanced artificial skin model, and tension-release artificial skin model, tissues were harvested on the seventh day of air layer culture, and total RNA was collected from each tissue in the same manner as in Example 1, and real-time PCR was performed. The genes analyzed were COL1A1, COL1A2, FBN1, ELN, and MMP1, which are genes related to the function of the dermis, i.e., ECM synthesis function, and KGF, which is a gene related to the turnover function of the epidermis, i.e., epidermal keratinocyte proliferation control gene. The measured expression value of each gene was corrected by the ΔΔCT method, with GAPDH set as an endogenous gene expression control.

[0061] Furthermore, a portion of the collected tissue was immediately fixed in 4% paraformaldehyde phosphate buffer, and paraffin section specimens were prepared using standard methods. Immunohistochemical staining for type 1 collagen, which is involved in ECM synthesis function, and Ki67, a proliferation marker of epidermal keratinocytes, was performed using the following procedures.

[0062] COL1 Immunohistochemical staining was performed using rabbit anti-human type 1 collagen antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength 470 nm) and COL1 expression (excitation wavelength 594 nm, observation wavelength 620 nm) were observed using a confocal microscope.

[0063] Ki67 Immunohistochemical staining was performed using rat anti-human ki67 antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rat IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the nuclei (excitation wavelength: 350 nm, observation wavelength 470 nm) and Ki67 expression (excitation wavelength 594 nm, observation wavelength 620 nm) were observed using a confocal microscope. In addition, the number of Ki67-positive cells was visually counted.

[0064] Furthermore, a portion of the collected tissue was immediately fixed in 4% paraformaldehyde phosphate buffer, frozen section specimens were prepared by standard methods, and the three-dimensional structure of type 1 collagen fibers was photographed by second harmonic generation imaging according to the method of Lynch, B. et al. The orientation of fibroblast nuclei was evaluated by ellipsoidal approximation of the fluorescent nuclear image using Image J and measuring the inclination of the principal axis. The orientation of type 1 collagen in the dermis was evaluated by two-dimensional fast Fourier transform analysis using the FFT function of Image J and the Oval profile plug-in.

[0065] Figure 4 is a plot of the expression levels of genes related to dermal function, i.e., ECM synthesis function, in each artificial skin model. In the tension-balanced skin model, which was stimulated with tension balance compared to the tension-free bell-shaped artificial skin, the expression of COL1A1, COL1A2, FBN1, and ELN, which are genes that code for the constituent fibrous proteins of ECM, increased, and the expression of MMP1, a collagen degrading enzyme, decreased, indicating that the tension-balanced stimulation improved the ECM synthesis function. In addition, compared to the tension-release model, the expression of COL1A1, COL1A2, FBN1, and ELN, which are genes that code for the constituent fibrous proteins of ECM, decreased, and the expression of MMP1, a collagen degrading enzyme, increased, indicating that the release of tension balance reduced the ECM synthesis function.

[0066] Figure 5 shows the immunohistochemical staining images of type 1 collagen in each artificial skin model. Compared to the tension-free bell-shaped artificial skin, the tension-balanced skin model, which was stimulated with tension balance, showed increased expression of type 1 collagen, indicating that tension-balanced stimulation improves type 1 collagen synthesis function. Furthermore, the tension-release model showed decreased expression of type 1 collagen compared to the tension-balanced model, indicating that release of tension balance reduces type 1 collagen synthesis function.

[0067] Figure 6 is a plot of the epidermal turnover function in each artificial skin model, i.e., the expression level of KGF, an epidermal keratinocyte proliferation control gene. Compared to the tension-free bell-shaped artificial skin, the tension-balanced skin model, which was stimulated with tension balance, showed increased expression of KGF, suggesting that tension-balanced stimulation promotes epidermal keratinocyte proliferation and thus promotes turnover.

[0068] Figure 7 shows the immunohistochemical staining images of the proliferation cell marker Ki67 in each artificial skin model. Figure 8 also plots the results of counting the number of Ki67-positive cells. Compared to the tension-free bell-shaped artificial skin, the tension-balanced skin model, which was stimulated with tension balance, showed an increased number of Ki67-expressing cells, indicating that tension-balanced stimulation promotes the proliferation of epidermal keratinocytes. Furthermore, the number of Ki67-positive cells in the tension-release model was reduced compared to the tension-balanced model, indicating that releasing tension balance inhibits the proliferation of epidermal keratinocytes.

[0069] Figure 9 shows the morphology of the fibroblast nuclei, cell membrane, cytoskeleton, and collagen fibers in each artificial skin model. Figure 10 shows the results of an analysis of the orientation of the nuclei. Compared to the tension-free bell-shaped artificial skin, the tension-balanced skin model showed an orientation in which the nuclei were aligned in the horizontal direction, the same as the tension direction. This orientation was lost when tension was released, and the structure changed to one similar to that of the bell-shaped artificial skin. Figure 11 shows the results of an analysis of the orientation of the collagen fibers using a two-dimensional fast Fourier transform. In the tension-free bell-shaped artificial skin, aggregates of collagen fibers with no orientation were observed, while in the tension-balanced skin model stimulated by tension-balanced stimulation, the collagen fibers were aligned in the horizontal direction, the same as the tension direction. This orientation was lost when tension was released, and the structure changed to one similar to that of the bell-shaped artificial skin.

[0070] As described above, in the tension-balanced artificial skin model, improved ECM synthesis function, improved type 1 collagen synthesis function, promotion of epidermal keratinocyte proliferation and the resulting promotion of turnover, promotion of epidermal keratinocyte proliferation, and alignment of collagen fibers in the horizontal direction along the same direction as the tension direction were confirmed. In the tension-balanced artificial skin model, which reflects actual good skin conditions, tension balance promotes the expression of mechanical stress signaling molecules (ITGA2, MRTFA, ACTA2 (αSMA)), and it is believed that the above-mentioned improvements in ECM synthesis function, improved type 1 collagen synthesis function, promotion of epidermal keratinocyte proliferation and the resulting promotion of turnover, promotion of epidermal keratinocyte proliferation, and alignment of collagen fibers in the horizontal direction along the tension direction observed in normal skin tissue are maintained.

[0071] [Example 3] Screening of materials that activate mechanical stress signaling and evaluation of their skin functionality For the three models of bell-shaped artificial skin, tension-balanced artificial skin model, and tension-released artificial skin model prepared in the same manner as in Example 1, after three days of air layer culture, the medium was replaced with a drug evaluation medium containing 1% fetal bovine serum, 1% penicillin / streptomycin, 5 μg / ml insulin (FUJIFILM Wako Pure Chemical Industries, Ltd.), and 1 μM hydrocortisone (FUJIFILM Wako Pure Chemical Industries, Ltd.), and pre-cultured for 48 hours. After that, the medium was replaced with a drug evaluation medium containing 10 μM ATRA (all-trans retinoic acid), a mechanical stress activation material, and cultured for 6-48 hours.

[0072] After completion of the culture, the tissues were harvested. Total RNA was collected from each tissue in the same manner as in Example 1, and real-time PCR was performed. The genes whose expression was analyzed were mechanical stress signaling genes MRTFA and ACTA2, and ECM synthesis function-related genes COL1A1 and HAS3. The measured expression value of each gene was corrected by the ΔΔCT method, with GAPDH set as an endogenous gene expression control.

[0073] Furthermore, a portion of the collected tissue was immediately fixed in 4% paraformaldehyde phosphate buffer, paraffin sections were prepared by standard methods, and immunohistochemical staining for type 1 collagen was performed according to the following procedure.

[0074] Type 1 collagen Immunohistochemical staining was performed using rabbit anti-human type 1 collagen antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Invitrogen). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength: 470 nm) and type 1 collagen expression (excitation wavelength: 594 nm, observation wavelength: 620 nm) were observed using a confocal microscope.

[0075] 12 is a plot of the change in MRTFA gene expression in each artificial skin model in response to ATRA administration. A significant increase in MRTFA gene expression was confirmed only in the tension-balanced artificial skin model by ATRA administration.

[0076] Figure 13 plots the changes in COL1A1 and HAS3 gene expression in each artificial skin model in response to ATRA administration. For the COL1A1 gene, ATRA administration significantly promoted gene expression only in the tension-balanced artificial skin compared to the control group. For the HAS3 gene, ATRA administration significantly promoted gene expression compared to all control groups, with the expression promotion ratio being the highest in the tension-balanced artificial skin.

[0077] Immunohistochemical staining of type 1 collagen in the tension-balanced skin model is shown in Figure 14. Compared with the control group, the expression level of type 1 collagen was increased in the ATRA-administered group, confirming that collagen fiber formation was promoted.

[0078] As described above, by using the promotion of expression of mechanical stress signaling genes in skin cells as an indicator, it is possible to find a material (ATRA in this example) that promotes collagen fiber formation under conditions close to the in vivo environment. In particular, it was confirmed that more efficient evaluation / screening is possible by using a tension-balanced artificial skin model that reflects actual skin conditions.

[0079] [Example 4] Evaluation of skin functionality using mechanical stress signaling-inhibiting materials For the three models of bell-shaped artificial skin, tension-balanced artificial skin model, and tension-released artificial skin model prepared in the same manner as in Example 1, after three days of air layer culture, the medium was replaced with a drug evaluation medium containing 1% fetal bovine serum, 1% penicillin / streptomycin, 5 μg / ml insulin (FUJIFILM Wako Pure Chemical Industries, Ltd.), and 1 μM hydrocortisone (FUJIFILM Wako Pure Chemical Industries, Ltd.), and precultured for 48 hours. After that, the medium was replaced with a drug evaluation medium containing 10 nM Y-27632 (R&D Systems Co., Ltd.), a mechanical stress suppression material, and cultured for 6 to 48 hours.

[0080] After completion of the culture, the tissues were harvested. Total RNA was collected from each tissue in the same manner as in Example 1, and real-time PCR was performed. The genes whose expression was analyzed were COL1A1, COL1A2, FBN1, ELN, and MMP1, which are genes related to ECM synthesis function. The measured expression value of each gene was corrected by the ΔΔCT method, with GAPDH set as an endogenous gene expression control.

[0081] Furthermore, a portion of the collected tissue was immediately fixed in 4% paraformaldehyde phosphate buffer, paraffin section specimens were prepared by the usual method, and immunohistochemical staining of mechanical stress signaling molecules (ITGA2, MRTFA, αSMA) and type 1 collagen was performed. For mechanical stress signaling molecules (ITGA2, MRTFA, αSMA), the same procedure as in Example 1 was performed, and for type 1 collagen, the following procedure was used. In addition, the actin cytoskeleton and cell membrane were observed by the following procedure.

[0082] Type 1 collagen Immunohistochemical staining was performed using rabbit anti-human type 1 collagen antibody (Abcam.plc) and Alexa Fluor 594-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the total amount and localization of nuclei (excitation wavelength: 350 nm, observation wavelength 470 nm) and type 1 collagen expression (excitation wavelength 594 nm, observation wavelength 620 nm) were observed using a confocal microscope.

[0083] Actin cytoskeleton and cell membrane Fluorescent tissue staining was performed using Alexa594-labeled phalloidin (Thermo Fisher Scientific Co., Ltd.) and Alexa488-labeled WGA (Thermo Fisher Scientific Co., Ltd.). The nuclei of the stained tissue were stained with Hoechst 33342 (Thermo Fisher Scientific Co., Ltd.), and the nuclei (excitation wavelength: 350 nm, observation wavelength: 470 nm), actin cytoskeleton (excitation wavelength: 594 nm, observation wavelength: 620 nm), and cell membrane (excitation wavelength: 488 nm, observation wavelength: 535 nm) were observed using a confocal microscope.

[0084] Figure 15 shows immunohistochemical staining images of mechanical stress signal molecules in each artificial skin model after administration of Y27632. It was confirmed that administration of Y27632 to the tension balance model reduced the expression levels of ITGA2, MRTFA, and αSMA to the same extent as the tension release model.

[0085] Figure 16 shows the results of image analysis of the nuclear translocation rate of MRTFA. The nuclear translocation rate of MRTFA was significantly decreased by administration of Y27632, confirming the inhibitory effect of Y27632 on mechanical stress signals.

[0086] Figure 17 plots the changes in gene expression of COL1A1, COL1A2, FBN1, ELN, and MMP1 in each artificial skin model after ATRA administration. Administration of Y27632 to the tension balance model significantly suppressed gene expression of COL1A1, COL1A2, FBN1, and ELN, which are involved in ECM synthesis, to a level similar to that of tension-free artificial skin. Furthermore, administration of Y27632 significantly increased gene expression of MMP1, which is involved in ECM degradation, to a level similar to that of tension-free artificial skin.

[0087] The immunohistochemical staining image of type 1 collagen is shown in Figure 18. The expression level of type 1 collagen in the tension-balanced artificial skin group was reduced to the same level as in the tension-release model, confirming that collagen fiber formation was suppressed.

[0088] Fluorescent staining images of the cytoskeleton and cell membrane are shown in Figure 19. It was confirmed that the orientation of the cytoskeleton, cell membrane, and nucleus in the direction of tension in the tension-balanced artificial skin group was reduced to the same level as in the tension-release model, and the orientation of the tissue was disrupted.

[0089] As described above, by using the suppression of expression of mechanical stress signaling genes in skin cells as an indicator, it is possible to find materials (Y27632 in this example) that act to suppress collagen fiber formation, promote ECM degradation, and reduce tissue orientation. In particular, it was confirmed that more efficient evaluation / screening is possible by using a tension-balanced artificial skin model that reflects actual skin conditions. [Industrial Applicability]

[0090] According to the method for evaluating / screening tissue morphology / tissue function control agents of the present invention, it is possible to find health care materials and pharmaceutical materials that can transmit signals to cells that constitute tissues equivalent to signals obtained by mechanical stress under normal conditions in the living body. The materials found by the method of the present invention can appropriately control the tissue morphology and tissue function of the living body, and are therefore expected to be effective against symptoms caused by reduced mechanical stress. For example, they are useful for treating aesthetic deterioration such as wrinkles, sagging, age spots, loss of transparency, and enlarged pores due to aging, as well as scars, pregnancy-related and keloid skin fibrosis, and epithelial cell cancer.

Claims

1. applying a test substance to a tissue or cell capable of expressing a mechanical stress signaling molecule; and assessing the expression, activity, or subcellular localization of mechanical stress signaling molecules in the tissue or cells; The present invention is characterized in that it comprises The mechanical stress signaling molecule is ITGA2, MRTFA, or ACTA2 (αSMA); A method for evaluating / screening a tissue morphology / tissue function control agent, wherein the tissue or cell capable of expressing the mechanical stress signaling molecule is skin tissue, epidermal tissue, dermal tissue, epidermal cells (including epidermal keratinocytes), dermal cells (including dermal fibroblasts), cultures thereof (including established cell lines), or an artificial skin model composed of any of these.

2. 2. The method for evaluating / screening a tissue morphology / tissue function controlling agent according to claim 1, wherein the tissue morphology / tissue function controlling agent is a skin function controlling agent.

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