WO2023243447A1 - Cosmetic method to control formation of circular collagen - Google Patents

Cosmetic method to control formation of circular collagen Download PDF

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Publication number
WO2023243447A1
WO2023243447A1 PCT/JP2023/020684 JP2023020684W WO2023243447A1 WO 2023243447 A1 WO2023243447 A1 WO 2023243447A1 JP 2023020684 W JP2023020684 W JP 2023020684W WO 2023243447 A1 WO2023243447 A1 WO 2023243447A1
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collagen
skin
dermal matrix
expression
wnt16
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PCT/JP2023/020684
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French (fr)
Japanese (ja)
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智暢 江連
伸彦 大野
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株式会社 資生堂
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F7/00—Heating or cooling appliances for medical or therapeutic treatment of the human body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H23/00—Percussion or vibration massage, e.g. using supersonic vibration; Suction-vibration massage; Massage with moving diaphragms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61H—PHYSICAL THERAPY APPARATUS, e.g. DEVICES FOR LOCATING OR STIMULATING REFLEX POINTS IN THE BODY; ARTIFICIAL RESPIRATION; MASSAGE; BATHING DEVICES FOR SPECIAL THERAPEUTIC OR HYGIENIC PURPOSES OR SPECIFIC PARTS OF THE BODY
    • A61H7/00—Devices for suction-kneading massage; Devices for massaging the skin by rubbing or brushing not otherwise provided for
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to the technical field of suppressing skin aging.
  • Sagging skin has a major impact on the appearance of one's age, and maintaining skin firmness and improving sagging is a major cosmetic issue.
  • Skin firmness is mainly due to the thickness of the dermal layer, and firm skin contains abundant elastic fibers such as collagen and elastin that fill the dermal layer.
  • collagen and elastin that fill the dermal layer.
  • the function of dermal fibroblasts declines and the production of elastic fibers decreases, and matrix metalloproteinases are activated and degrade elastic fibers. This causes the dermal layer to thin, causing sagging or moss.
  • the amount of collagen tends to decrease with age, but the amount of collagen is also affected by skin care, exposure to ultraviolet rays, and diet.
  • sagging and moss do not necessarily depend only on the amount of collagen in the dermis.
  • the amount of collagen does not necessarily decrease, and it is thought that the symptoms of sagging are affected by some influence other than the amount of collagen.
  • Other known causes besides the amount of collagen include weakening of facial muscles and increase in subcutaneous fat.
  • conventional beauty methods have focused on increasing collagen and elastic fibers by activating dermal fibroblasts, or suppressing the decomposition of collagen and elastic fibers.
  • Patent Document 1 JP 2012-144499 and Patent Document 2: Publication No. 2013-014555.
  • the purpose of this study is to clarify the internal structure of the skin related to sagging, and to provide a new beauty method to prevent and improve sagging.
  • the present inventors conducted intensive research to elucidate the internal structure of the skin that causes facial sagging, and found that in the dermis of the face, structures with elasticity are not spread evenly, and there are areas with high elasticity, It was found that there were scattered areas with low elasticity. After conducting a more detailed analysis, we found that there was a ring-shaped part with high elasticity surrounding the part with low elasticity, and the collagen arranged in a ring contracted toward the center. It was found that it causes tension on the skin and prevents it from sagging. We have also found that by applying mechanical stimulation and/or thermal stimulation to hair follicles, the formation of dermal matrix can be promoted and the formation of annularly arranged collagen can be controlled.
  • the invention therefore relates to: [1] A cosmetic method comprising promoting the formation of dermal matrix and/or controlling the formation of circular collagen in facial skin by mechanical stimulation and/or thermal stimulation of hair follicles. [2] The cosmetic method according to item 1, wherein the annular collagen refers to collagen formed in an annular shape around the hair follicle. [3] The cosmetic method according to item 1 or 2, which improves skin firmness by forming circular collagen. [4] The cosmetic method according to any one of items 1 to 3, wherein mechanical stimulation to the hair follicle is applied by bringing a probe into contact with the skin.
  • the dermal matrix is at least one selected from the group consisting of tenascin C, versican, and periostin.
  • the circular collagen is collagen formed in a circular shape around the hair follicle.
  • FIG. 1A is a heat map showing the difference in tension in a horizontal section of the dermis when the skin is deformed and analyzed. Points with strong tension (orange to red in the heat map) are shown with dotted arrows, and points with weak tension (green to light blue in the heat map) are shown with solid arrows.
  • FIG. 1B shows a heat map of the difference in tension in a horizontal section of the dermis and its surrounding tissue superimposed.
  • FIG. 2 shows enlarged photographs of horizontal sections of the dermis of facial skin specimens obtained from young and elderly subjects using X-ray CT.
  • Figure 3 shows that expression of tenascin-C was increased in fibroblasts by humoral factors secreted from hair follicle epithelial cells.
  • FIG. 10A is a graph showing changes in circular collagen diameter when organ culture is performed with thermal stimulation applied.
  • FIG. 10B is an immunostaining photograph showing the expression of Wnt16 and versican in organ culture with thermal stimulation applied.
  • FIG. 10C is a graph showing changes in the expression level of Wnt16 in human outer root sheath cells cultured under thermal stimulation.
  • the present invention provides a cosmetic method that includes promoting the formation of dermal matrix and/or controlling the formation of cyclic collagen in facial skin by mechanical stimulation and/or thermal stimulation of hair follicles.
  • the dermal matrix and fibers do not exist uniformly, but that a dermal matrix region exists just below the hair follicle, and collagen is formed in a ring shape surrounding the dermal matrix. ( Figure 1).
  • the dermal matrix can be determined by measuring the expression of contained components such as fibronectin, tenascin C, versican, and periostin. The diameter of the circular collagen increases depending on the amount of dermal matrix.
  • the amount of circular collagen can be measured based on morphology and/or collagen as a component. While there is a positive correlation between the amount of circular collagen and tension, there is an inverse correlation between the amount of circular collagen and sagging.
  • the tension in the plane direction differs depending on whether the collagen has a circular collagen structure or exists uniformly (FIG. 2 and Table 1). Therefore, when cyclic collagen is lost due to aging, it becomes impossible to maintain tension in the plane direction, and the skin as a whole is pulled downward without being able to resist gravity, resulting in sagging. Furthermore, there is a correlation between the diameter of the circular collagen and the diameter of the microhair, and the thicker the diameter of the microhair, the larger the diameter of the circular collagen (FIG. 6).
  • Stromal components are produced by fibroblasts.
  • the present inventors have shown that humoral factors from hair follicle epithelial cells increase the amount of dermal matrix produced (FIG. 3).
  • humoral factors secreted from hair follicle cells such as Wnt16, activate fibroblasts in the perifollicular and deep regions of the hair follicle, leading to the activation of fibroblasts in the dermal matrix. Production increases. Activated fibroblasts also increase collagen production. During the maturation process, the produced collagen changes its structure into a circular collagen that surrounds the dermal matrix.
  • Mechanical stimulation to the hair follicle tissue may be applied by any method as long as it is applied so as to deform the shape of the hair follicle.
  • mechanical stimulation of hair follicular tissue can be achieved with a device that includes a probe that contacts the skin.
  • the mechanical stimulus it is preferable to apply a stretching stimulus that stretches the skin.
  • the stretching stimulus is applied by moving a probe applied to the skin in a horizontal direction with respect to the skin surface while it is in contact with the skin.
  • it can be applied by moving two or more probes to be applied to the skin so as to separate them while they are in contact with the skin.
  • Stretching stimulation can be applied by moving the probe, more specifically by vibration. Mechanical stimulation allows the promotion of dermal matrix formation and/or the control of circular collagen formation.
  • the formation of circular collagen is promoted, which improves firmness.
  • At least one action selected from activation of hair follicle cells, promotion of dermal matrix formation, and control of circular collagen formation by mechanical stimulation is a performance indication of a device that applies mechanical stimulation, especially stretching stimulation, on packages, catalogs, It may be displayed on promotional materials, manuals, the screen of the device, etc., or may be used to advertise the provision of services using such devices.
  • the beauty method according to the present invention may be provided as a service in cosmetic medicine, beauty treatment, etc., or may be provided by a device that applies mechanical stimulation.
  • the beauty method according to the present invention may be displayed on a catalog, promotional material, instruction manual, or monitor screen.
  • the cosmetic method according to the present invention may be displayed together with its mechanism of action and/or effects.
  • Another aspect of the present invention also relates to a method of evaluating cyclic collagen based on the amount of dermal matrix.
  • Cyclic collagen differs from normal collagen fibers in that it has a structure formed by aggregation of collagen fibers, so it cannot be measured by measuring the amount of collagen protein or mRNA. Cyclic collagen can be evaluated based on the shape of aggregated collagen fibers by tissue staining, etc., but such evaluation methods lack quantitative properties.
  • cyclic collagen is formed so as to surround the dermal matrix containing proteoglycans, there is a relationship in which as the amount of dermal matrix increases, the amount of cyclic collagen also increases. Therefore, cyclic collagen can be evaluated based on the amount of dermal matrix.
  • annular collagen annular collagen formed around the hair follicle or in the subfollicular region can be evaluated.
  • the amount of dermal matrix can be determined by measuring the expression level of any component of the dermal matrix, such as tenascin C, fibronectin, versican, periostin, etc. Tenascin C, versican, or periostin is preferably used.
  • the expression level of protein and/or mRNA can be measured using techniques well known in the art, such as immunological techniques or quantitative PCR, respectively.
  • cyclic collagen By evaluating cyclic collagen, current or future skin tightness can be determined. It is possible to predict future sagging, wrinkles, and moss caused by skin tension. As an example, when the amount of circular collagen is lower than a predetermined threshold value, there is a high probability that sag will be formed even if no sag is currently formed.
  • the threshold value can be appropriately determined by large-scale investigation of the amount of cyclic collagen in living organisms.
  • a threshold value can be set based on the relationship between sagging and the amount of annular collagen in the skin using a sagging determination method known in this technical field such as cutometer or photo judgment. Further, by conducting a follow-up survey after a predetermined period of time, it becomes possible to set the threshold value more accurately.
  • the present invention also relates to a method of screening for a cyclic collagen production promoter using dermal matrix protein and/or Wnt16 expression as an indicator in a fibroblast culture.
  • the screening method of the present invention more specifically includes the following: cultivating a fibroblast culture in a medium containing the candidate component; measuring the expression of dermal matrix protein and/or Wnt16 in the fibroblast culture after culturing; and comparing the measured dermal matrix protein and/or Wnt16 expression with a control dermal matrix protein and/or Wnt16 expression.
  • candidate components can be screened as cyclic collagen production promoters.
  • the screening method may include a step of selecting a candidate component as a cyclic collagen production promoter when dermal matrix protein and/or Wnt16 expression is increased.
  • the cyclic collagen production promoter thus screened can promote cyclic collagen production by enhancing dermal matrix protein and/or Wnt16 expression.
  • the cyclic collagen production promoter can also be called a sagging improving agent.
  • As the dermal matrix protein at least one selected from the group consisting of tenascin C, versican, and periostin can be used.
  • the control dermal matrix protein and/or Wnt16 expression can be used to express dermal matrix protein and/or Wnt16 in fibroblast cultures that differ only in that they do not contain the candidate component.
  • a threshold value may be set based on the expression of dermal matrix protein and/or Wnt16 in the control group, or culture and measurement steps may be performed in parallel.
  • the expression of dermal matrix protein and/or Wnt16 may be the protein amount or mRNA amount of dermal matrix protein and/or Wnt16 in fibroblast culture, and can be expressed by the present technical field such as immunological method or quantitative PCR, respectively. It can be measured using a well-known method.
  • any library of cosmetic materials, food materials, pharmaceutical materials, etc. can be used.
  • a library a compound library, an extract library, etc. may be used.
  • compounds and extracts included in each library commercially available compounds and extracts may be used, or synthesized compounds and prepared extracts may be used.
  • Example 1 Reconstruction of skin dynamics The excess skin was deformed, images were acquired with X-ray CT, skin dynamics were reconstructed on a computer, and the height of tension in the horizontal section of the skin was measured ( Figure 1A). Furthermore, in order to investigate the causes of high and low tension regions in the horizontal section of the dermis, we observed the shallow and deep regions of the dermis in the horizontal section ( Figure 1B). Regions of low tension were shown to be surrounded by regions of high tension. It was also shown that the region of low tension was the lower region of the hair follicle.
  • Example 2 Changes in circular collagen due to aging Facial skin specimens were obtained from excess facial skin of young people (under 40 years old) and elderly people (over 60 years old). A cylindrical skin section several millimeters in diameter was cut from the skin specimen and photographed using X-ray CT ( Figure 2). In skin specimens from young people, the dermal matrix region and the circular collagen surrounding the dermal matrix region could be clearly distinguished, whereas in skin specimens from elderly patients, it was difficult to distinguish between the dermal matrix region and the circular collagen, and the collagen had an amorphous shape. (A typical example is shown in Figure 2).
  • Example 3 Relationship between cyclic collagen and skin tension and sagging
  • the amount of cyclic collagen was quantified using a hair follicle condition grading method.
  • the tension in the cheek area of the subject's face was measured using a Cutometer.
  • the degree of sagging in the cheeks of the subject's face was evaluated with photographs.
  • Statistical analysis was performed on the state of circular collagen and the degree of tension and sagging of the cheeks using Spearman's correlation coefficient. The results are shown in the table below.
  • Example 4 Induction of dermal matrix by hair follicle cells Hair follicle epithelial cells and fibroblasts were seeded in a Boyden chamber, and the influence of hair follicle epithelial cells on fibroblasts was investigated. More specifically, fibroblasts (established from human biopsy skin) were seeded in the upper chamber at 5,000 cells/ml in DMEM medium, and hair follicle epithelial cells (established from human biopsy skin) were seeded in DMEM medium. The cells were seeded at 250,000 cells/ml in the lower chamber and cultured at 37°C under a CO 2 atmosphere for 48 hours. Fibroblasts in the upper chamber after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen).
  • RNA extraction kit Qiagen
  • a reverse transcription reaction was performed using a Superscript kit (Invitrogen) and subjected to quantitative PCR.
  • a control (Cont.) was prepared similarly, differing only in that the lower chamber did not contain hair follicle epithelial cells.
  • the following primer set was used for quantitative PCR.
  • the expression level of TenascinC is shown in FIG.
  • Example 5 Production of dermal matrix by hair follicle stimulation Stretching stimulation was applied to cylindrically cut skin sections (young subjects and elderly subjects). Organ culture was performed in DMEM medium at 37° C. in a 5% CO 2 atmosphere for 7 days while applying stretching stimulation. The skin samples after culture were fixed with acetone and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 ⁇ m were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 hours, the primary antibodies were anti-tenascin C antibody (Abcam), anti-versican antibody (Abcam), and anti-periostin antibody (Abcam) diluted x200.
  • a blocking solution 1% BSA
  • FIG. 4 It was shown that tenascin-C is expressed in the area from around the hair follicle to under the hair follicle. Furthermore, after stretching stimulation, it was shown that the expression of tenascin-C increased in the area from around the hair follicle to under the hair follicle.
  • Example 6 Relationship between fine hair and collagen on the body and face Skin specimens were obtained from excess skin from the body and from the face, and examined using an X-ray CT machine (sold by Zeiss). It was subjected to measurement and a three-dimensional structure was constructed. In particular, fine hair and collagen in the dermis were visualized (FIGS. 5A and B). From the three-dimensional construct, the amount of collagen contained in the dermis was determined and compared between the body and face ( Figure 5C). Next, the diameter of microhairs and the diameter of circular collagen in the excess skin were measured and plotted on a graph (FIG. 6). The density of fine hair was higher on the face than on the body. Collagen content was high in the body, and collagen was uniformly present in the dermis.
  • Example 7 Factors that induce proteoglycan layers Screening was performed for factors that induce proteoglycan layers in fibroblast cultures. More specifically, fibroblasts (established from human biopsied skin) were seeded in a 24-well plate culture vessel at 1 ⁇ 10 4 cells/ml in DMEM medium supplemented with 10% FBS. 1 ⁇ g/ml of Wnt16 (R&D Co., Ltd.) was added to the medium and cultured at 37° C. in a CO 2 atmosphere for 48 hours. The fibroblasts after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen). A reverse transcription reaction was performed using a Super script kit (Invitrogen) and subjected to quantitative PCR. The following primer set was used for quantitative PCR. The expression levels of periostin and versican are shown in FIG. Addition of Wnt16 increased the expression levels of periostin and versican.
  • Example 8 Expression of Wnt16 around hair follicles in young and elderly subjects Facial skin specimens were obtained from excess facial skin of young people (under 40 years old) and elderly people (over 60 years old). A cylindrical skin section with a diameter of several millimeters was cut from the skin specimen, fixed with acetone, and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 ⁇ m were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 h, they were incubated overnight at 4°C using a x200 dilution of anti-Wnt16 antibody (LS-bio) as the primary antibody, followed by Envision (1% BSA).
  • a blocking solution 1% BSA
  • LS-bio anti-Wnt16 antibody
  • FIG. 8A the expression of Wnt16 was evaluated using 4-level grading, and the results are shown in FIG. 8B.
  • Wnt16 expression around hair follicles decreased.
  • Example 10 Effects of thermal stimulation
  • Abdominal skin specimens young subjects under 40 years of age cut into cylinders were subjected to organ culture in a DMEM medium. Thermal stimulation was applied at 42°C for 60 minutes, and then culture was performed at 37°C in a 5% atmosphere for 7 days. Skin samples after organ culture were fixed with acetone and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 ⁇ m were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 hours, they were incubated overnight using anti-versican antibody (Abcam) and anti-Wnt16 antibody (anti-Wnt16 antibody) diluted 1:200 as primary antibodies.
  • Abcam anti-versican antibody
  • anti-Wnt16 antibody anti-Wnt16 antibody
  • human outer root sheath cells were obtained from fine hair sites in the dermis and cultured in kSFM medium at 42°C for 60 minutes. As a control, the cells were cultured at 37°C. Human outer root sheath cells after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen). A reverse transcription reaction was performed using a Superscript kit (Invitrogen) and subjected to quantitative PCR. A control (Cont.) was prepared similarly, differing only in that the lower chamber did not contain hair follicle epithelial cells. The following primer set was used for quantitative PCR. The expression level of Wnt16 is shown in FIG. 10C.

Abstract

The purpose is to clarify the internal structures of the skin related to sagging and to provide a novel cosmetic method for preventing and improving sagging. Firmness of the skin was discovered to be due to circular collagen in the dermis, and it was discovered that the formation of circular collagen can be controlled through promoting formation of dermal matrix by applying mechanical stimulation to hair follicles. Based on the above findings, provided are a cosmetic method that includes promoting formation of dermal matrix and/or controlling formation of circular collagen by mechanical stimulation of hair follicles and a method for evaluating circular collagen formation based on dermal matrix weight.

Description

環状コラーゲンの形成を制御する美容方法Cosmetic method to control the formation of circular collagen
　本発明は、肌の老化を抑制するための技術分野に関する。 The present invention relates to the technical field of suppressing skin aging.
　肌のたるみは見た目年齢への影響が大きく、肌のハリを維持し、たるみを改善することは、美容上の大きな課題である。肌のハリが失われることで、たるみ又はこけが現れ、これにより老けた印象を与える。肌のハリは、主に真皮層の厚さに起因しており、ハリのある肌には、真皮層を充填するコラーゲンやエラスチンといった弾性線維が豊富に含まれている。加齢や紫外線への曝露などの影響により、真皮線維芽細胞の機能が低下して弾性線維の産生が減少するとともに、マトリクスメタロプロテイナーゼが活性化し弾性線維を分解することが知られている。これにより真皮層が菲薄化し、たるみ、又はこけが生じる。加齢によりコラーゲン量は減少傾向を示すが、コラーゲン量はスキンケアや、紫外線への暴露量、食生活によっても影響を受ける。 Sagging skin has a major impact on the appearance of one's age, and maintaining skin firmness and improving sagging is a major cosmetic issue. As the skin loses its firmness, sagging or moss appears, giving the appearance of aging. Skin firmness is mainly due to the thickness of the dermal layer, and firm skin contains abundant elastic fibers such as collagen and elastin that fill the dermal layer. It is known that due to the effects of aging and exposure to ultraviolet rays, the function of dermal fibroblasts declines and the production of elastic fibers decreases, and matrix metalloproteinases are activated and degrade elastic fibers. This causes the dermal layer to thin, causing sagging or moss. The amount of collagen tends to decrease with age, but the amount of collagen is also affected by skin care, exposure to ultraviolet rays, and diet.
　一方、たるみやこけは必ずしも真皮中におけるコラーゲン量にのみ依存するわけではない。たるみを引き起こしている高齢対象者において、必ずしもコラーゲン量が減少しているわけではなく、コラーゲン量以外の何らかの影響によりたるみの症状が影響されることが考えられる。コラーゲン量以外の原因としては、表情筋の衰えや皮下脂肪の増大なども知られている。従来の美容方法では、たるみの改善を達成するために、真皮線維芽細胞の賦活化によりコラーゲンや弾性線維等を増加させること、或いはコラーゲンや弾性線維等の分解を抑制することに注力してきており、真皮線維芽細胞の賦活作用や、コラーゲン産生促進作用又は分解抑制作用を有する成分が発見され、化粧料に応用されている（特許文献１：特開２０１２－１４４４９９号公報及び特許文献２：特開２０１３－０１４５５５号公報）。 On the other hand, sagging and moss do not necessarily depend only on the amount of collagen in the dermis. In elderly subjects who experience sagging, the amount of collagen does not necessarily decrease, and it is thought that the symptoms of sagging are affected by some influence other than the amount of collagen. Other known causes besides the amount of collagen include weakening of facial muscles and increase in subcutaneous fat. In order to improve sagging, conventional beauty methods have focused on increasing collagen and elastic fibers by activating dermal fibroblasts, or suppressing the decomposition of collagen and elastic fibers. , ingredients that have an activation effect on dermal fibroblasts, a collagen production promoting effect, or a degradation suppressing effect have been discovered and have been applied to cosmetics (Patent Document 1: JP 2012-144499 and Patent Document 2: Publication No. 2013-014555).
特開２０１２－１４４４９９号公報Japanese Patent Application Publication No. 2012-144499 特開２０１３－０１４５５５号公報Japanese Patent Application Publication No. 2013-014555
　たるみに関わる皮膚内部構造を明らかにし、たるみを予防、改善するための新規の美容方法を提供することを目的とする。 The purpose of this study is to clarify the internal structure of the skin related to sagging, and to provide a new beauty method to prevent and improve sagging.
　本発明者らが、顔のたるみを引き起こす皮膚内部構造を解明すべく鋭意研究を行ったところ、顔の真皮において、弾性を有する構造が均一に広がっているわけではなく、弾性の高い部位と、弾性の低い部位とが散在していることを見出した。より詳細に解析を行ったところ、弾性の低い部分の周囲を取り囲むように環状に弾性の高い部分が存在していることを見出し、環状に配置されたコラーゲンが、中心方向に収縮することで、皮膚に対する張力を引き起こし、たるみを防止していることを見出した。また、毛包に対し機械刺激及び／又は温熱刺激を付与することで、真皮基質の形成が促進され、そして環状に配置されたコラーゲンの形成を制御できることを見出した。
　そこで、本発明は以下に関する：
［１］　顔の皮膚において、毛包への機械刺激及び／又は温熱刺激により、真皮基質の形成の促進及び／又は環状コラーゲンの形成を制御することを含む、美容方法。
［２］　前記環状コラーゲンとは、毛包周囲に環状に形成されたコラーゲンをいう、項目１に記載の美容方法。
［３］　環状コラーゲンの形成により皮膚のハリを改善する、項目１又は２に記載の美容方法。
［４］　毛包への機械刺激が、プローブを皮膚に接触させることにより付与される、項目１～３のいずれか一項に記載の美容方法。
［５］　毛包への機械刺激が、２以上のプローブを皮膚に接触させ、プローブ間の距離を広げる方向に動かすことにより付与される、項目１～４のいずれか一項に記載の美容方法。
［６］　機械刺激が、伸展刺激である、項目１～５のいずれか一項に記載の美容方法。
［７］　毛包への温熱刺激が、昇温されたプローブを皮膚に接触させることにより付与される、項目１～６のいずれか一項に記載の美容方法。
［８］　機械刺激に加えて、前記プローブを昇温させて温熱刺激を付与する、項目４～６のいずれか一項に記載の美容方法。
［９］　真皮基質量に基づいて環状コラーゲン形成を評価する方法。
［１０］　前記真皮基質が、テネイシンＣ、バーシカン、及びペリオスチンからなる群から選ばれる少なくとも１つである、項目９に記載の方法。
［１１］　環状コラーゲンが、毛包周囲に環状に形成されたコラーゲンである、項目９又は１０に記載の方法。
［１２］　項目９～１１のいずれか一項に記載の環状コラーゲン形成を評価する方法により、現在又は将来の肌のハリを評価する方法。
［１３］　線維芽細胞培養物においてテネイシンＣ、バーシカン、及びペリオスチンからなる群から選ばれる少なくとも１つの発現を指標とした環状コラーゲン産生促進剤のスクリーニング方法。
［１４］　候補成分を含む培地で線維芽細胞培養物を培養する工程、
　培養後の線維芽細胞培養物において基質タンパク質又はＷｎｔ１６の発現を測定する工程、及び
　測定された基質タンパク質又はＷｎｔ１６の発現を、対照の基質タンパク質又はＷｎｔ１６の発現と比較する工程
　を含み、基質タンパク質又はＷｎｔ１６の発現が亢進した場合に、候補成分を環状コラーゲン産生促進剤としてスクリーニングする、項目１３に記載のスクリーニング方法。
［１５］　前記線維芽細胞培養物が、皮膚器官の器官培養により得られた培養物である、項目１３又は１４に記載のスクリーニング方法。 The present inventors conducted intensive research to elucidate the internal structure of the skin that causes facial sagging, and found that in the dermis of the face, structures with elasticity are not spread evenly, and there are areas with high elasticity, It was found that there were scattered areas with low elasticity. After conducting a more detailed analysis, we found that there was a ring-shaped part with high elasticity surrounding the part with low elasticity, and the collagen arranged in a ring contracted toward the center. It was found that it causes tension on the skin and prevents it from sagging. We have also found that by applying mechanical stimulation and/or thermal stimulation to hair follicles, the formation of dermal matrix can be promoted and the formation of annularly arranged collagen can be controlled.
The invention therefore relates to:
[1] A cosmetic method comprising promoting the formation of dermal matrix and/or controlling the formation of circular collagen in facial skin by mechanical stimulation and/or thermal stimulation of hair follicles.
[2] The cosmetic method according to item 1, wherein the annular collagen refers to collagen formed in an annular shape around the hair follicle.
[3] The cosmetic method according to item 1 or 2, which improves skin firmness by forming circular collagen.
[4] The cosmetic method according to any one of items 1 to 3, wherein mechanical stimulation to the hair follicle is applied by bringing a probe into contact with the skin.
[5] The cosmetic method according to any one of items 1 to 4, wherein the mechanical stimulation to the hair follicle is applied by bringing two or more probes into contact with the skin and moving in a direction to increase the distance between the probes. .
[6] The beauty method according to any one of items 1 to 5, wherein the mechanical stimulation is a stretching stimulation.
[7] The cosmetic method according to any one of items 1 to 6, wherein the thermal stimulus to the hair follicle is provided by bringing a heated probe into contact with the skin.
[8] The cosmetic method according to any one of items 4 to 6, wherein in addition to mechanical stimulation, thermal stimulation is applied by increasing the temperature of the probe.
[9] A method for evaluating circular collagen formation based on the amount of dermal matrix.
[10] The method according to item 9, wherein the dermal matrix is at least one selected from the group consisting of tenascin C, versican, and periostin.
[11] The method according to item 9 or 10, wherein the circular collagen is collagen formed in a circular shape around the hair follicle.
[12] A method for evaluating current or future skin firmness by the method for evaluating circular collagen formation according to any one of items 9 to 11.
[13] A method for screening a cyclic collagen production promoter using as an indicator the expression of at least one selected from the group consisting of tenascin C, versican, and periostin in a fibroblast culture.
[14] Cultivating a fibroblast culture in a medium containing a candidate component,
measuring the expression of the substrate protein or Wnt16 in the fibroblast culture after culturing; and comparing the measured expression of the substrate protein or Wnt16 with a control expression of the substrate protein or Wnt16, 14. The screening method according to item 13, wherein candidate components are screened as cyclic collagen production promoters when Wnt16 expression is increased.
[15] The screening method according to item 13 or 14, wherein the fibroblast culture is a culture obtained by organ culture of a skin organ.
　環状コラーゲンの形成を制御することで、肌のたるみを抑制することが可能となる。 By controlling the formation of cyclic collagen, it is possible to suppress skin sagging.
図１Ａは、皮膚を変形して解析した際の、真皮水平断面における張力の違いを示すヒートマップである。張力が強い箇所（ヒートマップの橙色～赤色）を点線の矢印で示し、張力が弱い箇所（ヒートマップの緑～水色）を実線の矢印で示す。図１Ｂは、真皮水平断面の張力の違いのヒートマップと、その周辺組織を重ね合わせて示す。FIG. 1A is a heat map showing the difference in tension in a horizontal section of the dermis when the skin is deformed and analyzed. Points with strong tension (orange to red in the heat map) are shown with dotted arrows, and points with weak tension (green to light blue in the heat map) are shown with solid arrows. FIG. 1B shows a heat map of the difference in tension in a horizontal section of the dermis and its surrounding tissue superimposed. 図２は、Ｘ線－ＣＴにより若齢被験者及び高齢被験者から取得された顔の皮膚標本の真皮水平断面を拡大して示した写真を示す。FIG. 2 shows enlarged photographs of horizontal sections of the dermis of facial skin specimens obtained from young and elderly subjects using X-ray CT. 図３は、毛包上皮細胞から分泌される液性因子により線維芽細胞においてテネイシンＣの発現が増大したことを示す。Figure 3 shows that expression of tenascin-C was increased in fibroblasts by humoral factors secreted from hair follicle epithelial cells. 図４は、皮膚器官培養物における、伸展刺激を付与しなかった場合のテイネシンＣの発現（Ａ）、伸展刺激を付与した場合のテネイシンＣの発現（Ｂ）、伸展刺激を付与した場合のバーシカンの発現（Ｃ）、及び伸展刺激を付与した場合のペリオスチンの発現（Ｄ）を示す写真である。対照と比較して、伸展刺激を付与した皮膚器官培養物においてテネイシンＣの発現量が増大すること、さらにバーシカン及びペリオスチンの発現量が増大することを示す。Figure 4 shows the expression of tenascin C in the skin organ culture when no stretch stimulus was applied (A), the expression of tenascin C when a stretch stimulus was applied (B), and the expression of versicin when a stretch stimulus was applied. (C) and the expression of periostin when a stretching stimulus is applied (D). It is shown that the expression level of tenascin C is increased in the skin organ culture subjected to stretch stimulation, as compared to the control, and the expression level of versican and periostin is also increased. 図５は、体部（Ａ）と顔部（Ｂ）における真皮において、微細毛及びコラーゲンを立体構築して示した画像である。図５（Ｃ）は、体部と顔部の真皮におけるコラーゲン量を示すグラフである。FIG. 5 is an image showing three-dimensional structure of fine hair and collagen in the dermis of the body (A) and face (B). FIG. 5(C) is a graph showing the amount of collagen in the dermis of the body and face. 図６は、微細毛の直径と、環状コラーゲンの直径との関係を示すグラフである。FIG. 6 is a graph showing the relationship between the diameter of microhair and the diameter of circular collagen. 図７は、Ｗｎｔ１６が真皮線維芽細胞においてペリオスチン及びバーシカンの発現を促進することを示すグラフである。FIG. 7 is a graph showing that Wnt16 promotes the expression of periostin and versican in dermal fibroblasts. 図８Ａは、若齢者及び高齢者の真皮におけるＷｎｔ１６の発現を調べた免疫染色写真である。図８Ｂは、免疫染色写真におけるＷｎｔ１６発現を定量して示したグラフである。FIG. 8A is an immunostaining photograph examining the expression of Wnt16 in the dermis of young and elderly subjects. FIG. 8B is a graph showing quantitative expression of Wnt16 in immunostaining photographs. 図９Ａは伸展刺激を付与して器官培養した場合の、環状コラーゲン直径の変化を示すグラフである。図９Ｂは、伸展刺激を付与して器官培養した場合のＷｎｔ１６及びバーシカンの発現を示す免疫染色写真である。FIG. 9A is a graph showing changes in circular collagen diameter when organ culture is performed with stretching stimulation applied. FIG. 9B is an immunostaining photograph showing the expression of Wnt16 and versican when organ culture was performed with stretching stimulation applied. 図１０Ａは温熱刺激を付与して器官培養した場合の環状コラーゲン直径の変化を示すグラフである。図１０Ｂは、温熱刺激を付与して器官培養した場合のＷｎｔ１６及びバーシカンの発現を示す免疫染色写真である。図１０Ｃは温熱刺激を付与して培養されたヒト外毛根鞘細胞におけるＷｎｔ１６発現量の変化を示すグラフである。FIG. 10A is a graph showing changes in circular collagen diameter when organ culture is performed with thermal stimulation applied. FIG. 10B is an immunostaining photograph showing the expression of Wnt16 and versican in organ culture with thermal stimulation applied. FIG. 10C is a graph showing changes in the expression level of Wnt16 in human outer root sheath cells cultured under thermal stimulation.
　本発明は、顔の皮膚において、毛包への機械刺激及び／又は温熱刺激により、真皮基質の形成の促進及び/又は環状コラーゲンの形成を制御することを含む、美容方法を提供する。 The present invention provides a cosmetic method that includes promoting the formation of dermal matrix and/or controlling the formation of cyclic collagen in facial skin by mechanical stimulation and/or thermal stimulation of hair follicles.
　環状コラーゲンとは、真皮層、特に真皮の網状層において、糖タンパク質及びプロテオグリカンに富む真皮基質領域を環状に取り囲む構造をとったコラーゲンをいう。真皮網状層を皮膚表面に対して水平面で解析すると、真皮基質領域は、主に毛包直下に略円形の形状で存在し、その直径は１００～１，０００μｍとなる。プロテオグリカンに富む領域を取り囲むようにコラーゲン領域が取り囲んでおり、コラーゲン領域の環状構造の直径は１００～１，０００μｍとなる。環状コラーゲンは、コラーゲンを検出し、環状をとっている形態から判別することができる。 Cyclic collagen refers to collagen that has a structure in the dermal layer, particularly the reticular layer of the dermis, that surrounds the dermal matrix region rich in glycoproteins and proteoglycans in a ring shape. When the dermal reticular layer is analyzed in a plane horizontal to the skin surface, the dermal matrix region mainly exists directly under the hair follicle in a substantially circular shape, and its diameter is 100 to 1,000 μm. A collagen region surrounds the proteoglycan-rich region, and the diameter of the annular structure of the collagen region is 100 to 1,000 μm. Cyclic collagen can be identified by detecting collagen and from its circular shape.
　真皮基質は、糖タンパク質及びプロテオグリカンから構成された真皮層に存在するゲル状の物質をいう。真皮基質と、コラーゲンやエラスチンなどの線維とから間質成分が構成され、さらに細胞成分と一緒になって真皮層を構成する。真皮基質単独では、張力をほとんど発生させない。真皮基質に存在する糖タンパク質としては、フィブロネクチン、テネイシン、バーシカン、ペリオスチンなどが主であり、プロテオグリカンとしてはグリコサミノグリカン、ヒアルロン酸が主に含まれる。従来、間質成分において、真皮基質と線維の分布についての知見はなく、線維と真皮基質とは一様に分布していると考えられていた。一方、本発明者らの研究により、真皮基質と線維とは一様に存在するのではなく、毛包直下に真皮基質領域が存在し、真皮基質を取り囲むように環状にコラーゲンが形成することが示された（図１）。真皮基質については、含まれる成分、例えばフィブロネクチン、テネイシンＣ、バーシカン、ペリオスチンなどの発現を測定することにより決定することができる。環状コラーゲンの直径は、真皮基質の量に応じて大きくなる。 The dermal matrix refers to a gel-like substance that exists in the dermal layer and is composed of glycoproteins and proteoglycans. The interstitial component is composed of the dermal matrix and fibers such as collagen and elastin, which together with cellular components constitute the dermal layer. The dermal matrix alone generates little tension. Glycoproteins present in the dermal matrix mainly include fibronectin, tenascin, versican, periostin, etc., and proteoglycans mainly include glycosaminoglycan and hyaluronic acid. Conventionally, there was no knowledge about the distribution of dermal matrix and fibers in the interstitial component, and it was thought that fibers and dermal matrix were uniformly distributed. On the other hand, research by the present inventors has revealed that the dermal matrix and fibers do not exist uniformly, but that a dermal matrix region exists just below the hair follicle, and collagen is formed in a ring shape surrounding the dermal matrix. (Figure 1). The dermal matrix can be determined by measuring the expression of contained components such as fibronectin, tenascin C, versican, and periostin. The diameter of the circular collagen increases depending on the amount of dermal matrix.
　環状に形成されたコラーゲンは、その中心に向かって張力を発生させる。通常、各毛包の下側に、１つの真皮基質領域と、真皮基質領域を取り囲む環状コラーゲンが存在する。各環状コラーゲンがその中心に向かって発揮する張力によって、肌全体として面方向に対する張りのある状態を達成することができる。加齢に伴い環状コラーゲンの形態は失われる（図２）。また、体部と、顔部とを比較すると、体部における微細毛は、顔部における微細毛よりも密度が低く、真皮のコラーゲンは体部ではほぼ一様に高含量で存在する。顔部では微細毛を取り囲むように存在して環状コラーゲンを形成する一方、コラーゲン量は少なかった（図５）。環状コラーゲン量は形態及び/又は成分としてのコラーゲンに基づいて測定することができる。環状コラーゲンの量と張力との間には正の相関性がある一方、環状コラーゲン量と、たるみとの間には逆相関性がある。環状コラーゲン構造をとるか、一様に存在するかにより面方向に対する張りは異なる（図２及び表１）。したがって、加齢によって環状コラーゲンが失われると、面方向への張りが維持できなくなり、重力に抗らえずに皮膚全体が下方に引っ張られたるみを生じる。また、環状コラーゲンの直径と、微細毛の直径とは相関関係があり、微細毛の直径が太いほど、環状コラーゲンの直径も大きくなる（図６）。対象の顔における環状コラーゲンが失われているか否かは、たるみを元に計測されてもよいし、皮膚試料から真皮基質を構成する糖タンパク質量又は発現を計測することにより決定することができる。環状コラーゲンが失われている対象に対しては、機械刺激及び／又は温熱刺激を付与することにより、Ｗｎｔ１６の発現又は真皮基質を構成する糖タンパク質の発現を介して、環状コラーゲンを増大させることができる。 The ring-shaped collagen generates tension toward its center. Typically, on the underside of each hair follicle there is one dermal matrix area and a ring of collagen surrounding the dermal matrix area. The tension exerted by each annular collagen toward its center makes it possible to achieve a state of tension in the surface direction of the skin as a whole. With aging, the morphology of circular collagen is lost (Figure 2). Furthermore, when comparing the body and face, the density of fine hair on the body is lower than that on the face, and collagen in the dermis is almost uniformly present in a high content on the body. On the face, while surrounding microhairs and forming circular collagen, the amount of collagen was small (Figure 5). The amount of circular collagen can be measured based on morphology and/or collagen as a component. While there is a positive correlation between the amount of circular collagen and tension, there is an inverse correlation between the amount of circular collagen and sagging. The tension in the plane direction differs depending on whether the collagen has a circular collagen structure or exists uniformly (FIG. 2 and Table 1). Therefore, when cyclic collagen is lost due to aging, it becomes impossible to maintain tension in the plane direction, and the skin as a whole is pulled downward without being able to resist gravity, resulting in sagging. Furthermore, there is a correlation between the diameter of the circular collagen and the diameter of the microhair, and the thicker the diameter of the microhair, the larger the diameter of the circular collagen (FIG. 6). Whether or not circular collagen has been lost in the subject's face may be measured based on sagging, or it can be determined by measuring the amount or expression of glycoproteins constituting the dermal matrix from a skin sample. For subjects that have lost cyclic collagen, applying mechanical stimulation and/or thermal stimulation can increase cyclic collagen through the expression of Wnt16 or the expression of glycoproteins that constitute the dermal matrix. can.
　間質成分は、線維芽細胞により産生される。本発明者らにより毛包上皮細胞からの液性因子によって、真皮基質の産生量が増大することが示された（図３）。理論に限定されることを意図するものではないが、毛包細胞から分泌された液性因子、一例としてＷｎｔ１６により、毛包周囲及び毛包の深部の領域で線維芽細胞が活性化し、真皮基質生産量が増大する。また活性化された線維芽細胞は、コラーゲン産生も増大する。産生されたコラーゲンは成熟する過程で真皮基質を取り囲む環状コラーゲンへと構造を変化させる。したがって、環状コラーゲンが失われた皮膚において真皮基質生産量が増大する環境下では、コラーゲン産生量も増大し、成熟することで環状コラーゲンが再生される。さらに毛包に伸展刺激を付与すると、毛包周辺の真皮線維芽細胞において、真皮基質の生産が高くなることが示された（図４）。理論に限定されることを意図するものではないが、毛包組織に対し機械刺激及び／又は温熱刺激が付与されることにより、毛包細胞は活性化され、液性因子の産生量が増大し、その結果真皮基質の生産が高くなると考えられる。真皮基質であるペリオスチン及びバーシカンは、Ｗｎｔ１６のシグナル伝達経路を活性化することで発現増大される（図７）。また、若齢皮膚では、毛包周囲においてＷｎｔ１６の発現が高い一方、老齢皮膚では毛包周囲におけるＷｎｔ１６発現が低下する（図８）。ここで、毛包組織に対し伸展刺激が付与されると、毛包周囲のＷｎｔ１６の発現が増大し、また真皮基質であるバーシカンの発現も増大する（図９Ｂ）。Ｗｎｔ１６の発現及び／又はバーシカンの発現の増大に合わせて、環状コラーゲン量も増大する（図９Ａ）。さらに、毛包組織に対し温熱刺激が付与されると、毛包周囲のＷｎｔ１６の発現が増大し、また真皮基質であるバーシカンの発現も増大する（図１０Ｂ，Ｃ）。Ｗｎｔ１６の発現及び／又はバーシカンの発現の増大に合わせて、環状コラーゲン量も増大する（図１０Ａ）。したがって、本発明の毛包への伸展刺激及び／又は温熱刺激を付与する美容方法により、真皮基質の産生が促進され、及び／又は環状コラーゲンの形成が促進される。これにより、たるみ、シワ又はこけの予防、及びハリの改善がもたらされる。 Stromal components are produced by fibroblasts. The present inventors have shown that humoral factors from hair follicle epithelial cells increase the amount of dermal matrix produced (FIG. 3). Without intending to be limited by theory, humoral factors secreted from hair follicle cells, such as Wnt16, activate fibroblasts in the perifollicular and deep regions of the hair follicle, leading to the activation of fibroblasts in the dermal matrix. Production increases. Activated fibroblasts also increase collagen production. During the maturation process, the produced collagen changes its structure into a circular collagen that surrounds the dermal matrix. Therefore, in an environment where the amount of dermal matrix production increases in the skin where cyclic collagen has been lost, the amount of collagen produced also increases, and as the cyclic collagen matures, cyclic collagen is regenerated. Furthermore, it was shown that when a stretching stimulus was applied to the hair follicle, the production of dermal matrix increased in the dermal fibroblasts surrounding the hair follicle (Figure 4). Without intending to be limited by theory, it is believed that by applying mechanical and/or thermal stimulation to hair follicular tissue, hair follicle cells are activated and the production of humoral factors is increased. , it is thought that the production of dermal matrix will increase as a result. The expression of periostin and versican in the dermal matrix is increased by activating the Wnt16 signal transduction pathway (FIG. 7). Furthermore, in young skin, Wnt16 expression is high around hair follicles, while in aged skin, Wnt16 expression is decreased around hair follicles (FIG. 8). Here, when a stretching stimulus is applied to the hair follicle tissue, the expression of Wnt16 around the hair follicle increases, and the expression of versican, which is a dermal matrix, also increases (FIG. 9B). In accordance with the increase in Wnt16 expression and/or versican expression, the amount of circular collagen also increases (FIG. 9A). Furthermore, when thermal stimulation is applied to the hair follicle tissue, the expression of Wnt16 around the hair follicle increases, and the expression of versican, which is a dermal matrix, also increases (FIGS. 10B and C). In accordance with the increase in Wnt16 expression and/or versican expression, the amount of circular collagen also increases (FIG. 10A). Therefore, the cosmetic method of the present invention that applies stretching stimulation and/or thermal stimulation to hair follicles promotes the production of dermal matrix and/or the formation of circular collagen. This prevents sagging, wrinkles or moss, and improves firmness.
　毛包組織に対する機械刺激は、毛包の形状を変形させるように付与されれば任意の手法により付与されてもよい。一例として、毛包組織に対する機械刺激は、皮膚に接触するプローブを備える機器により達成することができる。機械刺激としては、皮膚を伸展させる伸展刺激を付与することが好ましい。伸展刺激は、皮膚に適用するプローブを皮膚に当てた状態で皮膚表面に対し水平方向に動かすことにより付与される。さらに、皮膚に適用する２本以上のプローブを皮膚に当てた状態で、プローブ間を引き離すように移動させることで付与することができる。伸展刺激はプローブの移動、より具体的に振動により付与されうる。機械刺激により、真皮基質の形成の促進及び/又は環状コラーゲンの形成の制御が可能になる。具体的に環状コラーゲンの形成が促進され、これによりハリが改善する。機械刺激による毛包細胞の活性化、真皮基質の形成の促進、及び環状コラーゲンの形成制御から選ばれる少なくとも１の作用は、機械刺激、特に伸展刺激を与える機器の性能表示として、パッケージ、カタログ、販促資料、説明書、機器の画面等に表示してもよいし、かかる機器を用いたサービス提供の宣伝に用いられてもよい。 Mechanical stimulation to the hair follicle tissue may be applied by any method as long as it is applied so as to deform the shape of the hair follicle. As an example, mechanical stimulation of hair follicular tissue can be achieved with a device that includes a probe that contacts the skin. As the mechanical stimulus, it is preferable to apply a stretching stimulus that stretches the skin. The stretching stimulus is applied by moving a probe applied to the skin in a horizontal direction with respect to the skin surface while it is in contact with the skin. Furthermore, it can be applied by moving two or more probes to be applied to the skin so as to separate them while they are in contact with the skin. Stretching stimulation can be applied by moving the probe, more specifically by vibration. Mechanical stimulation allows the promotion of dermal matrix formation and/or the control of circular collagen formation. Specifically, the formation of circular collagen is promoted, which improves firmness. At least one action selected from activation of hair follicle cells, promotion of dermal matrix formation, and control of circular collagen formation by mechanical stimulation is a performance indication of a device that applies mechanical stimulation, especially stretching stimulation, on packages, catalogs, It may be displayed on promotional materials, manuals, the screen of the device, etc., or may be used to advertise the provision of services using such devices.
　毛包組織に対する温熱刺激は、皮膚表面から温熱が付与されれば任意の手法により付与されてもよい。一例として、皮膚に接触するプローブを体温以上、例えば３８℃以上、４０℃以上、４２℃以上、又は４５℃以上の設定温度にて接触させることが好ましい。火傷を誘発させない観点から、４３℃以下が好ましい。温熱刺激の温度は、外界の温度に応じて適宜設定することもできる。温熱刺激は、一定温度で付与されてもよいし、温度を変化させて付与されてもよい。温熱刺激は、通常、５分～６０分間にわたって適用される。温熱刺激は、機械刺激と同時に付与されてもよい。 Thermal stimulation to the hair follicle tissue may be applied by any method as long as heat is applied from the skin surface. As an example, it is preferable to bring the probe into contact with the skin at a set temperature of at least body temperature, for example, at least 38°C, at least 40°C, at least 42°C, or at least 45°C. From the viewpoint of not inducing burns, the temperature is preferably 43°C or lower. The temperature of the thermal stimulus can also be appropriately set depending on the temperature of the outside world. Thermal stimulation may be applied at a constant temperature or may be applied while changing the temperature. Thermal stimulation is typically applied for 5 to 60 minutes. Thermal stimulation may be applied simultaneously with mechanical stimulation.
　本発明に係る美容方法は、美容医療、美容整体などにおいてサービスとして提供されてもよいし、機械刺激を付与する機器により提供されてもよい。本発明に係る美容方法は、カタログ、販促資料、説明書、モニター画面上に表示されてもよい。本発明に係る美容方法は、その作用メカニズム及び／又は効果と共に表示されてもよい。 The beauty method according to the present invention may be provided as a service in cosmetic medicine, beauty treatment, etc., or may be provided by a device that applies mechanical stimulation. The beauty method according to the present invention may be displayed on a catalog, promotional material, instruction manual, or monitor screen. The cosmetic method according to the present invention may be displayed together with its mechanism of action and/or effects.
　本発明の別の態様では、真皮基質の量に基づいて環状コラーゲンを評価する方法にも関する。環状コラーゲンはコラーゲン線維が集合して形成した構造の点で、通常のコラーゲン線維とは異なることから、コラーゲンタンパク量やｍＲＮＡ量の測定では測定することができない。環状コラーゲンは、組織染色等によりコラーゲン線維が集合した形状に基づき評価することができるが、かかる評価方法は定量性に欠ける。一方、環状コラーゲンはプロテオグリカンを含む真皮基質を取り囲むように形成されることから、真皮基質量が増大すると、環状コラーゲン量も増大するという関係を有する。したがって、真皮基質量に基づき、環状コラーゲンを評価することができる。環状コラーゲンとしては、毛包周囲又は毛包下領域に形成された環状コラーゲンを評価することができる。真皮基質量は、真皮基質の任意の成分、例えばテネイシンＣ、フィブロネクチン、バーシカン、ペリオスチンなどの発現量を測定することにより決定することができ、テネイシンＣ、バーシカン、又はペリオスチンを用いることが好ましい。発現量は、タンパク質及び/又はｍＲＮＡを、それぞれ免疫学的手法又は定量的ＰＣＲ等の本技術分野に周知の手法を用いて測定することができ。 Another aspect of the present invention also relates to a method of evaluating cyclic collagen based on the amount of dermal matrix. Cyclic collagen differs from normal collagen fibers in that it has a structure formed by aggregation of collagen fibers, so it cannot be measured by measuring the amount of collagen protein or mRNA. Cyclic collagen can be evaluated based on the shape of aggregated collagen fibers by tissue staining, etc., but such evaluation methods lack quantitative properties. On the other hand, since cyclic collagen is formed so as to surround the dermal matrix containing proteoglycans, there is a relationship in which as the amount of dermal matrix increases, the amount of cyclic collagen also increases. Therefore, cyclic collagen can be evaluated based on the amount of dermal matrix. As the annular collagen, annular collagen formed around the hair follicle or in the subfollicular region can be evaluated. The amount of dermal matrix can be determined by measuring the expression level of any component of the dermal matrix, such as tenascin C, fibronectin, versican, periostin, etc. Tenascin C, versican, or periostin is preferably used. The expression level of protein and/or mRNA can be measured using techniques well known in the art, such as immunological techniques or quantitative PCR, respectively.
　環状コラーゲンを評価することで、現在又は将来の肌の張りを決定することができる。将来の肌の張りに起因するたるみ、しわ、コケを予測することができる。一例として、環状コラーゲン量が、所定の閾値よりも低い場合に、現時点でたるみが形成していない場合であっても、たるみが形成する蓋然性が高い。閾値は、生体における環状コラーゲン量の大規模調査により適宜決定することができる。キュートメーターや写真判定などの本技術分野に周知のたるみ判定手法を用いてたるみと皮膚の環状コラーゲン量との関係に基づいて、閾値を設定することができる。さらに所定の期間を空けて追跡調査を行うことで、より正確に閾値を設定することが可能になる。 By evaluating cyclic collagen, current or future skin tightness can be determined. It is possible to predict future sagging, wrinkles, and moss caused by skin tension. As an example, when the amount of circular collagen is lower than a predetermined threshold value, there is a high probability that sag will be formed even if no sag is currently formed. The threshold value can be appropriately determined by large-scale investigation of the amount of cyclic collagen in living organisms. A threshold value can be set based on the relationship between sagging and the amount of annular collagen in the skin using a sagging determination method known in this technical field such as cutometer or photo judgment. Further, by conducting a follow-up survey after a predetermined period of time, it becomes possible to set the threshold value more accurately.
　本発明の更なる態様では、本発明は、線維芽細胞培養物において真皮基質タンパク質及び／又はＷｎｔ１６発現を指標とした環状コラーゲン産生促進剤のスクリーニング方法にも関する。本発明のスクリーニング方法は、より具体的に以下の：
　候補成分を含む培地で線維芽細胞培養物を培養する工程、
　培養後の線維芽細胞培養物において真皮基質タンパク質及び／又はＷｎｔ１６の発現を測定する工程、及び
　測定された真皮基質タンパク質及び／又はＷｎｔ１６発現を、対照の真皮基質タンパク質及び／又はＷｎｔ１６発現と比較する工程
　を含み、真皮基質タンパク質及び／又はＷｎｔ１６発現が亢進した場合に、候補成分を環状コラーゲン産生促進剤としてスクリーニングすることができる。スクリーニング方法は、真皮基質タンパク質及び／又はＷｎｔ１６発現が亢進した場合に、候補成分を環状コラーゲン産生促進剤として選択する工程を含んでもよい。こうしてスクリーニングされた環状コラーゲン産生促進剤は、真皮基質タンパク質及び／又はＷｎｔ１６発現を亢進することにより、環状コラーゲン産生促進することができる。環状コラーゲン産生促進剤は、たるみ改善剤ともいうことができる。真皮基質タンパク質としては、テネイシンＣ、バーシカン、及びペリオスチンからなる群から選ばれる少なくとも１を利用することができる。 In a further aspect of the present invention, the present invention also relates to a method of screening for a cyclic collagen production promoter using dermal matrix protein and/or Wnt16 expression as an indicator in a fibroblast culture. The screening method of the present invention more specifically includes the following:
cultivating a fibroblast culture in a medium containing the candidate component;
measuring the expression of dermal matrix protein and/or Wnt16 in the fibroblast culture after culturing; and comparing the measured dermal matrix protein and/or Wnt16 expression with a control dermal matrix protein and/or Wnt16 expression. When dermal matrix protein and/or Wnt16 expression is increased, candidate components can be screened as cyclic collagen production promoters. The screening method may include a step of selecting a candidate component as a cyclic collagen production promoter when dermal matrix protein and/or Wnt16 expression is increased. The cyclic collagen production promoter thus screened can promote cyclic collagen production by enhancing dermal matrix protein and/or Wnt16 expression. The cyclic collagen production promoter can also be called a sagging improving agent. As the dermal matrix protein, at least one selected from the group consisting of tenascin C, versican, and periostin can be used.
　対照の真皮基質タンパク質及び／又はＷｎｔ１６発現は、候補成分を含まない点でのみ異なる線維芽細胞培養物における真皮基質タンパク質及び／又はＷｎｔ１６の発現を使用することができる。対照群は、予め実験が行われて、対照群の真皮基質タンパク質及び／又はＷｎｔ１６の発現に基づいて閾値を設定してもよいし、並行して培養及び測定工程が行われてもよい。真皮基質タンパク質及び／又はＷｎｔ１６の発現は、線維芽細胞培養物における真皮基質タンパク質及び／又はＷｎｔ１６のタンパク質量又はｍＲＮＡ量であってもよく、それぞれ免疫学的手法又は定量的ＰＣＲ等の本技術分野に周知の手法を用いて測定することができる。 The control dermal matrix protein and/or Wnt16 expression can be used to express dermal matrix protein and/or Wnt16 in fibroblast cultures that differ only in that they do not contain the candidate component. For the control group, an experiment may be conducted in advance and a threshold value may be set based on the expression of dermal matrix protein and/or Wnt16 in the control group, or culture and measurement steps may be performed in parallel. The expression of dermal matrix protein and/or Wnt16 may be the protein amount or mRNA amount of dermal matrix protein and/or Wnt16 in fibroblast culture, and can be expressed by the present technical field such as immunological method or quantitative PCR, respectively. It can be measured using a well-known method.
　本発明のスクリーニング方法に用いる候補成分は、化粧品素材、食品素材、医薬品素材などの任意のライブラリーを使用することができる。かかるライブラリーとしては、化合物ライブラリー、エキスライブラリーなどを使用してもよい。各ライブラリーに含まれる化合物及びエキスは、市販の化合物及びエキスを使用してもよいし、合成された化合物及び調製されたエキスを使用してもよい。 As the candidate component used in the screening method of the present invention, any library of cosmetic materials, food materials, pharmaceutical materials, etc. can be used. As such a library, a compound library, an extract library, etc. may be used. As the compounds and extracts included in each library, commercially available compounds and extracts may be used, or synthesized compounds and prepared extracts may be used.
　本明細書において言及される全ての文献はその全体が引用により本明細書に取り込まれる。 All documents mentioned herein are incorporated by reference in their entirety.
　以下に説明する本発明の実施例は例示のみを目的とし、本発明の技術的範囲を限定するものではない。本発明の技術的範囲は特許請求の範囲の記載によってのみ限定される。本発明の趣旨を逸脱しないことを条件として、本発明の変更、例えば、本発明の構成要件の追加、削除及び置換を行うことができる。 The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is limited only by the claims. Changes in the present invention, such as additions, deletions, and substitutions of constituent elements of the present invention, may be made without departing from the spirit of the present invention.
実施例１：皮膚動態の再構築
　余剰皮膚について皮膚を変形し、Ｘ線ＣＴで画像を取得し、皮膚動態をコンピューター上に再構築を行い、皮膚の水平断面における張力の高さを測定した（図１Ａ）。また真皮の水平断面において張力が高い領域、低い領域の原因を探るため、真皮の水平断面より浅部領域および深部領域を観察した（図１Ｂ）。張力の低い領域が、張力の高い領域に取り囲まれていることが示された。また、張力の低い領域は毛包の下側領域であることが示された。 Example 1: Reconstruction of skin dynamics The excess skin was deformed, images were acquired with X-ray CT, skin dynamics were reconstructed on a computer, and the height of tension in the horizontal section of the skin was measured ( Figure 1A). Furthermore, in order to investigate the causes of high and low tension regions in the horizontal section of the dermis, we observed the shallow and deep regions of the dermis in the horizontal section (Figure 1B). Regions of low tension were shown to be surrounded by regions of high tension. It was also shown that the region of low tension was the lower region of the hair follicle.
実施例２：加齢による環状コラーゲンの変化
　若齢者（４０歳未満）及び高齢者（６０歳以上）の顔の余剰皮膚から、顔面皮膚標本を得た。皮膚標本から数ミリ径の円柱状皮膚切片を切り出し、Ｘ線　ＣＴを用いて撮影を行った（図２）。若齢者の皮膚標本では真皮基質領域と真皮基質領域を取り囲む環状コラーゲンとが明瞭に区別できた一方、高齢者の皮膚標本では真皮基質領域と環状コラーゲンとが識別が難しく、コラーゲンは不定形であった（図２に代表例を示す）。 Example 2: Changes in circular collagen due to aging Facial skin specimens were obtained from excess facial skin of young people (under 40 years old) and elderly people (over 60 years old). A cylindrical skin section several millimeters in diameter was cut from the skin specimen and photographed using X-ray CT (Figure 2). In skin specimens from young people, the dermal matrix region and the circular collagen surrounding the dermal matrix region could be clearly distinguished, whereas in skin specimens from elderly patients, it was difficult to distinguish between the dermal matrix region and the circular collagen, and the collagen had an amorphous shape. (A typical example is shown in Figure 2).
実施例３：環状コラーゲンと、皮膚の張力およびたるみとの関連
　３０名の女性被験者（３０～５０代）において、環状コラーゲンの量を毛包の状態のグレーディング手法により数値化した。また、被験者の顔の頬部における張力をＣｕｔｏｍｅｔｅｒを用いて測定した。また、被験者の顔の頬部におけるたるみの程度を写真評価した。環状コラーゲンの状態と、頬の張力およびたるみの程度について、Ｓｐｅａｍａｎ’ｓ　ｃｏｒｒｅｌａｔｉｏｎ　ｃｏｅｆｆｉｃｉｅｎｔを用いて統計解析を行った。結果を下記表に示す。
Example 3: Relationship between cyclic collagen and skin tension and sagging In 30 female subjects (30s to 50s), the amount of cyclic collagen was quantified using a hair follicle condition grading method. In addition, the tension in the cheek area of the subject's face was measured using a Cutometer. In addition, the degree of sagging in the cheeks of the subject's face was evaluated with photographs. Statistical analysis was performed on the state of circular collagen and the degree of tension and sagging of the cheeks using Spearman's correlation coefficient. The results are shown in the table below.
実施例４：毛包細胞による真皮基質の誘導
　ボイデンチャンバーに毛包上皮細胞および線維芽細胞播種し、毛包上皮細胞による線維芽細胞への影響を調べた。より具体的に線維芽細胞（ヒト生検皮膚より樹立）をＤＭＥＭ培地中で５，０００個／ｍｌで上部チャンバーに播種し、毛包上皮細胞（ヒト生検皮膚より樹立）をＤＭＥＭ培地中で下部チャンバーに２５０，０００個／ｍｌで播種し、３７℃、ＣＯ２雰囲気下で４８時間培養した。培養後の上部チャンバーの線維芽細胞を回収し、ＲＮＡ抽出キット（Ｑｉａｇｅｎ）を用いてｍＲＮＡを抽出した。Ｓｕｐｅｒ　ｓｃｒｉｐｔキット　（Ｉｎｖｉｔｒｏｇｅｎ）を用いて逆転写反応を行ない、定量的ＰＣＲに供した。対照として下部チャンバーに毛包上皮細胞を含まない点でのみ異なる対照（Ｃｏｎｔ．）を同様に調製した。定量的ＰＣＲには、下記のプライマーセットを用いた。ＴｅｎａｓｃｉｎＣの発現量を図３に示す。
Example 4: Induction of dermal matrix by hair follicle cells Hair follicle epithelial cells and fibroblasts were seeded in a Boyden chamber, and the influence of hair follicle epithelial cells on fibroblasts was investigated. More specifically, fibroblasts (established from human biopsy skin) were seeded in the upper chamber at 5,000 cells/ml in DMEM medium, and hair follicle epithelial cells (established from human biopsy skin) were seeded in DMEM medium. The cells were seeded at 250,000 cells/ml in the lower chamber and cultured at 37°C under a CO 2 atmosphere for 48 hours. Fibroblasts in the upper chamber after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen). A reverse transcription reaction was performed using a Superscript kit (Invitrogen) and subjected to quantitative PCR. A control (Cont.) was prepared similarly, differing only in that the lower chamber did not contain hair follicle epithelial cells. The following primer set was used for quantitative PCR. The expression level of TenascinC is shown in FIG.
実施例５：毛包刺激による真皮基質の産生
　円柱状に切り出された皮膚切片（若齢対象と及び高齢対象）に伸展刺激を付与した。伸展刺激を付与しながら、７日間、ＤＭＥＭ培地中で３７℃５％ＣＯ２雰囲気下で器官培養を行なった。培養後の皮膚試料を、アセトンで固定し、ＰＢＳで洗浄した。次いでパラフィン包埋を行い、５μｍの厚さの皮膚切片を取得した。皮膚切片をブロッキング溶液１％　ＢＳＡ）で０．５時間インキュベートした後に、１次抗体としてｘ２００倍希釈の抗テネイシンＣ抗体（Ａｂｃａｍ社）、抗バーシカン抗体（Ａｂｃａｍ社）、抗ペリオスチン抗体（Ａｂｃａｍ社）をそれぞれ用いて一晩、４℃でインキュベートし、次いでＥｎｖｉｓｉｏｎ（ＤＡＫＯ）で発色し、組織染色切片を調製し、顕微鏡にて観察を行った（図４）。毛包周囲から毛包下の領域において、テネイシンＣが発現していることが示された。また、伸展刺激後では、毛包周囲から毛包下の領域においてテネイシンＣの発現が増大することが示された。 Example 5: Production of dermal matrix by hair follicle stimulation Stretching stimulation was applied to cylindrically cut skin sections (young subjects and elderly subjects). Organ culture was performed in DMEM medium at 37° C. in a 5% CO 2 atmosphere for 7 days while applying stretching stimulation. The skin samples after culture were fixed with acetone and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 μm were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 hours, the primary antibodies were anti-tenascin C antibody (Abcam), anti-versican antibody (Abcam), and anti-periostin antibody (Abcam) diluted x200. were incubated overnight at 4° C., and then colored with Envision (DAKO) to prepare tissue-stained sections and observed under a microscope (FIG. 4). It was shown that tenascin-C is expressed in the area from around the hair follicle to under the hair follicle. Furthermore, after stretching stimulation, it was shown that the expression of tenascin-C increased in the area from around the hair follicle to under the hair follicle.
実施例６：体部及び顔部における微細毛とコラーゲンとの関係
　体部由来の余剰皮膚及び顔部由来の余剰皮膚から皮膚標本を取得し、Ｘ線ＣＴ機器（販売元：Zeiss社）での計測に供し、３次元構造を構築した。特に真皮における微細毛及びコラーゲンを可視化して示した（図５Ａ、Ｂ）。３次元構築物から、真皮中に含まれるコラーゲン量を決定し、体部及び顔部で比較した（図５Ｃ）。次に、余剰皮膚において微細毛の直径と、環状コラーゲンの直径をそれぞれ測定し、グラフにプロットした（図６）。体部に比べて顔部では、微細毛の密度が高かった。体部ではコラーゲン含量が高く、真皮中において一様にコラーゲンが存在した。一方、顔部ではコラーゲン含量は少なく、微細毛の密度が高いことに起因して微細毛を取り囲むようにコラーゲンが形成されるが、コラーゲンは一様に存在している訳でなく、微細毛を取り囲みつつ濃淡が存在する。そして環状コラーゲンの直径と、微細毛の直径とは相関関係を示した（Ｐ＜０．００１、Ｒ＝０．７３）。 Example 6: Relationship between fine hair and collagen on the body and face Skin specimens were obtained from excess skin from the body and from the face, and examined using an X-ray CT machine (sold by Zeiss). It was subjected to measurement and a three-dimensional structure was constructed. In particular, fine hair and collagen in the dermis were visualized (FIGS. 5A and B). From the three-dimensional construct, the amount of collagen contained in the dermis was determined and compared between the body and face (Figure 5C). Next, the diameter of microhairs and the diameter of circular collagen in the excess skin were measured and plotted on a graph (FIG. 6). The density of fine hair was higher on the face than on the body. Collagen content was high in the body, and collagen was uniformly present in the dermis. On the other hand, the collagen content in the facial area is low, and due to the high density of fine hairs, collagen is formed to surround the fine hairs, but collagen is not uniformly present and does not cover fine hairs. There are shades of darkness surrounding it. There was a correlation between the diameter of circular collagen and the diameter of microhair (P<0.001, R=0.73).
実施例７：プロテオグリカン層を誘導する因子
　線維芽細胞の培養物において、プロテオグリカン層を誘導する因子についてスクリーニングを行った。より具体的に線維芽細胞（ヒト生検皮膚より樹立）を、１０％ＦＢＳ添加ＤＭＥＭ培地中で１×１０4Ｃｅｌｌｓ／ｍｌで２４穴プレート培養容器に播種した。培地に１μｇ/ｍｌのＷｎｔ１６（Ｒ＆Ｄ社）を添加し、３７℃ＣＯ2雰囲気下で４８時間培養を行った。培養後の線維芽細胞を回収し、ＲＮＡ抽出キット（Ｑｉａｇｅｎ）を用いてｍＲＮＡを抽出した。Ｓｕｐｅｒ　ｓｃｒｉｐｔキット（Ｉｎｖｉｔｒｏｇｅｎ）を用いて逆転写反応を行ない、定量的ＰＣＲに供した。定量的ＰＣＲには、下記のプライマーセットを用いた。ペリオスチン及びバーシカンの発現量を図７に示す。Ｗｎｔ１６を添加することにより、ペリオスチン及びバーシカンの発現量が増大した。 Example 7: Factors that induce proteoglycan layers Screening was performed for factors that induce proteoglycan layers in fibroblast cultures. More specifically, fibroblasts (established from human biopsied skin) were seeded in a 24-well plate culture vessel at 1×10 4 cells/ml in DMEM medium supplemented with 10% FBS. 1 μg/ml of Wnt16 (R&D Co., Ltd.) was added to the medium and cultured at 37° C. in a CO 2 atmosphere for 48 hours. The fibroblasts after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen). A reverse transcription reaction was performed using a Super script kit (Invitrogen) and subjected to quantitative PCR. The following primer set was used for quantitative PCR. The expression levels of periostin and versican are shown in FIG. Addition of Wnt16 increased the expression levels of periostin and versican.
実施例８：若齢者及び高齢者の毛包周囲におけるＷｎｔ１６の発現
　若齢者（４０歳未満）及び高齢者（６０歳以上）の顔の余剰皮膚から、顔面皮膚標本を得た。皮膚標本から数ミリ径の円柱状皮膚切片を切り出し、アセトンで固定し、ＰＢＳで洗浄した。次いでパラフィン包埋を行い、５μｍの厚さの皮膚切片を取得した。皮膚切片をブロッキング溶液１％　ＢＳＡ）で０．５時間インキュベートした後に、１次抗体としてｘ２００倍希釈の抗Ｗｎｔ１６抗体（ＬＳ－ｂｉｏ社）を用いて一晩、４℃でインキュベートし、次いでＥｎｖｉｓｉｏｎ（ＤＡＫＯ）で発色し、組織染色切片を調製し、顕微鏡にて観察を行った（図８Ａ）。この結果について、Ｗｎｔ１６の発現を４段階のグレーディングで評価を行い、結果を図８Ｂに示す。若齢者の皮膚標本において、毛包周囲においてＷｎｔ１６の高発現がみられた一方、老齢者の皮膚標本では、毛包周囲のＷｎｔ１６発現が低下した。 Example 8: Expression of Wnt16 around hair follicles in young and elderly subjects Facial skin specimens were obtained from excess facial skin of young people (under 40 years old) and elderly people (over 60 years old). A cylindrical skin section with a diameter of several millimeters was cut from the skin specimen, fixed with acetone, and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 μm were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 h, they were incubated overnight at 4°C using a x200 dilution of anti-Wnt16 antibody (LS-bio) as the primary antibody, followed by Envision (1% BSA). DAKO) to prepare tissue-stained sections and observe them under a microscope (FIG. 8A). Regarding this result, the expression of Wnt16 was evaluated using 4-level grading, and the results are shown in FIG. 8B. In skin specimens from young people, high expression of Wnt16 was observed around hair follicles, while in skin specimens from old people, Wnt16 expression around hair follicles decreased.
実施例９：伸展刺激による影響
　円柱状に切り出された腹部皮膚標本（４０未満若齢対象）に伸展刺激を付与した。伸展刺激を付与しながら、７日間、ＤＭＥＭ培地中で３７℃５％ＣＯ２雰囲気下で器官培養を行なった。器官培養後の皮膚試料を、アセトンで固定し、ＰＢＳで洗浄した。次いでパラフィン包埋を行い、５μｍの厚さの皮膚切片を取得した。皮膚切片をブロッキング溶液１％　ＢＳＡ）で０．５時間インキュベートした後に、１次抗体としてｘ２００倍希釈の抗バーシカン抗体（Ａｂｃａｍ社）、抗Ｗｎｔ１６抗体（ＬＳ－ｂｉｏ社）をそれぞれ用いて一晩、４℃でインキュベートし、次いでＥｎｖｉｓｉｏｎ（ＤＡＫＯ）で発色し、組織染色切片を調製し、顕微鏡にて観察を行った（図９Ｂ）。毛包周囲において、伸展刺激により、バーシカン及びＷｎｔ１６の発現が増大したことが示された。環状コラーゲンの直径を、プロテオグリカン層のサイズとして計測した（図９Ａ）（Ｎ＝３、＊＊：Ｐ＜０．０１）。 Example 9: Effects of Stretching Stimulation Stretching stimulation was applied to abdominal skin specimens (young subjects under 40) cut into cylindrical shapes. Organ culture was performed in DMEM medium at 37° C. in a 5% CO 2 atmosphere for 7 days while applying stretching stimulation. Skin samples after organ culture were fixed with acetone and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 μm were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 hours, they were incubated overnight using anti-versican antibody (Abcam) and anti-Wnt16 antibody (LS-bio) diluted 1:200 as primary antibodies, respectively. The cells were incubated at 4° C., then colored using Envision (DAKO), and tissue-stained sections were prepared and observed under a microscope (FIG. 9B). It was shown that stretch stimulation increased the expression of versican and Wnt16 around the hair follicle. The diameter of the circular collagen was measured as the size of the proteoglycan layer (FIG. 9A) (N=3, **: P<0.01).
実施例１０：温熱刺激による影響
　円柱状に切り出された腹部皮膚標本（４０未満若齢対象）をＤＭＥＭ培地中で器官培養を行った。４２℃で６０分間の温熱刺激を付与し、次いで７日間、３７℃５％雰囲気下で培養を行った。器官培養後の皮膚試料を、アセトンで固定し、ＰＢＳで洗浄した。次いでパラフィン包埋を行い、５μｍの厚さの皮膚切片を取得した。皮膚切片をブロッキング溶液１％　ＢＳＡ）で０．５時間インキュベートした後に、１次抗体としてｘ２００倍希釈の抗バーシカン抗体（Ａｂｃａｍ社）、抗Ｗｎｔ１６抗体（抗Ｗｎｔ１６抗体）をそれぞれ用いて一晩、４℃でインキュベートし、次いでＥｎｖｉｓｉｏｎ（ＤＡＫＯ）で発色し、組織染色切片を調製し、顕微鏡にて観察を行った（図１０Ｂ）。毛包周囲において、温熱刺激により、バーシカン及びＷｎｔ１６の発現が増大したことが示された。環状コラーゲンの直径を、プロテオグリカン層のサイズとして計測した（図１０Ａ）（Ｎ＝３、＊＊：Ｐ＜０．０１）。 Example 10: Effects of thermal stimulation Abdominal skin specimens (young subjects under 40 years of age) cut into cylinders were subjected to organ culture in a DMEM medium. Thermal stimulation was applied at 42°C for 60 minutes, and then culture was performed at 37°C in a 5% atmosphere for 7 days. Skin samples after organ culture were fixed with acetone and washed with PBS. Paraffin embedding was then performed, and skin sections with a thickness of 5 μm were obtained. After incubating the skin sections in a blocking solution (1% BSA) for 0.5 hours, they were incubated overnight using anti-versican antibody (Abcam) and anti-Wnt16 antibody (anti-Wnt16 antibody) diluted 1:200 as primary antibodies. The cells were incubated at 0.degree. C., then colored using Envision (DAKO), and tissue-stained sections were prepared and observed under a microscope (FIG. 10B). It was shown that thermal stimulation increased the expression of versican and Wnt16 around the hair follicle. The diameter of the circular collagen was measured as the size of the proteoglycan layer (FIG. 10A) (N=3, **: P<0.01).
　次いで、ヒト外毛根鞘細胞を真皮の微細毛部位から取得し、ｋＳＦＭ培地で４２℃で６０分間培養を行った。対照として、３７℃で培養した。培養後のヒト外毛根鞘細胞を回収し、ＲＮＡ抽出キット（Ｑｉａｇｅｎ）を用いてｍＲＮＡを抽出した。Ｓｕｐｅｒ　ｓｃｒｉｐｔキット　（Ｉｎｖｉｔｒｏｇｅｎ）を用いて逆転写反応を行ない、定量的ＰＣＲに供した。対照として下部チャンバーに毛包上皮細胞を含まない点でのみ異なる対照（Ｃｏｎｔ．）を同様に調製した。定量的ＰＣＲには、下記のプライマーセットを用いた。Ｗｎｔ１６の発現量を図１０Ｃに示す。
Next, human outer root sheath cells were obtained from fine hair sites in the dermis and cultured in kSFM medium at 42°C for 60 minutes. As a control, the cells were cultured at 37°C. Human outer root sheath cells after culture were collected, and mRNA was extracted using an RNA extraction kit (Qiagen). A reverse transcription reaction was performed using a Superscript kit (Invitrogen) and subjected to quantitative PCR. A control (Cont.) was prepared similarly, differing only in that the lower chamber did not contain hair follicle epithelial cells. The following primer set was used for quantitative PCR. The expression level of Wnt16 is shown in FIG. 10C.

Claims (15)

  1. 　顔の皮膚において、毛包への機械刺激及び／又は温熱刺激により、真皮基質の形成の促進及び/又は環状コラーゲンの形成を制御することを含む、美容方法。 A cosmetic method comprising promoting the formation of dermal matrix and/or controlling the formation of circular collagen in facial skin by mechanical stimulation and/or thermal stimulation of hair follicles.
  2. 　前記環状コラーゲンとは、毛包周囲に環状に形成されたコラーゲンをいう、請求項１に記載の美容方法。 The cosmetic method according to claim 1, wherein the annular collagen refers to collagen formed in an annular shape around hair follicles.
  3. 　前記環状コラーゲンの形成により皮膚のハリを改善する、請求項１に記載の美容方法。 The cosmetic method according to claim 1, wherein the formation of the circular collagen improves skin firmness.
  4. 　毛包への機械刺激が、プローブを皮膚に接触させることにより付与される、請求項１に記載の美容方法。 The cosmetic method according to claim 1, wherein the mechanical stimulation to the hair follicle is applied by bringing a probe into contact with the skin.
  5. 　毛包への機械刺激が、２以上のプローブを皮膚に接触させ、プローブ間の距離を広げる方向に動かすことにより付与される、請求項４に記載の美容方法。 The cosmetic method according to claim 4, wherein the mechanical stimulation to the hair follicle is applied by bringing two or more probes into contact with the skin and moving them in a direction that increases the distance between the probes.
  6. 　機械刺激が、伸展刺激である、請求項５に記載の美容方法。 The beauty method according to claim 5, wherein the mechanical stimulation is a stretching stimulation.
  7. 　毛包への温熱刺激が、昇温されたプローブを皮膚に接触させることにより付与される、請求項１に記載の美容方法。 The cosmetic method according to claim 1, wherein the thermal stimulus to the hair follicle is applied by bringing a heated probe into contact with the skin.
  8. 　機械刺激に加えて、前記プローブを昇温させて温熱刺激を付与する、請求項４～６のいずれか一項に記載の美容方法。 The cosmetic method according to any one of claims 4 to 6, wherein in addition to mechanical stimulation, thermal stimulation is applied by increasing the temperature of the probe.
  9. 　真皮基質量に基づいて環状コラーゲン形成を評価する方法。 A method for evaluating circular collagen formation based on the amount of dermal matrix.
  10. 　前記真皮基質が、テネイシンＣ、ペリオスチン及びバーシカンからなる群から選ばれる少なくとも１である、請求項９に記載の方法。 The method according to claim 9, wherein the dermal matrix is at least one selected from the group consisting of tenascin C, periostin, and versican.
  11. 　環状コラーゲンが、毛包周囲に環状に形成されたコラーゲンである、請求項９に記載の方法。 The method according to claim 9, wherein the circular collagen is collagen formed in a circular shape around the hair follicle.
  12. 　請求項９～１１のいずれか一項に記載の環状コラーゲン形成を評価する方法により、現在又は将来の肌のハリを評価する方法。 A method for evaluating current or future skin firmness by the method for evaluating circular collagen formation according to any one of claims 9 to 11.
  13. 　線維芽細胞培養物において真皮基質タンパク質及び／又はＷｎｔ１６の発現を指標とした環状コラーゲン産生促進剤のスクリーニング方法。 A method for screening a cyclic collagen production promoter using dermal matrix protein and/or Wnt16 expression as an indicator in a fibroblast culture.
  14. 　候補成分を含む培地で線維芽細胞培養物を培養する工程、
    　培養後の線維芽細胞培養物において真皮基質タンパク質及び／又はＷｎｔ１６の発現を測定する工程、及び
    　測定された真皮基質タンパク質及び／又はＷｎｔ１６発現を、対照の真皮基質タンパク質及び／又はＷｎｔ１６発現と比較する工程
    　を含み、真皮基質タンパク質及び／又はＷｎｔ１６発現が亢進した場合に、候補成分を環状コラーゲン産生促進剤としてスクリーニングする、請求項１３に記載のスクリーニング方法。     cultivating a fibroblast culture in a medium containing the candidate component;
    measuring the expression of dermal matrix protein and/or Wnt16 in the fibroblast culture after culturing; and comparing the measured dermal matrix protein and/or Wnt16 expression with a control dermal matrix protein and/or Wnt16 expression. The screening method according to claim 13, comprising the step of screening the candidate component as a cyclic collagen production promoter when dermal matrix protein and/or Wnt16 expression is increased.
  15. 　前記線維芽細胞培養物が、皮膚器官の器官培養により得られた培養物である、請求項１３又は１４に記載のスクリーニング方法。 The screening method according to claim 13 or 14, wherein the fibroblast culture is a culture obtained by organ culture of a skin organ.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007520285A (en) * 2004-02-06 2007-07-26 バロレ，ダニエル Method and apparatus for treating mammalian tissue
JP2021508453A (en) * 2017-12-20 2021-03-11 アラーガン、インコーポレイテッドＡｌｌｅｒｇａｎ，Ｉｎｃｏｒｐｏｒａｔｅｄ Botulinum toxin cell-binding domain polypeptide and usage for skin rejuvenation
WO2021167097A1 (en) * 2020-02-19 2021-08-26 株式会社 資生堂 Method for improving aging by activating arrector pili muscle cells

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007520285A (en) * 2004-02-06 2007-07-26 バロレ，ダニエル Method and apparatus for treating mammalian tissue
JP2021508453A (en) * 2017-12-20 2021-03-11 アラーガン、インコーポレイテッドＡｌｌｅｒｇａｎ，Ｉｎｃｏｒｐｏｒａｔｅｄ Botulinum toxin cell-binding domain polypeptide and usage for skin rejuvenation
WO2021167097A1 (en) * 2020-02-19 2021-08-26 株式会社 資生堂 Method for improving aging by activating arrector pili muscle cells

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