Beauty methods

Abstract

The present invention aims to provide a method for proliferating stem cells and their derived cells, thereby solving cosmetic problems such as wrinkles and sagging skin. Stem cells and their derived cells can be proliferated by providing mechanical stimulation to skin containing sebaceous glands.

Description

[Technical Field]

[0001] The present invention relates to a method for increasing stem cells and their induced cells, and to a cosmetic method utilizing the same. [Background technology]

[0002] The dermis is mainly composed of interstitial and cellular components, and also contains blood vessels and nerves. The dermis is constantly affected by external factors such as temperature changes, ultraviolet radiation, and physical stimuli, as well as internal factors such as stress and aging. This alters the cellular activity of dermal fibroblasts contained in the dermal layer. Since dermal fibroblasts produce interstitial components that are involved in skin elasticity and firmness, a decrease in cellular activity leads to thinning of the dermis, loss of elasticity, causing wrinkles and sagging, which are major cosmetic problems. To date, research has been conducted on ingredients that inhibit the activity of enzymes that break down interstitial components, such as heparanase and matrix metalloproteinase, as drugs to improve wrinkles, sagging, and firmness (Patent Document 1: JP 2016-169238, Patent Document 2: JP 2012-144499). Furthermore, research is being conducted on components that can increase the cellular activity of dermal fibroblasts, and various dermal fibroblast activators have been developed (Patent Document 3: Japanese Patent Publication No. 2006-262806). [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] Japanese Patent Publication No. 2016-169238 [Patent Document 2] Japanese Patent Publication No. 2012-144499 [Patent Document 3] Japanese Patent Publication No. 2006-262806 [Overview of the project] [Problems that the invention aims to solve]

[0004] Our research has shown that once dermal fibroblasts experience a decline in cellular activity due to aging or other factors, it is difficult to restore their activity. Therefore, we concluded that, rather than trying to enhance the cellular activity of dermal fibroblasts that have already experienced a decline in activity, inducing the differentiation of new dermal fibroblasts would be more effective in solving cosmetic problems such as wrinkles and sagging. [Means for solving the problem]

[0005] The inventors of this invention diligently experimented with methods to induce differentiation from stem cells into dermal fibroblasts, and surprisingly discovered that applying mechanical stimulation to the area around the sebaceous glands, where many stem cells are present, promotes the division of stem cells and induces new dermal fibroblasts.

[0006] Therefore, the present invention relates to the following: [1] A method for proliferating stem cells or induced cells thereof around sebaceous glands, comprising applying mechanical stimulation to a skin sample when organ culturing a skin sample containing sebaceous glands taken from the skin. [2] The method according to item 1, wherein the mechanical stimulus is an extension stimulus. [3] A cosmetic method comprising applying mechanical stimulation to skin that is prone to sagging, wrinkles, and loss of firmness, thereby causing the proliferation of stem cells or induced cells surrounding the sebaceous glands. [4] The cosmetic method described in item 3, wherein the sebaceous gland density in the skin is such that it accounts for 10-80% of the skin area. [5] The cosmetic method according to item 3 or 4, wherein the mechanical stimulation is stretching, pressing, or suction on the skin. [6] Measurement section for measuring sebaceous gland density, and Mechanical stimulation application part A beauty device comprising the following: applying mechanical stimulation to a skin area with a high density of sebaceous glands, thereby causing the proliferation of stem cells or induced cells surrounding the sebaceous glands. [Brief explanation of the drawing]

[0007] [Figure 1]Figure 1 shows fibroblasts in the dermis of young and elderly subjects, observed and remodeled using a three-dimensional electron microscope (SBF-SEM). [Figure 2] Figure 2 shows photographs of cheek skin samples from young subjects (in their 20s) and elderly subjects (in their 80s) after tissue staining. [Figure 3] Figure 3 shows photographs of skin samples from the cheek of an elderly subject (in their 80s), showing the normal dermal layer and the area around the sebaceous gland after staining for stem cells. [Figure 4] Figure 4A shows photographs of CD54-positive stem cells stained and cultured in skin samples, both when no stretching stimulation was applied (control) and when stretching stimulation was applied. Figure 4B shows photographs of collagen stained and cultured in skin samples, both when no stretching stimulation was applied and when stretching stimulation was applied. [Figure 5] Figure 5A shows photographs of the nasolabial folds before and after stretch stimulation of the cheeks. Figure 5B is a graph showing that the wrinkles in the nasolabial folds improved with stretch stimulation. [Figure 6] Figure 6 shows micrographs of cells cultured for 2 days after adding 2.5 ml of cell suspensions prepared at concentrations of 0.25 × 10⁴ cells / ml, 0.5 × 10⁴ cells / ml, 1.0 × 10⁴ cells / ml, 2.0 × 10⁴ cells / ml, and 4.0 × 10⁴ cells / ml, indicating the degree of contact at each concentration. [Figure 7] Figure 7 is a graph comparing the expression levels of type I collagen at each level of contact. [Figure 8] Figure 8 shows the gene expression suppression effect of siRNA on each gene. [Figure 9] Figure 9 shows the changes in type I collagen expression levels when gene expression is suppressed by siRNA targeting each gene. [Figure 10]Figure 10 shows the changes in cell proliferation rate (A) and gene expression of p21 (B) when the gene expression of cadherin 2 is suppressed in cultured dermal fibroblasts. [Figure 11] Figure 11 shows the morphological changes when the gene expression of cadherin 2 is suppressed in cultured dermal fibroblasts. By suppressing the gene expression of cadherin 2 (Knockout), cell - cell adhesion was no longer observed, the shape became rounded, and staining of β - Gal, an age marker, was observed.

Embodiments for Carrying out the Invention

[0008] The present invention relates to a method for growing stem cells or their induced cells. This method includes a step of applying mechanical stimulation to a skin sample containing sebaceous glands collected from the skin during organ culture of the skin sample.

[0009] There are stem cells capable of differentiating into dermal fibroblasts around the sebaceous glands. These stem cells are characterized by CD54 expression. When organ - culturing a skin sample containing sebaceous glands and applying mechanical stimulation to the skin sample for culturing, the stem cells around the sebaceous glands increase. The cells increased in this way may maintain their stemness or may be induced cells differentiated from stem cells. Examples of induced cells differentiated from stem cells include dermal progenitor cells and dermal fibroblasts. Since the stem cells present in the dermis can differentiate into dermal fibroblasts, they can also be called dermal stem cells.

[0010] Mechanical stress applied to skin samples or skin refers to physical stimuli that exert mechanical action. Mechanical stress is not limited to stimuli applied through machines such as probes or actuators, but may be applied by any means that exert mechanical action. Examples of mechanical stress include at least one of pressing, suction, compression, and stretching. Mechanical stress may be applied parallel to the surface of the skin sample or skin, i.e., transversely, or perpendicular to the surface, i.e., longitudinally. The intensity of the mechanical stress can be arbitrarily set within a range that does not damage, tear, or destroy the skin sample or skin. As an example, it is preferable to apply compression or stretching stimuli to the skin sample so that a deformation of about 10% to 50% occurs in the skin sample. The deformation is preferably 20% to 40%, and more preferably about 30%.

[0011] Sebaceous glands are organs that produce sebum and are located in association with hair follicles. Sebaceous glands are distributed throughout almost the entire body except for the palms and soles of the feet. Therefore, the skin sample in this invention may be obtained from any part of the skin, or in particular from the skin of seborrheic areas where sebaceous glands are well-developed. Examples of seborrheic areas include the forehead, nasal wings, nasolabial folds, scalp, sternum, armpits, abdomen, and external genitalia. In another embodiment, the area from which the sample should be obtained can be selected based on the proportion of the sebaceous gland area in the epidermis. For example, a sample may be obtained from skin where sebaceous glands occupy 10-80% of the skin area. The sebaceous gland area is preferably 30-80%, and more preferably 50-80%. The size of the skin sample can be arbitrarily selected, for example, 100 mm. 2 ~10000mm 2 Sections with a surface area and depth to the dermis can be used. From the viewpoint of reducing invasiveness, 500 mm 2 The following is preferable: 300mm 2 The following is more preferable. On the other hand, from the viewpoint of obtaining a sufficient amount of stem cells or their induced cells, 100 mm 2 The above is preferable, 500mm 2 The above is even more preferable.

[0012] Organ culture of skin samples can be performed according to standard procedures, for example, by the method described in Journal of Dermatological Science 74 (2014) 236-241. Mechanical stimulation, especially stretching stimulation, may be applied before or during culture. The intensity of the mechanical stimulation can be appropriately selected depending on the size of the skin sample. As an example, stretching stimulation is applied by fixing one end of the skin sample with tweezers or similar tools and pulling it. Mechanical stimulation may be applied for the entire duration or part of the organ culture period.

[0013] Stem cells or their induced cells, which are proliferated in the organ culture process, can be isolated or separated by cutting or enzymatic treatment of the cultured organ. The separated cells can be further cultured or injected directly into the dermis of the original target without further culture.

[0014] In another aspect, the present invention relates to a cosmetic method that involves applying mechanical stimulation to the skin, thereby facilitating the proliferation of stem cells or their induced cells around the sebaceous glands. In this cosmetic method, stem cells or their induced cells around the sebaceous glands proliferate, and these cells differentiate into dermal progenitor cells and dermal fibroblasts. Therefore, the cosmetic method of the present invention can promote an increase in dermal fibroblasts in the dermis, thereby promoting the production of interstitial components. Thus, the cosmetic method of the present invention can also be described as a method for promoting interstitial component production, or a method for improving wrinkles, sagging, and firmness of the skin.

[0015] The beauty method of the present invention is applicable to individuals who are concerned with wrinkles and sagging, and those who have lost firmness. Sagging can be determined by visual assessment. It is preferable to apply the beauty method of the present invention to individuals whose Ur / Uf ratio is 6 or less, preferably 5 or less, and more preferably 4 or less, in measurements by visual assessment, and / or to individuals whose Ur / Uf ratio is 0.8 or less, preferably 0.7 or less, and more preferably 0.6 or less, in measurements using a cute meter.

[0016] The mechanical stimulation applied to the skin may, for example, be at least one of the following: pressing, suction, compression, or stretching. The mechanical stimulation may be applied parallel to the skin surface, i.e., transversely, or vertically, i.e., longitudinally. The mechanical stimulation can usually be applied for a period of 1 minute or more, more preferably for 3 minutes or more, and even more preferably for 5 minutes or more. There is no particular upper limit, but from the viewpoint of ensuring the simplicity of the method, it is preferably within 1 hour, more preferably within 30 minutes, and even more preferably within 15 minutes. Mechanical stimulation can be applied to the skin using a device equipped with components such as probes and actuators applied to apply mechanical stimulation. As an example of such a device, the actuator described in Japanese Patent Publication No. 2011-505897 can be used. Furthermore, in one embodiment, this cosmetic method may include facial exercises and massage. Facial exercises may include puffing out the cheeks or widening the eyes. Facial massages may include massage using hands or rollers.

[0017] The present invention's cosmetic method can promote the proliferation of stem cells or their induced cells surrounding the sebaceous glands. Therefore, it may further include a step of observing stem cells or their induced cells surrounding the sebaceous glands before and after the application of mechanical stimulation. It is preferable to observe the stem cells or their induced cells immunohistochemically 24 hours or more, preferably 48 hours or more, after the application of mechanical stimulation. Instead of observing stem cells or their induced cells, the process may include steps to confirm improvements in wrinkles, sagging, and skin firmness. Improvements in wrinkles and sagging can be measured using known measuring instruments (e.g., Japanese Patent Publication No. 2017-064391). Skin firmness can be measured using a dermatometer or a cutemeter.

[0018] In this invention, mechanical stimulation is preferably applied to areas with a high density of sebaceous glands. 2 The above is preferable, and more preferably 400 pieces / cm 2Applying mechanical stimulation to the area described above is preferable in that it promotes the proliferation of stem cells or their induced cells around the sebaceous glands.

[0019] In a further aspect of the present invention, Sebaceous gland detection section, and Mechanical stimulation application part The invention also relates to a beauty device that applies mechanical stimulation to a skin area with a high density of sebaceous glands, thereby causing the proliferation of stem cells or induced cells surrounding the sebaceous glands.

[0020] The sebaceous gland detection unit is, for example, a microscope, and can detect the location and number of sebaceous glands. By applying mechanical stimulation to areas with a high density of sebaceous glands using the mechanical stimulation application unit, the proliferation of stem cells or their induced cells surrounding the sebaceous glands can be promoted.

[0021] Examples of mechanical stimulation include at least one of the following: pressing, suction, and stretching. The mechanical stimulation attachment unit comprises components such as probes and actuators applied to provide these mechanical stimuli. A pressing probe can provide mechanical stimulation to the skin by pressing the skin vertically. A suction probe can provide mechanical stimulation to the skin by applying negative pressure when in contact with the skin. A stretching probe can provide mechanical stimulation to the skin by having one or more contact points in contact with the skin move horizontally relative to the skin surface.

[0022] Dermal fibroblasts are fibroblasts present in the dermis that produce stromal components within the dermis. Therefore, dermal fibroblasts induced from stem cells can help replenish stromal components in the dermis that have decreased due to external or internal factors. In fact, enhanced collagen production was observed near stem cells surrounding the sebaceous glands that were increased by mechanical stimulation (Figure 4B).

[0023] On the other hand, research by the present inventors has revealed that dermal fibroblasts present in the dermis of elderly individuals differ in morphology from those present in the dermis of young individuals, and that, contrary to previous findings, dermal fibroblasts form adhesions with each other (Figure 1). Specifically, dermal fibroblasts in young individuals have protrusions and adhere to other fibroblasts, while fibroblasts in elderly individuals are spherical with few protrusions and have little or no adhesion to other fibroblasts. It is thought that in dermal fibroblasts of elderly individuals, the loss of cell-to-cell adhesion leads to a decrease in cell activity, i.e., the production of interstitial components. Research by the present inventors has shown that the amount of collagen produced changes in response to intercellular adhesion of dermal fibroblasts (Figure 7), and furthermore, that cadherin 2 is involved in such intercellular adhesion (Figure 9), indicating that intercellular adhesion contributes to the production capacity of interstitial components, including collagen production, i.e., cell activity. Although not intended to be purely theoretical, it is thought that the stem cells surrounding the sebaceous glands, increased by mechanical stimulation according to the present invention, differentiate into dermal fibroblasts as they proliferate. In that case, since there are many dermal fibroblasts in the surrounding area, there is also a high degree of cell-cell adhesion, which leads to an increase in dermal fibroblasts with high cell activity around the sebaceous glands, thereby increasing collagen production (Figure 4B), and it is thought that this is what actually improved wrinkles in vivo (Figure 5).

[0024] Conventionally, for cosmetic purposes, particularly to improve wrinkles, sagging, and firmness, researchers have searched for drugs that can activate dermal fibroblasts, and various components, including extracts, have been selected. On the other hand, it may be difficult to re-establish adhesion in fibroblasts that have lost their projections and thus lost cell adhesion. Therefore, the inventors conceived of stimulating stem cells that exist as a source of dermal fibroblasts, rather than activating already existing dermal fibroblasts, and found that the number of stem cells or their induced cells around the sebaceous glands can be increased by mechanical stimulation (Figure 4). New dermal fibroblasts induced from stem cells have projections and can form adhesion with other fibroblasts. New dermal fibroblasts induced from stem cells have excellent production capacity for interstitial components, including collagen, by adhering to other fibroblasts (Figure 7).

[0025] The interstitial components of the dermis are mainly composed of collagen fibers, elastic fibers, and matrix. Therefore, by enhancing the cellular activity of dermal fibroblasts, the production of at least one, preferably two, and more preferably all of the components of collagen fibers, elastic fibers, and matrix increases.

[0026] Collagen fibers are composed of collagen. Approximately 20 types of collagen molecules are known, differing in the molecular structure of the α-chain. In this invention, any type of collagen may be used, but type I, type III, type V, type IV, type VII, and type 17 collagen, which are mainly present in the dermis, are preferred, more preferably type I, type III, and type V collagen, and most preferably type I collagen, which accounts for about 80% in the dermis. The amount of collagen fiber production can be determined by measuring the amount of collagen produced or expressed. The amount of collagen produced changes depending on the degree of adhesion of dermal fibroblasts. Therefore, even in the skin of elderly people, new fibroblasts induced from stem cells maintain cell adhesion, resulting in high production of interstitial components, including collagen.

[0027] The main component of elastic fibers is elastin, with fibrillin further surrounding the elastin. The amount of elastic fibers produced can be determined by measuring the production or expression level of at least one of elastin or fibrillin.

[0028] The matrix, primarily the extracellular matrix, is composed of glycoproteins and proteoglycans. Glycoproteins are proteins containing sugars, such as fibronectin in the dermis. Fibronectin binds to cell surface proteins and functions as a cell scaffold, and can also bind to other macromolecules such as collagen. Proteoglycans are large molecules in which glycosaminoglycans are bound to an axial protein, and in the dermis, hyaluronic acid and dermatan sulfate are the main glycosaminoglycans. Proteoglycans primarily play a role in water retention in the dermis.

[0029] All references made herein are incorporated herein by citation in their entirety.

[0030] The embodiments of the present invention described below are for illustrative purposes only and do not limit the technical scope of the invention. The technical scope of the invention is limited solely by the claims. Modifications to the invention, such as additions, deletions, and substitutions of constituent elements of the invention, can be made without departing from the spirit of the invention. [Examples]

[0031] Observation using a 3D microscope Skin sections from young subjects (20 years old) and elderly subjects (80 years old) were treated with conductive resin and subjected to 3D electron microscopy (SBF-SEM) observation. The 3D remodeled image is shown (Figure 1). The sample preparation and observation methods using SBF-SEM are described below.

[0032] Serial Blockface Scanning Electron Microscope (SBF-SEM) Human skin samples were fixed in a buffer solution of 2% glutaraldehyde and 2% paraformaldehyde at 4°C for several days. The samples were treated with phosphate-buffered saline containing 2% osmium tetroxide (Nisshin EM, Tokyo, Japan) and 1.5% potassium ferrocyanide (Wako Pure Chemical Industries, Ltd.) at 4°C for 1 hour, then with 1% thiocarbohydrazide (Sigma, St. Louis, Mo, USA) at room temperature for 20 minutes, then with 2% osmium tetroxide aqueous solution at room temperature for 30 minutes, then with 2% uranyl acetate solution at 4°C for more than 12 hours, and finally at room temperature for 1 hour. The samples were incubated in a solution of 0.67% lead nitrate (pH 5.0-5.5, TAAB, Berkshire, UK) and 0.03 ml-aspartic acid (Nacalai Tesque, Kyoto, Japan) at 65°C for 30 minutes. Next, the samples were dehydrated in a stepwise ethanol series, treated with dehydrated acetone, and impregnated with Quetol812 epoxy resin (Nisshin EM, Tokyo, Japan) at 35°C. They were then embedded in Quetol812 containing Kejen black powder (Nguyen et al. Sci Rep. 2016). The resin was incubated at 70°C for over 7 nights to ensure polymerization. SBF-SEM observation was performed using a field emission scanning electron microscope (Merlin, Carl Zeiss, Oberkochen, Germany) equipped with a 3View chamber ultramicrostrome system (Gatan, Pleasanton, CA). A continuous image sequence was acquired at 80 nm steps over a depth of 60 μm with a width of 80-90 μm × 80-90 μm (11-12 nm / pixel). The continuous images were processed using FIJI (https: / / fiji.sc / ). Segmentation and 3D reconstruction were performed using Amira (Maxnet Co., Ltd, Tokyo, Japan).

[0033] Observation of skin sections Skin sections obtained from the cheeks of young and elderly women were stained with Wang Gieson stain and observed under a microscope (Figure 2). Photographs were taken of the areas containing sebaceous glands (Figure 2). In young women, the staining intensity was high, indicating a high amount of collagen in the dermis, while in elderly women, the staining intensity in the dermis was low, indicating a low amount of collagen. On the other hand, the area surrounding the sebaceous glands showed a high amount of collagen (white arrows).

[0034] Skin sections obtained from the cheeks of elderly individuals were fixed with acetone, reacted with an anti-CD54 antibody, and then stem cells were identified using Emvisoin (DAKO). Images were taken of the entire dermis and the area around the sebaceous glands. While stem cells are present in the skin of elderly individuals, their quantity is extremely small throughout the dermis, whereas a large number of stem cells are found around the sebaceous glands (Figure 3: the black dotted line in the figure indicates the boundary of the sebaceous gland, and the arrow indicates the stem cell).

[0035] The above experiments suggest that in elderly individuals, dermal fibroblasts in the dermis lose their adhesion to other cells, leading to a decrease in cellular activity (collagen production). On the other hand, the studies also suggest that even in the skin of elderly individuals, there are many stem cells around the sebaceous glands, which serve as a source of dermal fibroblasts and contribute to the increased collagen levels around the sebaceous glands.

[0036] Organ culture experiment Two 10mm square human skin sections were obtained. One section was cultured without compression, while the other section was immersed in DMEM medium containing 10% FBS, under vertical pressure to deform it approximately 30% in the longitudinal direction, and cultured for 7 days in a 5% CO2 atmosphere at 37°C.

[0037] The skin sample sections after culturing were fixed with acetone, and the stem cells were visualized using anti-CD54 antibody with Emvisoin (DAKO). The results are shown in Fig. 4A. In the skin samples given compressive stimulation, it was observed that the stem cells or the cells induced from the stem cells were proliferating. Further, the skin sample sections after culturing were fixed with acetone, reacted with anti-type I collagen antibody and visualized using Emvisoin (DAKO). The results are shown in Fig. 4B. In the skin samples given compressive stimulation, it was shown that the collagen production was increased around the sebaceous glands.

[0038] The effects of mechanical stimulation In 8 female subjects (in their 40s), the exercise of puffing out the cheeks was performed once a day for 10 minutes, and stretching stimulation was given to the cheeks. The experiment was carried out over 2 months. Before the experiment and after 1 month of the experiment, photographs were taken (Fig. 5A), and the degree of cheek sagging was visually evaluated (Fig. 5B). Improvement was seen between before and after the experiment.

[0039] From the above experiments, it was shown that when mechanical stimuli such as stretching stimulation and compressive stimulation are applied to the skin containing sebaceous glands, the stem cells or the induced cells from the stem cells proliferate, and it was shown that the stretching stimulation actually contributes to the improvement of wrinkles in vivo.

[0040] Culture experiment Human primary cultured fibroblasts were obtained from human skin samples according to a conventional method. The number of cells was counted, and in DMEM medium (containing 10% FBS), 0.25×10 4 cells (non-contact), 0.5×10 4 cells (mild contact), 1.0×10 4 cells (moderate contact), 2.0×10 4 cells (high contact), and 4.0×10 4 cells (excessive contact) were prepared. Such cell suspensions were dropped at 2.5 ml per well into a 6-well plate (manufactured by Falcon) and cultured for 2 days in an atmosphere of 37°C and 5% CO2. The microscopic photographs of the cells after culturing are shown in Fig. 6.

[0041] Changes in gene expression due to differences in the degree of adhesion Cells were collected from cultures with varying degrees of contact and subjected to microarray analysis. This revealed that type I collagen expression changed depending on the degree of cell contact (data not shown). Therefore, the expression level of type I collagen was determined by real-time PCR using the primers described below (Figure 7). The expression of the GAPDH gene was used as an internal standard to determine the expression level. [Table 1]

[0042] The experiments described above demonstrated that changes in the degree of adhesion lead to changes in the expression of collagen, one of the interstitial components, in response to intercellular adhesion.

[0043] Identification of adhesion factors involved in adhesion and affecting collagen expression. To determine the factors contributing to type I collagen expression, the expression of cell adhesion proteins was suppressed in cultured fibroblasts using siRNA. siRNAs for CDH2, CDH11, and CDH13 were obtained from Qiagen. These siRNAs were used according to standard procedures to suppress the expression of each gene in cultured dermal fibroblasts. (1 × 10⁻⁶) 4 Place 0.5 ml of cell suspension with a cell density of [cells / ml] into a 24-well plate (area: 2 cm²). 2 The cells were seeded in each well of the 37°C 5% CO2 atmosphere and cultured for 2 days. Cultured fibroblasts were collected, and the gene expression of each cell adhesion protein was determined by real-time PCR, confirming the suppression of the expression of the target protein (Figure 8). Next, when the expression of type I collagen was measured in the collected cells, it was shown that suppressing the gene expression of CDH2 reduced the expression level of type I collagen (Figure 9).

[0044] Dermal fibroblasts with suppressed cadherin 2 expression and control dermal fibroblasts were each measured in 1 × 10⁻¹⁶ units. 4 Place 0.5 ml of cell suspension with a cell density of [cells / ml] into a 24-well plate (area: 2 cm²). 2Cells were seeded in each well of the cultured cells and cultured for 2 days at 37°C in a 5% CO2 atmosphere. The cultured cells were counted and their proliferation was compared (Figure 10A). In addition, the gene expression of p21, an inhibitory protein of the CDK family, was measured for each cultured cell by RT-PCR using the following primers (Figure 10B). Furthermore, cells were stained with X-Gal according to β-galactosidase activity using a cell senescence assay kit (Biovision) and photographed under a microscope (Figure 11). In control cells, cultured dermal fibroblasts had a flattened shape and cell adhesion was observed, but suppression of cadherin 2 gene expression (knockout) reduced cell adhesion, and consequently, the cell shape became more spherical compared to the control. In addition, intracellular β-galactosidase activity, an indicator of cell senescence, was elevated in dermal fibroblasts with suppressed cadherin 2 gene expression. [Table 2]

[0045] In dermal fibroblasts with suppressed cadherin 2 gene expression, both collagen gene expression and cell proliferation were suppressed. Furthermore, in dermal fibroblasts with suppressed cadherin 2 gene expression, the gene expression of p21, which causes cell cycle arrest by cyclin suppression, increased, and intracellular β-galactosidase activity, an indicator of cellular aging, was also shown to be high. These results indicate that dermal fibroblasts form intercellular contacts via the cell adhesion protein cadherin 2, and that the loss of such contacts leads to cellular aging and decreased cellular activity.

Claims

1. A method for proliferating, isolating, or separating stem cells or induced cells thereof around sebaceous glands, comprising applying mechanical stimulation to a skin sample when organ culturing a skin sample containing sebaceous glands collected from the skin.

2. The method according to claim 1, wherein the mechanical stimulus is an extension stimulus.

3. The method according to claim 1 or 2, comprising the step of cutting an organ cultured skin sample to separate the cells.

4. The method according to any one of claims 1 to 3, comprising the step of enzymatically treating an organ cultured skin sample to separate cells.

5. A cosmetic method for those concerned about wrinkles, sagging, or loss of firmness, which includes the following: A process for measuring sebaceous gland density in skin that is prone to sagging, wrinkles, and loss of firmness, and A process of applying mechanical stimulation to skin areas with a high density of sebaceous glands. A cosmetic method (excluding medical procedures) that includes, and includes, the proliferation of stem cells or induced cells surrounding the sebaceous glands by mechanical stimulation.

6. The cosmetic method according to claim 5, wherein the sebaceous gland density in the skin is 10 to 80%.

7. The cosmetic method according to claim 5 or 6, wherein the mechanical stimulation is stretching, pressing, or suction on the skin.

8. A measuring unit for measuring sebaceous gland density, and Mechanical stimulation application part A beauty device comprising the following: applying mechanical stimulation to a skin area with a high density of sebaceous glands, thereby causing the proliferation of stem cells or induced cells surrounding the sebaceous glands.

Images