201235662 六、發明說明： 【發明所屬之技術領域】 本發明係關於人類皮膚的壓力蓄積度之評價方法，以 及敏感性肌膚之評價方法。 【先前技術】 人類的皮膚時常與外界接觸’並受到各種刺激。該刺 激的具代表性之例子係如紫外線。紫外線係即使在未於外 觀上對肌膚造成太大變化之情形下’仍會誘發皮膚細胞之 DNA斷裂或膠原蛋白之改質等，而成為皮膚老化的原因。 紫外線係經常持續對肌膚造成壓力（stress)。此外，對女 性而言，洗臉或化妝等日常行為亦會對皮膚造成刺激，該 等刺激會作為潛在壓力而蓄積於肌膚。而且，對於該等刺 激（壓力）之抵抗性若會因某種原因減少，即定義為敏感性 肌膚。此種皮膚之外部刺激的蓄積度於本案中係稱為皮膚 的壓力蓄積度。另一方面，敏感性肌膚係指雖無明顯的皮 膚病變’但容易引起不利、有害的反應之肌膚。而且，關 於此種敏感性肌膚的原因，據稱為壓力的蓄積若達到一定 程度’即提升肌膚的感受性。此外，敏感性肌膚對外部刺 激的抵抗性係較健康肌膚低，可說是容易發生皮膚問題的 肌膚。近年來，意識到敏感性肌膚的女性有增加的傾向’ 於化妝品等方面亦期望有刺激較少者。 在化妝品的販售現場，係由美容專家進行問診，評價 肌膚壓力的蓄積狀況和是否為敏感性肌膚’而選擇對肌膚 造成的壓力（刺激）少之化妝品。然而，近年來由於通訊踩 201235662 ;判定肌膚 場的販售方法係逐漸普及，因此’期 膚的程产進且即使並非專家亦可對敏感性肌 直：ΐ: 評價。此者若為可行，則即使不依靠 二二沴’亦可評價使用者肌膚的壓力狀態和是否為 =:=:自行選擇適當的化妝品。此外，可簡單 •又k 0使用者的肌膚之化妝品或化學焕膚 (chenucal peeling)劑，藉此’避免皮膚的炎症等肌膚問 通或田】作帛@可更為提高化妝品之護膚作用或化學煥膚 之施行等的有益性。因此，正尋求不依靠問診而仍能評價 皮膚之外部刺激暴露過程（肌膚壓力的蓄積度），並且客觀 地判別是否為敏感性肌膚之方法。 在專利文獻1中係揭示於皮膚塗佈化妝料後，將剝離 出的皮膚角質層回收並進行觀察，以評價皮膚的敏感性之 方法。然而’該方法需對對母種化妝品全都進行檢查，此 外，無法評價使用者的肌膚壓力蓄積度。 專利文獻2中係揭示於皮膚塗佈類辣椒素（capsinoid) 等刺激性物質，以評價是否為敏感性肌膚之方法。然而， 使用類辣椒素等刺激性強之物質的試驗方法，對被驗者而 言係為不佳。 專利文獻3係揭示測定臉部角質層之鈣衛蛋白 (calprotectin)存在量，評價是否為敏感性肌膚之方法。 非專利文獻1、2則記載，因熱休克蛋白（heat shock protein，亦即HSP)27會對於屬於界面活性劑之月桂基硫 酸鈉（SDS)所引起的皮膚刺激產生反應’而於皮膚增加。 4 323933 201235662 [先前技術文獻] [專利文獻] [專利文獻1]日本特開平10-295648號公報 [專利文獻2]日本特開2005-296007號公報 [專利文獻3]日本專利第4469762號公報 [非專利文獻] [非專利文獻 1] Ingeborg L.A. Boxman，et al.， Experimental Dermatology, 2002, 11, 509-517.201235662 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a method for evaluating the degree of pressure accumulation of human skin, and a method for evaluating sensitive skin. [Prior Art] Human skin is often in contact with the outside world and is subjected to various stimuli. A representative example of this stimuli is ultraviolet light. Ultraviolet rays cause DNA rupture or collagen modification of skin cells even if they do not cause too much change in the appearance of the skin, and cause skin aging. Ultraviolet rays often continue to stress the skin. In addition, for women, daily activities such as washing or makeup can also cause irritation to the skin, and these stimuli accumulate as a potential pressure on the skin. Moreover, if the resistance to such irritations (pressure) is reduced for some reason, it is defined as sensitive skin. The accumulation of external stimuli of such skin is referred to herein as the degree of pressure accumulation of the skin. On the other hand, sensitive skin refers to a skin that has no obvious skin lesions but is likely to cause adverse and harmful reactions. Moreover, the cause of such sensitive skin is said to increase the sensitivity of the skin if it is accumulated to a certain extent. In addition, sensitive skin is less resistant to external irritations than healthy skin, and it is a skin that is prone to skin problems. In recent years, women who are aware of sensitive skin have an increasing tendency to be less irritated in cosmetics and the like. At the sales site of cosmetics, a cosmetic expert conducts a consultation to evaluate the accumulation of skin pressure and whether it is a sensitive skin, and selects a cosmetic that has less stress (stimulation) on the skin. However, in recent years, because of the communication treading 201235662; the method of selling the skin field is gradually popularized, so the process of the skin is progressing and even if it is not an expert, the sensitivity can be straightened: ΐ: Evaluation. If this is feasible, the pressure state of the user's skin can be evaluated and whether it is =:=: choose the appropriate cosmetics by yourself. In addition, it can be used as a cosmetic or chemical cure for the skin of the user, so as to avoid skin inflammation or other skin problems. The beneficial effects of the implementation of chemical rejuvenation. Therefore, it is seeking to evaluate the external stimuli exposure process of the skin (the accumulation of skin pressure) without relying on the consultation, and objectively discriminate whether it is a sensitive skin. Patent Document 1 discloses a method in which a peeled skin cuticle is recovered and observed after skin application of a cosmetic to evaluate skin sensitivity. However, this method requires inspection of all the mother cosmetics, and in addition, the user's skin pressure accumulation cannot be evaluated. Patent Document 2 discloses a method of evaluating whether or not a skin is coated with a stimulating substance such as capsinoid to evaluate whether it is sensitive skin. However, the test method using a substance such as capsaicin which is highly irritating is not good for the subject. Patent Document 3 discloses a method for measuring the amount of calprotectin present in the stratum corneum of the face and evaluating whether it is a sensitive skin. Non-Patent Documents 1 and 2 describe that heat shock protein (i.e., HSP) 27 is increased in the skin by reacting with skin irritation caused by sodium lauryl sulfate (SDS) which is a surfactant. [Patent Document 1] JP-A-2005-296007 [Patent Document 3] Japanese Patent No. 4469762 [Publication] [Patent Document 1] [Patent Document 1] Non-Patent Document] [Non-Patent Document 1] Ingeborg LA Boxman, et al., Experimental Dermatology, 2002, 11, 509-517.
[非專利文獻 2] Ingeborg L.A. Boxman，et al.， British Journal of Dermatology, 2002, 146, 777-785. 【發明内容】 (發明欲解決之課題） 如上所述，正期望有評價肌膚壓力蓄積度，並客觀地 評價是否為敏感性肌膚之方法。該評價方法期望為並非以 外科方式取出皮膚而對皮膚造成刺激等對使用者造成負擔 之方法。本發明之課題係提供一種使用生化指標之新穎評 價方法，該方法不會對使用者造成負擔，而可評價肌膚的 壓力蓄積度，並且可簡便而確實地評價是否為敏感性肌膚。 (解決課題之手段） 本發明為以下構成。 (1) 一種人類皮膚的壓力蓄積度之評價方法，其係將皮膚-角 質層之熱休克蛋白（HSP)27表現量（expression level)、與預先或同時測定之正常人類皮膚的HSP27 表現量進行對比。 5 323933 201235662 (2) —種敏感性肌膚之評價方法，其係將皮膚角質層之 HSP27表現量、與預先或同時測定之正常人類皮膚的 HSP27表現量進行對比。 (3) 如（1)或（2)所述之評價方法，其具備下述步驟： 採取步驟，係採取皮膚角質層； 測定步驟，係測定前述採取步驟所採取之角質層細 胞的HSP27表現量；以及 比較步驟，係將前述測定步驟所測定之HSP27表現 量與健康肌膚的皮膚角質層之HSP27表現量進行比較。 (4) 如（1)或（2)所述之評價方法，其具備下述步驟： 採取步驟，係採取被驗者之評價對象部位的角質 層； 測定步驟，係測定前述採取步驟所採取之角質層的 HSP27表現量；以及 比較步驟，係將前述測定步驟所測定之HSP27表現 量與具有健康肌膚者之前述評價對象部位之角質層的 HSP27表現量進行比較。 (5) —種人類皮膚的壓力蓄積度之評價方法，其係將皮膚角 質層之HSP27表現量、與預先或同時測定之標本群組皮 膚的HSP27表現量之分布進行相對比較。 (6) —種敏感性肌膚之評價方法，其係將皮膚角質層之 HSP27表現量、與預先或同時測定之標本群組皮膚的 HSP27表現量之分布進行相對比較。 (7) 如（5)或（6)所述之評價方法，其具備下述步驟： 6 323933 201235662 採取步驟，係採取被驗者之評價對象部位的角質 層； 測定步驟，係測定前述採取步驟所採取之角質層的 HSP27表現量；以及 比較步驟，係將前述測定步驟所測定之HSP27表現 量與標本群組之前述評價對象部位之角質層的HSP27 表現量之分布進行比較。 (8)如（3)、（4)及（7)中任一項所述之評價方法，其中，採 取皮膚角質層之步驟係藉由膠帶剝離（tape stripping) 法而進行者。 (發明之效果） 依據本發明之評價方法，可藉由測定角質層之HSP27 表現量而評價肌膚壓力蓄積度。此外，以該HSP27存在量 作為指標，可簡便地評價肌膚是否為敏感性肌膚。另外， 由於本發明之評價方法係以膠帶剝離法等簡便的操作方法 來採取評價試料，故對被驗者之負擔為少，任何人皆可簡 單地進行評價。此外，因為係生化試驗方法，由任何人進 行測定皆會得到相同的結果，故不需如以往般藉由與專家 面談的方式進行諮詢。 【實施方式】 以下詳細說明本發明。 本發明之評價方法之特徵係以皮膚角質層的HSP27存 在量作為指標。 HSP27係已知作為熱休克蛋白質家族之一員而為分子 7 323933 201235662 量27KD之蛋白質，並且具有作為分子伴護蛋白（molecular chaperon)之功能。已知會隨著免疫壓力’而增加在皮膚之 基因表現。 本發明中之敏感性肌膚係指雖無明顯的皮膚病變，但 容易引起不利、有害的反應之肌膚。此外，其對於外部刺 激的抵抗性係較健康肌膚低，可說是容易發生皮膚問題的 肌膚。另一方面，健康肌膚係指不會表現如上述之敏感性 肌膚的性質而為健康的正常肌膚。在敏感性肌膚中，已知 會有使經皮水分蒸散量（TEWL ; transepidermal water loss) 提高且使高頻傳導度（角質層水分量）降低等之傾向。此 外’敏感性肌膚會有發生斑疹或肌膚粗糙之情形。 角質層係位於皮膚最上層之組織，具有從來自體外之 異物或刺激而保護皮膚之功能。 本發明之評價對象部位，只要是可取得角質層之部 分’即可包含任意部位，主要部位可列舉如顏面、頸部、 上臂部。可依照以往的方法，而得到取自該等部位之皮膚 之角質層。然而，如前所述，由於以外科方式取出皮膚等 方法係會對使用者造成負擔，故以膠帶剝離、刮擦等簡便 地得到角質層之方法為較佳。 如此所準備之各試料之HSP27表現量，係可藉由以往 已知的方法進行測定。例如，可依據與對於HSP27之抗體 的反應’使用酵素免疫分析法（enZyme immunoassay)、放 射免疫分析法（radioimmunoassay)、西方墨點法（western blotting method)等方法。 8 323933 201235662 從各試料中’將hSP27經由各種本身即為已知的生化 方法，例如凍結解凍法（fa。. zing and thawing method)、 超音波震碎法、均質法等而調製可溶性區分Ο· fraction)。卒取後’迅迷地進行測定。 由於蓄積有壓力的肌膚或_性肌膚，其角質層之 HSP27表現s係明顯較健康肌膚高，故以角質層之Hsp27 表現量多寡作為指標，即可簡便地評價壓力蓄積度或肌膚 是否為敏感性肌膚。本發明所使用之角質層，可使用膠帶 剝離等藉由角質膠帶而僅採取角質層的表 法來進行採取。此外，本發明可在不對皮; 化學物質等刺激下’測定HSP27的表現量。因此，可在不 對使用者造成負擔下’評價使用者的㈣是否為敏感性肌 膚。特別是可簡單地選擇較適合使用者的肌膚之化妝品或 化學煥膚劑’藉此，可避免皮膚的炎症等肌膚問題或副作 用，而可更為發揮化妝品之護膚作用或有兴陡 /本發明之評價方法係由下述步驟所構:：採取步驟’ :採取角質層；測定步驟’係測定前 :質層的·表現量；以及比較 定:r:表現量與健康肌膚的角質層濱7表 現里進行比較。或者是，本發明之評僧 所構成：採取步驟，係採取角質層；測定二㈣下 述採取步驟所採取之角質層的Hsm表現^驟以 驟，係將前制定㈣所測定之HSP s ^ 之角質層之·27表現量的分布^表現量與標本群組 疋订比較。以下說明此評 323933 9 201235662 價方法。 首先，將如上述所測定之HSP27表現量與健康肌膚的 角質層之HSP27表現4進行錄。此外，亦可測定被驗者 之某評價對象部位的角質層之HSP27表現量，將所測定之 HSP27表現量與具有健康肌膚者之同—評價對象部位的角 質層之HSP27表現量進行比較。具有健康肌膚者之角質層 的HSP27表現量’係藉由使用來自複數名具有健康肌膚者 之數據的平均值，而可進行更為客觀的評價。 或者是’亦可測定被驗者之某評價對象部位的角質層 之HSP27表現量，將所測定之Hsp27表現量與標本群組之 同一評價對象部位之角質層HSP27表現量的分布進行比 較。藉由使用標本群組之Hsp27表現量的分布，而可進行 更為客觀的評價。 如此比較後之結果，當所測定之HSP27表現量顯著地 大於健康肌膚的HSP27表現量時，或是相較於標本群組之 HSP27表現罝之分布而判斷HSp27表現量為大時，即評價 是壓力之蓄積為大，並且可評價為屬於敏感性肌膚之可能 性冋。該健康肌膚的試料可從測定HSP27者採取，亦可私 與測定HSP27者為;者採取。此外，該標本群組 效:規模來隨機抽出，但亦較佳係因應目的: 依例如性別、年齢之差別來抽出標本群挺。 的仔 ，:卜:當被驗者之某評價對 表現讀著地大於具有健康 咖2 角質層之HSP27表現量時，/者之R —箱對象部位* %疋相較於標本群組之同—免 323933 10 201235662 價對象部位的角質層之HSP27表現量之分布而判斷Hsp27 表現量為大時，亦評價是壓力的蓄積為大。此外，當此數 值顯著為高時’則評價為具有敏感性肌膚。 亦即，當評價是壓力蓄積為高時，可依據所測定之 HSP27表現量之程度，而評價壓力蓄積度之程度。當所測 定之HSP27表現量係相較於健康肌膚之Hsp27表現量、或 標本群組的表現量之分布而被判斷為顯著多時，該肌膚^ 強烈表現出敏感性肌膚的性質，並且其對外界刺激的抵抗 =係相較於健康肌膚或標本群組之平均而為顯著低，非二 容易發生皮膚問題之可能性為高。此外，當所 吊 繼肌一表現量而僅為少： 矣規旦。疋之表現量係相較於標本群組之HSP27 :里=布而被判斷為略大於平均值時’該肌膚可被評 =謂==蓄Γ大於標本群組之平均值’但仍未達 肌膚。卜界刺激之抵抗性低而會發生皮膚問題的敏感性 但本發明並不限 ，針對具體之實施例進行說明 定於下述實施例。[Non-Patent Document 2] Ingeborg LA Boxman, et al., British Journal of Dermatology, 2002, 146, 777-785. SUMMARY OF THE INVENTION (Problems to be Solved by the Invention) As described above, it is desirable to evaluate the degree of skin pressure accumulation. And objectively evaluate whether it is a method of sensitive skin. This evaluation method is expected to be a method of not burdening the user, such as irritating the skin without surgically removing the skin. An object of the present invention is to provide a novel evaluation method using a biochemical index which does not burden a user, and which can evaluate the degree of pressure accumulation of the skin, and can easily and surely evaluate whether it is sensitive skin. (Means for Solving the Problem) The present invention has the following constitution. (1) A method for evaluating the degree of pressure accumulation of human skin by expressing the heat shock protein (HSP) 27 expression level of the skin-stratum layer and the HSP27 expression amount of normal human skin measured in advance or simultaneously. Compared. 5 323933 201235662 (2) A method for evaluating sensitive skin, which compares the amount of HSP27 expression in the stratum corneum of the skin with the amount of HSP27 expression of normal human skin measured in advance or simultaneously. (3) The evaluation method according to (1) or (2), comprising the steps of: taking a step of taking the stratum corneum of the skin; and measuring the HSP27 expression of the stratum corneum cells taken by the taking step And a comparison step of comparing the amount of HSP27 measured by the aforementioned measuring step with the amount of HSP27 expression of the stratum corneum of healthy skin. (4) The evaluation method according to (1) or (2), comprising the steps of: taking a step of taking a stratum corneum of a subject to be evaluated by the subject; and measuring a step of measuring the aforementioned taking step The HSP27 expression amount of the stratum corneum; and a comparison step of comparing the HSP27 expression amount measured in the measurement step with the HSP27 expression amount of the stratum corneum of the evaluation target site of the healthy skin. (5) A method for evaluating the degree of pressure accumulation of human skin by comparing the HSP27 expression amount of the skin keratin layer with the distribution of the HSP27 expression amount of the specimen group skin measured in advance or simultaneously. (6) A method for evaluating sensitive skin, which compares the amount of HSP27 expression in the stratum corneum of the skin with the distribution of HSP27 expression in the skin of the specimen group measured in advance or simultaneously. (7) The evaluation method according to (5) or (6), comprising the steps of: 6 323933 201235662 taking a step of taking a stratum corneum of a subject to be evaluated by the subject; and measuring the step of measuring the aforementioned steps The HSP27 expression amount of the stratum corneum to be taken; and a comparison step of comparing the HSP27 expression amount measured in the measurement step with the HSP27 expression amount of the stratum corneum of the specimen portion in the specimen group. (8) The evaluation method according to any one of (3), wherein the step of taking the stratum corneum of the skin is carried out by a tape stripping method. (Effects of the Invention) According to the evaluation method of the present invention, the skin pressure accumulation degree can be evaluated by measuring the amount of HSP27 expression in the stratum corneum. In addition, the amount of HSP27 present can be used as an indicator to easily evaluate whether the skin is sensitive skin. Further, in the evaluation method of the present invention, the sample is evaluated by a simple operation method such as a tape peeling method, so that the burden on the subject is small, and anyone can easily evaluate it. In addition, because of the biochemical test method, the same result can be obtained by any person, so it is not necessary to consult with an expert as before. [Embodiment] Hereinafter, the present invention will be described in detail. The evaluation method of the present invention is characterized by the amount of HSP27 present in the stratum corneum of the skin as an index. HSP27 is known as a member of the heat shock protein family and is a protein of 27 KD, which has a function as a molecular chaperon. It is known that the gene expression in the skin increases with the immune pressure. The sensitive skin in the present invention refers to a skin which is likely to cause an adverse and harmful reaction although it has no obvious skin lesions. In addition, its resistance to external irritations is lower than that of healthy skin, and it can be said to be a skin problem that is prone to skin problems. On the other hand, healthy skin refers to normal skin that does not exhibit the properties of sensitive skin as described above. In sensitive skin, it is known that the transepidermal water loss (TEWL) is increased and the high-frequency conductivity (the stratum corneum moisture content) is lowered. In addition, sensitive skin may have rash or rough skin. The stratum corneum is located in the uppermost layer of the skin and has the function of protecting the skin from foreign bodies or stimuli from outside the body. The evaluation target portion of the present invention may include any portion as long as it can obtain the stratum corneum portion. Examples of the main portion include a face, a neck, and an upper arm. The stratum corneum of the skin taken from these sites can be obtained according to a conventional method. However, as described above, since the method of surgically removing the skin or the like imposes a burden on the user, a method of easily obtaining the stratum corneum by tape peeling, scratching or the like is preferable. The amount of HSP27 expression of each of the samples prepared in this manner can be measured by a conventionally known method. For example, an enzyme immunoassay (enZyme immunoassay), a radioimmunoassay method, a western blotting method, or the like can be used depending on the reaction with an antibody against HSP27. 8 323933 201235662 From each sample, 'hSP27 is prepared by various biochemical methods known per se, such as fa.. zing and thawing method, ultrasonic shattering method, homogenization method, etc. Fraction). After the stroke, the measurement was performed eagerly. Because of the accumulation of stressed skin or _ skin, the HSP27 expression of the stratum corneum is significantly higher than that of healthy skin. Therefore, by using the amount of Hsp27 in the stratum corneum as an indicator, it is easy to evaluate whether the pressure accumulation or the skin is sensitive. Sexual skin. The stratum corneum used in the present invention can be taken by a method in which only the stratum corneum is taken by a keratin tape such as tape peeling. Further, in the present invention, the amount of expression of HSP27 can be measured without stimulating the skin; Therefore, it is possible to evaluate whether the user's (4) is a sensitive skin without burdening the user. In particular, it is possible to easily select a cosmetic or chemical rejuvenating agent that is more suitable for the skin of the user, thereby avoiding skin problems or side effects such as inflammation of the skin, and more to exert the skin care effect of the cosmetic or to be steep/inventive The evaluation method is constructed by the following steps: taking the step ': taking the stratum corneum; measuring step' is before the measurement: the performance of the quality layer; and comparing: r: the amount of expression and the stratum corneum of healthy skin 7 Compare performance. Alternatively, the evaluation of the present invention consists of taking steps to adopt the stratum corneum; measuring the Hsm performance of the stratum corneum taken by the following steps (4), and determining the HSP s determined by (4). The distribution of the expression level of the stratum corneum of the stratum corneum is compared with the specimen group. The following describes the 323933 9 201235662 price method. First, the HSP27 expression level measured as described above and the HSP27 expression 4 of the stratum corneum of healthy skin were recorded. Further, the amount of HSP27 expression in the stratum corneum of a certain evaluation target site of the subject can be measured, and the measured HSP27 expression amount can be compared with the HSP27 expression amount of the stratum corneum of the same evaluation site. The HSP27 expression amount of the stratum corneum of a healthy skin can be more objectively evaluated by using an average value of data from a plurality of healthy skin. Alternatively, the HSP27 expression amount of the stratum corneum of a certain evaluation target portion of the subject can be measured, and the measured Hsp27 expression amount is compared with the distribution of the stratum corneum HSP27 expression amount of the same evaluation target portion of the specimen group. A more objective evaluation can be made by using the distribution of the Hsp27 expression amount of the specimen group. As a result of this comparison, when the measured amount of HSP27 is significantly greater than the HSP27 expression of healthy skin, or when the expression of HSP27 is larger than the distribution of HSP27 of the specimen group, the evaluation is The accumulation of stress is large and can be evaluated as the possibility of being sensitive skin. The sample of the healthy skin can be taken from those who measure HSP27, or those who measure HSP27 privately. In addition, the specimen group effect: the scale is randomly selected, but it is also better for the purpose: to extract the specimens according to the difference between gender and age. Aberdeen, Bu: When a certain evaluation of the subject is greater than the performance of HSP27 with a healthy coffee 2 stratum corneum, the R-box object part * % 疋 is the same as the specimen group - 323933 10 201235662 The distribution of HSP27 expression in the stratum corneum of the price target part is judged to be large when the expression amount of Hsp27 is large, and it is also evaluated that the accumulation of pressure is large. In addition, when this value is significantly high, it is evaluated as having sensitive skin. That is, when the evaluation is that the pressure accumulation is high, the degree of pressure accumulation can be evaluated in accordance with the degree of the measured amount of HSP27 measured. When the measured amount of HSP27 is judged to be significantly more than the distribution of Hsp27 expression of healthy skin or the amount of expression of the specimen group, the skin strongly exhibits the properties of sensitive skin, and the pair thereof The resistance of external stimuli = is significantly lower than the average of healthy skin or specimen groups, and the possibility of skin problems is high. In addition, when the continuation of the muscles is only a small amount of performance: 矣规旦. The performance of 疋 is compared to the HSP27: 里=布 of the specimen group and is judged to be slightly larger than the average value. 'The skin can be judged = the == Γ is greater than the average of the specimen group' but still not reached skin. The sensitivity of the stimuli is low and the sensitivity of the skin problem may occur. However, the present invention is not limited thereto, and the description of the specific examples is set forth in the following examples.
[實施例1J ⑴皆々hf外線照射所致之皮膚壓力蓄積度之試驗 ⑴系外線所致之壓力負荷 π ® Μ Μ名健康的男性作為被驗者而實施試驗。 使用 Multini。ρ , TT1 (1 〇 ar Ultraviolet Simulator Model 601 ί Ο /™\ I Q y· 1 · 1叻ΐ公司製）作為紫外線照射装置，光源係使用 323933 11 201235662 150W氙燈。調整照射強度而使被驗者背部皮膚會出現誘發 色素沉積之紅斑，並照射紫外線（UVB)l 70秒。又，藉由比 較紫外線照射部位與非照射部位，確認紫外線所致之壓力 蓄積。在人工紫外線照射前及照射後第5曰、第11曰、第 17日’藉由膠帶剝離法（角質層檢驗片（horny layer checker) : Asahibiomed製）而採取皮膚角質層試樣。 (2) 皮膚角質層中的蛋白質之萃取方法 在均質化用管中採取0. l%SDS/PBS(-)200ul，於其中 裝入上述經膠帶剝離之角質層檢驗片，使用均質用研棒 (pestle)刮擦黏著面，而從膠帶由角質層中萃取出蛋白質。 (3) 皮膚角質層蛋白之定量 藉由 DC Protein Assay Kit(Bio-Rad 製），對角質層 萃取液中所含之蛋白量進行定量。 (4) 以西方墨點法進行HSP27之定量 使用XP PANTERA Gel(5-20%之梯度膠：DRC公司製）， 藉由SDS-PAGE而將1.5yg的角質層蛋白質分離後，將蛋 白質轉印到 PVDF 膜，以 StartingBlock Blocking Buffer (Thermo Scientific 公司製）進行阻斷（blocking)。將一 級抗體（HSP27 Polyclonal Antibody : Stressgen 公司製 造）稀釋 1000 倍，將二級抗體（Goat anti-mouse IgG: invitrogen公司製造）稀釋10000倍並使其反應後，使用 ECLplus(BD Bioscience 公司製造），以 LAS-4000mini(富 士軟片公司製造）進行檢測。以Science Lab 2004 Multi Gauge(富士軟片公司製造）將所得之譜帶（band)的發光強 12 323933 201235662 度進行數值化。依據預先以基因重組之HSP27蛋白質作為 標準品所作成之標準曲線，讀取發光信號的強度作為H s p 2 7 的蛋白質量，而進行評價。 (5)結果 經賦予紫外線所致之刺激（壓力）的被驗者的角質層 HSP27量測定結果係示於表！、第1圖。確認到紫外線係歷 經長時間而使壓力蓄積於皮膚。 [表1] UV照射後之HSP27之變化量 照射後之日數 UV(-) UV(+) 0曰 〇· 02±0. 00 — 5曰 0. 03±0. 03 0. 02+0. 02 11曰 0. 05±0. 04 0. 11+0. 12 17曰 0. 05+0. 05 0. 16+0. 24 ng/ // g蛋白質 因紫外線照射而於皮膚產生紅斑，HSP27係隨曰數增 加。此係評價為持續刺激而結果蓄積壓力者。 [實施例2] (實驗2)十二基硫酸鈉（SDS)所致之低刺激之壓力蓄積試驗 為了確認已知會使皮膚的HSP27增加之界面活性劑 SDS的刺激是否會作為壓力而蓄積，故以健康肌膚的男女8 名作為對象，進行以下試驗。 (1)壓力之負荷 323933 13 201235662 於2日内 曰 次、每隔1小時以10%SDS水溶液 '月1J •貝1]部，而作成肌膚粗链（壓力狀態）。又，僅 \ A的右前臂’以作為對照。此外，在確認左 二二X刀瘵散1係較處理前更為上昇後，開始進行以下 之測疋。 ⑵試料之採取與水分蒸散量之測定 在肶膚粗糙處理後（第〇曰）、隔曰、第3曰、第5曰、 2曰第14日測定水分蒸散量，並且藉由料剝離法採 取角質層試樣。又，若於笛 '第14日時，水分蒸散量之測定值 仍回復至肌膚粗輪處理前之測定值時，則將測定延長至 第21日、第28日。 、政量之/則疋係以Courage & Khazaka公司製[Example 1J (1) Test of skin pressure accumulation degree by external exposure of hf (1) Pressure load by external line π ® Μ A healthy male was tested as a subject. Use Multini. ρ , TT1 (1 〇 ar Ultraviolet Simulator Model 601 ί Ο /TM\ I Q y· 1 · 1 叻ΐ 叻ΐ) As the ultraviolet ray irradiation device, the light source is 323933 11 201235662 150W xenon lamp. The intensity of the irradiation was adjusted so that the erythema which induces pigmentation appeared on the back skin of the subject, and ultraviolet rays (UVB) were irradiated for 70 seconds. Further, by accumulating the ultraviolet ray irradiation portion and the non-irradiation portion, the pressure accumulation due to the ultraviolet ray is confirmed. Skin stratum corneum samples were taken by the tape stripping method (horny layer checker: Asahibiomed) before and after artificial ultraviolet irradiation. (2) Extraction method of protein in the stratum corneum of the skin In the homogenization tube, 0.1% of SDS/PBS(-) 200 ul was taken, and the above-mentioned tape-peeled cuticle test piece was placed therein, and a homogenized pestle was used. (pestle) scratches the adhesive surface and extracts the protein from the stratum corneum from the tape. (3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified by a DC Protein Assay Kit (manufactured by Bio-Rad). (4) Quantification of HSP27 by Western blotting method Using XP PANTERA Gel (5-20% gradient gel: manufactured by DRC), 1.5 yg of stratum corneum protein was separated by SDS-PAGE, and the protein was transferred. The PVDF membrane was blocked by StartingBlock Blocking Buffer (manufactured by Thermo Scientific). The primary antibody (HSP27 Polyclonal Antibody: manufactured by Stressgen) was diluted 1000-fold, and the secondary antibody (Goat anti-mouse IgG: manufactured by Invitrogen) was diluted 10,000-fold and reacted, and then ECLplus (manufactured by BD Bioscience) was used. LAS-4000mini (manufactured by Fujifilm Co., Ltd.) for testing. The intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (manufactured by Fujifilm Co., Ltd.) at 12 323,933, and 35,535,662 degrees. The intensity of the luminescent signal was read as the protein amount of H s p 2 7 based on a standard curve prepared by using the genetically recombined HSP27 protein as a standard. (5) Results The results of the measurement of the stratum corneum HSP27 in the subject with the stimulation (pressure) due to ultraviolet rays are shown in the table! Figure 1. It was confirmed that the ultraviolet rays accumulated in the skin over a long period of time. [Table 1] The amount of change in HSP27 after UV irradiation. The number of days after irradiation. UV(-) UV(+) 0曰〇· 02±0. 00 — 5曰0. 03±0. 03 0. 02+0. 02 11曰0. 05±0. 04 0. 11+0. 12 17曰0. 05+0. 05 0. 16+0. 24 ng/ // g protein produces erythema on the skin due to ultraviolet radiation, HSP27 As the number of turns increases. This department evaluates those who continue to stimulate and accumulate stress. [Example 2] (Experiment 2) Pressure accumulation test of low stimulation by sodium dodecyl sulfate (SDS) In order to confirm whether the stimulation of the surfactant SDS, which is known to increase the HSP27 of the skin, accumulates as pressure, The following tests were conducted for 8 men and women with healthy skin. (1) Pressure load 323933 13 201235662 In 2 days, every 10 hours, 10% SDS aqueous solution 'month 1J • shell 1' is used, and the skin is thick (pressure state). Again, only the right forearm of \A is used as a control. In addition, after confirming that the left two-two X-knife scatter 1 system is higher than before the treatment, the following test is started. (2) Determination of sample taken and water evapotranspiration The amount of water evapotranspiration was measured after rough skin treatment (No.), barrier, No. 3, No. 5, No. 2, and was taken by the material stripping method. Cuticle sample. In addition, if the measured value of the moisture evapotranspiration returns to the measured value before the skin roughing treatment on the 14th day of the flute, the measurement is extended to the 21st and 28th days. , the government / the system is based on Courage & Khazaka
Tewameter 進行涓il 定。A 併 >=* J ^ 角質層係藉由角質層檢驗片 (Asahibiomed 製）採取。 (3) 皮膚角質層中的蛋白質之萃取方法 以與實驗1相同之操作，在均質化用管中採取〇. 1% SDS/PBS(-)2GGu卜於其中裝人上述經膠帶剝離之角質層檢 驗片’使用均質用研棒刮擦黏著面，而從膠帶由角質層中 萃取出蛋白質。 (4) 皮膚角質層蛋白之定量 藉由 DC Protein Assay Kit(Bio-Rad 製），對角質層 萃取液中所含之蛋白量進行定量。 (5) 以西方墨點法進行HSP27之定量 使用1?卩八1^1以661(5-20%之梯度膠：〇1^：公司製）， 14 323933 201235662 藉由SDS-PAGE而將1. 5#g的角質層蛋白質分離後’將蛋 白質轉gp 至ij PVDF 膜，以 StartingBlock Blocking Buffer (Thermo Scient i f ic公司製）進行阻斷。將一級抗體（HSP27 Polyclonal Antibody : Stressgen 公司製）稀釋 1000 倍’ 將二級抗體（Goat anti-mouse IgG : invitrogen 公司製造） 豨釋10000倍並使其反應後，使用ECLplus(BD Bioscience 公司製造）’以LAS-4000mini(富士軟片公司製造）進行檢 測。以 Science Lab 2004 Multi Gauge(富 士軟片公司製 造）將所得之譜帶的發光強度進行數值化。依據預先以基因 重組之HSP27蛋白質作為標準品而作成的標準曲線，讀取 發光信號的強度作為HSP27的蛋白質量，而進行評價。 (6)結果 經賦予由SDS洗淨所致之刺激（壓力）的被驗者之角質 層、以及對照之HSP27量之測定結果係示於表2、第2圖。 此外，一併表示水分蒸散量之變化。 323933 201235662 [表2] SDS刺激所致之變化 處理後曰數 HSP27C水處理） ng/ jU g蛋白質 HSP27(SDS 處理） ng/ # g蛋白質 水分蒸散量（SDS處理） g/m2 · hr 0 0. 06±0· 05 0. 09+0. 11 5. 45+1.79 1 0. 05+0. 08 0.13+0. 32 13.95+1.81 3 0. 05±0. 05 0. 07+0. 12 Π. 56+1.21 7 0. 03+0. 05 0. 39+0. 35 8. 26+1.47 14 0. 05+0. 03 0. 63±1. 19 7- 06±2. 19 21 0. 05±0. 03 0.09+0. Π 6. 26+1.61 27 0. 03+0. 03 0. 03±0. 03 6.00+1.30 由SDS洗淨所致之皮膚水分的蒸散，係於第i日到達 峰值並緩慢地回復，但HSP27則增加至第14日為止。此係 評價為皮膚壓力蓄積者。 (實驗3)異位性皮膚炎患者的壓力蓄積度之評價 異位性皮膚炎患者的皮膚常持續受到來自外界的刺 激，皮膚經常處於壓力狀態。測定處於此種狀態之患者之 皮膚壓力蓄積度。 “" (1)皮膚角質層之採取方法 _對診斷為異位性皮膚炎之4名女性患者之呈現皮膚炎 的清部皮膚’使用角質檢驗片（2.5cmx2.5cm: Asahibiomed 製），進行膠帶剝離，採取角質層。另外，亦對未呈現皮膚 炎症狀之上臂部皮膚以相同操作進行取樣。 以相同操作’亦對3名健康女性之頸部皮膚、上臂部 323933 16 201235662 皮膚進行取樣，以作為對照。 (2) 皮膚角質層中的蛋白質之萃取方法 在均質化用管中採取l%SDS/PBS(-)200ul，於其巾裝 入上述經膠帶剝離之角質層檢驗片，以均質用研棒刮擦黏 著面，而從膠帶上的角質層萃取出蛋白質。 (3) 皮膚角質層蛋白之定量 藉由 DC Protein Assay Kit(Bio-Rad 製），對角質層 萃取液中所含之蛋白量進行定量。 (4) 以西方墨點法進行HSP27之定量 使用XP PANTERA Gel(5-20°/。之梯度膠：DRC公司製）， 藉由SDS-PAGE而將1.5#g的角質層蛋白質分離後，將蛋 白質轉印到 PVDF 膜，以 StartingBlock Blocking Buffer (Thermo Scientific公司製）進行阻斷。將一級抗體（Hsp27 Polyclonal Antibody : Stressgen 公司製）稀釋 1000 倍， 將二級抗體（Goat anti-mouse IgG : invitrogen 公司製造） 稀釋10000倍並使其反應後，使用ECLplus(BD Bioscience 公司製造）’以LAS-4000mini(富士軟片公司製造）進行檢 測。以 Science Lab 2004 Multi Gauge(富 士軟片公司製 造）將所得之譜帶的發光強度進行數值化。將發光信號的強 度作為HSP27之相對量而進行評價。 (5 )結果 異位性皮膚炎患者以及健康者之皮膚角質層之HSP27 量測定結果係示於表3、第3圖。 17 323933 201235662 [表3] 測定部位 HSP27發光強度 健康者臂部 841417±290490 健康者頸部 861255±517352 異位性皮膚炎患者（AD)臂部（非皮膚炎部位） 12175507+13096705 異位性皮膚炎患者（AD)頸部（皮膚炎部位） 27209365±2850449 如表3、第3圖所揭明，健康者之HSP27幾乎不存在 於角質層中，但從異位性皮膚炎患者之患部皮膚則檢測出 高濃度的HSP27。此外，從異位性皮膚炎患者的正常皮膚 中檢測出的HSP27雖未達呈現皮膚炎的部位之程度，但相 較於健康者而為較高濃度。由以上結果，可知經常受到壓 力的異位性皮膚炎患者之皮膚係蓄積有壓力。此外，可確 認到並非異位性皮膚炎之患部之皮膚的HSP27亦相對為 高。由以上結果，可確認到測定HSP27作為敏感性肌膚的 指標係屬有用。 [實施例3] (實驗3-2)測定常受到來自外界的壓力之顏面部與較未受 到壓力之前臂部或臀部皮膚的HSP27，評價蓄積於皮膚之 壓力的程度。 試驗方法 (1)試料之採取 藉由膠帶剝離法，從8名健康女性之臉頰、顎下、前 臂、臀部採取角質層試樣。1部位係採取4片。 18 323933 201235662 (2) 皮膚角質層中的蛋白質之萃取方法 在均質化用管中裝入0.5%chaps緩衝液 υυ以1 ’於 其中裝入上述經膠帶剝離之角質層檢驗片，藉由抬# ' 買而從 膠帶由角質層中萃取出蛋白質。從1部位所採取的4 4 λ 樣係一併以1管進行萃取，而作為1個試樣。 ° (3) 皮膚角質層蛋白之定量 藉由 DC Protein Assay Kit(Bio-Rad 製），對角質> 萃取液中所含之蛋白量進行定量。 (4) 以ELISA法進行HSP27之定量 以 Total human HSP27 DuoSet ELISA kit(R&D systems 製）測定角質層萃取液中所含之HSP27量。將結果作為每單 位蛋白量之HSP27量，示於表4、第4圖。 又，標示係以平均值±S. E.表示。 [表4] 皮膚部位之HSP27測定結果 部位 測定結果 臉頰 127. 6±30. 6 顎下 63. 85±37. 34 前臂 30. 02±10.44 臀部 9. 92+3. 12Tewameter makes 涓 il. A and >=* J ^ The stratum corneum was taken by a cuticle test strip (Asahibiomed). (3) Extraction method of protein in the stratum corneum of the skin In the same operation as in Experiment 1, 均. 1% SDS/PBS(-)2GGu was used to carry the above-mentioned stripped stratum corneum. The test piece 'scrape the adhesive surface with a pestle using a homogenizer, and the protein is extracted from the stratum corneum from the tape. (4) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified by a DC Protein Assay Kit (manufactured by Bio-Rad). (5) Quantitative use of HSP27 by Western blotting method 1? 卩8 1^1 to 661 (5-20% gradient gel: 〇1^: company), 14 323933 201235662 by SDS-PAGE After separation of the stratum corneum protein of 5#g, the protein was transferred to gp to ij PVDF membrane and blocked with StartingBlock Blocking Buffer (manufactured by Thermo Scientific). The primary antibody (HSP27 Polyclonal Antibody: manufactured by Stressgen) was diluted 1000-fold. After the secondary antibody (Goat anti-mouse IgG: manufactured by Invitrogen) was released 10,000 times and reacted, ECLplus (manufactured by BD Bioscience) was used. Detected by LAS-4000mini (manufactured by Fujifilm Co., Ltd.). The luminous intensity of the obtained band was quantified by Science Lab 2004 Multi Gauge (manufactured by Fujifilm Co., Ltd.). The intensity of the luminescent signal was read as the protein amount of HSP27 based on a standard curve prepared by using the genetically recombined HSP27 protein as a standard. (6) Results The results of measurement of the amount of HSP27 in the stratum corneum of the subject and the amount of HSP27 in the control which were given the stimulation (pressure) by SDS washing are shown in Table 2 and Fig. 2 . In addition, the change in the amount of moisture evapotranspiration is also indicated. 323933 201235662 [Table 2] Changes due to SDS stimulation After treatment HSP27C water treatment) ng / jU g protein HSP27 (SDS treatment) ng / # g protein moisture evapotranspiration (SDS treatment) g / m2 · hr 0 0. 06±0· 05 0. 09+0. 11 5. 45+1.79 1 0. 05+0. 08 0.13+0. 32 13.95+1.81 3 0. 05±0. 05 0. 07+0. 12 Π. 56+1.21 7 0. 03+0. 05 0. 39+0. 35 8. 26+1.47 14 0. 05+0. 03 0. 63±1. 19 7- 06±2. 19 21 0. 05± 0. 03 0.09+0. Π 6. 26+1.61 27 0. 03+0. 03 0. 03±0. 03 6.00+1.30 Evapotranspiration of skin moisture caused by SDS washing, peaking on the i-day And responded slowly, but HSP27 increased until the 14th. This system is evaluated as a skin pressure accumulator. (Experiment 3) Evaluation of pressure accumulation in patients with atopic dermatitis The skin of patients with atopic dermatitis often continues to be stimulated from the outside, and the skin is often under stress. The degree of skin pressure accumulation of the patient in this state was measured. "" (1) Method of taking the stratum corneum of the skin _ A keratin test piece (2.5 cm x 2.5 cm: Asahibiomed) for 4 female patients diagnosed with atopic dermatitis The tape was peeled off and the stratum corneum was taken. In addition, the skin of the upper arm without the skin inflammation was sampled in the same operation. The same skin was used to sample the skin of the neck, upper arm 323933 16 201235662 of three healthy women. As a control. (2) Extraction method of protein in the stratum corneum of the skin In the homogenization tube, 1% SDS/PBS(-) 200 ul was taken, and the above-mentioned tape-peeled stratum corneum test piece was placed in the towel to homogenize. The adhesive surface was scraped with a pestle and the protein was extracted from the stratum corneum on the tape. (3) Quantification of the stratum corneum protein by the DC Protein Assay Kit (manufactured by Bio-Rad), which is contained in the stratum corneum extract The amount of protein was quantified. (4) Quantification of HSP27 by Western blotting method Using XP PANTERA Gel (5-20°/gradient gel: manufactured by DRC), 1.5#g of stratum corneum was obtained by SDS-PAGE. After the protein is separated, the egg is placed The white matter was transferred to a PVDF membrane and blocked with StartingBlock Blocking Buffer (manufactured by Thermo Scientific). The primary antibody (Hsp27 Polyclonal Antibody: Stressgen) was diluted 1000-fold, and the secondary antibody (Goat anti-mouse IgG: invitrogen) Manufactured) After diluting 10,000 times and reacting it, it was tested with LAS-4000mini (manufactured by Fujifilm Co., Ltd.) using ECLplus (manufactured by BD Bioscience Co., Ltd.). The band obtained by Science Lab 2004 Multi Gauge (manufactured by Fujifilm Co., Ltd.) The intensity of the luminescence intensity was quantified. The intensity of the luminescence signal was evaluated as the relative amount of HSP 27. (5) The results of HSP27 measurement of the skin horny layer of patients with atopic dermatitis and healthy people are shown in Table 3, 3 Fig. 17 323933 201235662 [Table 3] Measurement site HSP27 Luminous intensity Healthy arm 841417±290490 Healthy person neck 861255±517352 Atopic dermatitis patient (AD) Arm (non-dermatitis site) 12175507+13096705 Patients with dermatitis (AD) neck (dermatitis site) 27209365±2850449 as shown in Table 3, Figure 3 Jieming, the healthy of HSP27 is hardly present in the stratum corneum, but the affected areas of skin of patients with atopic skin inflammation is detected a high concentration of HSP27. Further, although the HSP27 detected from the normal skin of a patient with atopic dermatitis did not reach the level of dermatitis, it was higher than that of the healthy person. From the above results, it was found that the skin of a patient with atopic dermatitis which is often subjected to pressure accumulated pressure. In addition, it was confirmed that the skin of the affected part of the atopic dermatitis was also relatively high in HSP27. From the above results, it was confirmed that the measurement of HSP27 as an indicator of sensitive skin is useful. [Example 3] (Experiment 3-2) The HSP27, which was subjected to pressure from the outside and the skin of the arm or the buttocks before the pressure, was measured, and the degree of pressure accumulated in the skin was evaluated. Test method (1) Sample preparation A stratum corneum sample was taken from the cheeks, underarms, forearms, and buttocks of eight healthy women by a tape peeling method. 1 part is taken in 4 pieces. 18 323933 201235662 (2) Extraction method of protein in the stratum corneum of the skin In a homogenization tube, 0.5% chaps buffer solution is placed, and 1' of the stripped stratum corneum test piece is placed therein, by raising # 'Buy and extract protein from the stratum corneum from the tape. The 4 4 λ sample taken from the 1 site was extracted by one tube and used as one sample. ° (3) Quantification of skin stratum corneum protein The amount of protein contained in the keratin> extract was quantified by DC Protein Assay Kit (manufactured by Bio-Rad). (4) Quantification of HSP27 by ELISA The amount of HSP27 contained in the stratum corneum extract was measured by Total Human HSP27 DuoSet ELISA kit (manufactured by R&D Systems). The results are shown in Table 4 and Figure 4 as the amount of HSP27 per unit protein amount. Further, the designation is expressed by the mean value ± S. E. [Table 4] HSP27 measurement results of skin parts Site measurement results Cheeks 127. 6±30. 6 颚 63. 85±37. 34 Forearm 30. 02±10.44 Hip 9. 92+3. 12
Pg/ V g蛋白質 如表4、第4圖之結果所揭明，HSP27之測定結果係在 曰常壓力高之顏面部（臉頰）為特別高，露出較少的臀部則 顯著為低。由以上結果可知，HSP27係可作為評價皮膚之 19 323933 201235662 曰常壓力之指標。 [實施例4] (實驗4)測定標本群組之角質層的Hsp27，作成直方圖 (histogram)。 試驗方法 (1) 試驗試料之採取 以Ik機抽選之445名女性作為對象，藉由膠帶剝離法 從臉頰採取角質層試樣。試樣係從1部位採取1片。 (2) 皮膚角質層中的蛋白質之萃取方法 在均質化用管中裝入RIPA緩衝液5〇〇ul，於其中裝入 上述經膠帶剝離之角質層檢驗片，藉由均質而從膠帶由角 質層中萃取出蛋白質（萃取液A)。另外，將萃取後之角質 層檢驗片移至別的均質化用管中，裝入含有1%SDS25〇mM Tris-HCL緩衝液500yl，藉由均質而從殘留於膠帶之角 質層中萃取出蛋白質（萃取液B)。萃取液A係用於HSP27 之疋量及角質層蛋白之定量，萃取液B係用於角質層蛋白 之定量。 (3) 皮膚角質層蛋白之定量 以BCA protein assay(Pierce製）對角質層萃取液中 所含之蛋白量進行定量。 以萃取液A與萃取液B之蛋白質量的總計作為總蛋白 質（total protein)量。 (4) 以ELISA法進行HSP27之定量 以 Total human HSP27 DuoSet ELISA kit(R&D systems 323933 20 201235662 製）測定角質層萃取液中所含之HSP27量。結果係對每單位 蛋白質量之HSP27量進行解析。標本群組之Hsp27表現量 之頻率分布表係示於表5,直方圖係示 [表5] 標本群組之HSP27之頻率分布表 數據區㈤ 頻度 0. 4 0 2. 5 22 5. 0 64 7. 5 79 10. 0 51 12. 5 34 15. 0 27 17. 5 26 20. 0 25 22. 5 17 25. 0 19 27. 5 11 30. 0 15 32. 5 11 35. 0 11 37. 5 12 40. 0 4 42. 5 3 45. 0 〇 47. 5 2 50.0 3 超過50 9 累積 0% 5% 19% 37% 49% 56% 62% 68% 74% 78% 82% 84% 88% 90% 93% 95% 96% 97% 97% 97% 98% 100% 323933 21 201235662 如表5、第5圖所示，HSP27之表現量係分布於從 0. 4pg/ # g總蛋白質至超過50pg/ /z g總蛋白質之值為止的 廣範圍。平均值為15. Opg/#g總蛋白質。相較於該頻率分 布而將HSP27表現量判斷為大之基準係可自由地設定，例 如，可將平均值15.0pg///g總蛋白質以上之值判斷為 HSP27表現量大，並且，由於10. Opg//zg總蛋白質之累積 頻率為約50%，故亦可將超過10. 0pg/#g總蛋白質之值 判斷為HSP27表現量為大。無論如何，皆可判斷為若HSP27 之表現量越大，則人類皮膚的壓力蓄積度越大。 又，實驗4之HSP27表現量為實驗3之臉頰部HSP27 表現量的1/10，此係因為在實驗4之蛋白質萃取方法中， 是於萃取包含HSP27之蛋白質後，使用含有1%SDS之50mM Tris-HCL緩衝液以萃取角質層之蛋白質，而與實驗3相 比，實驗4之總蛋白質之檢測量為增加之故。實驗3、實 驗4於HSP27萃取步驟並無差異，而HSP27之溶解性為高， 故以該步驟即能充分地萃取。另一方面，藉由實驗4所追 加之以SDS萃取之方法，雖可萃取出蛋白質的幾乎全部 量，但無法測定該萃取液中之HSP27。由於實驗3與實驗4 之萃取步驟為相異，故無法比較實驗3與實驗4之數據， 但可進行實驗3之數據之間的比較、實驗4之數據之間的 比較。 [實施例5] (實驗5)關於人類皮膚的壓力蓄積度的問卷調查結果與 HSP27量之對比 22 323933 201235662 從實驗4之標本群組中隨機抽出189名，實施關於人 類皮膚的壓力蓄積度之問卷調查，將其結果與HSP27表現 量進行對比。 (1) 肌膚的敏感性之問卷調查 讓被驗者選擇該名被驗者的肌膚是「非敏感」、「略為 敏感」、「敏感」這3種類中之任一者。 對被驗者提示下述敏感性的10個要素，讓被驗者自行 判斷係屬於非敏感（不敏感）、略為敏感、敏感之何者。 1 使用化妝品會泛紅或感到搔癢 2 高清涼感（涼爽性）的化妝品會與肌膚不合 3 含有乙醇的化妝品會與肌膚不合 4 含有香水或香料的化妝品會與肌膚不合 5 由於肌膚脆弱而慎選化妝品 6 會因按摩等物理性刺激而使肌膚的狀態變差 7 即使是在平常生活中，照到紫外線肌膚就容易泛紅、刺 痛 8出汗後會有搔癢感 9 化妝品或清潔劑會不合而造成泛紅或搔癢 10有曾因化妝品造成的肌膚問題而就醫之經驗 預測選擇「敏感」者之人類皮膚的壓力蓄積度為高， 選擇「非敏感」者之人類皮膚的壓力蓄積度為低。 (2) 肌膚的敏感性之問卷調查結果與HSP27量的關係 肌膚的敏感性之問卷調查結果與HSP27量的關係係示 於表6、第6圖。 23 323933 201235662 [表6] 問卷調查之回答 HSP27量平均值 (pg///g總蛋白質） 回答人數 (人） 非敏感 7.0 70 略為敏感 15. 7 94 敏感 18.8 18 以HSP27量10.0pg/；ag總蛋白質作為基準，若判定為 較基準值低者之人類皮膚的壓力蓄積度為低，較基準值高 者之人類皮膚的壓力蓄積度為高，則有對應於被驗者之自 我感覺之傾向。 (3) 室外活動經驗之問卷調查 讓被驗者針對室外活動經驗選擇「有」、「無」中之任 一者。 室外活動係例示如運動或園藝。 預測針對室外活動經驗而回答為「有」者之人類皮膚 的壓力蓄積度為高，回答為「無」者之人類皮膚的壓力蓄 積度為低。 (4) 室外活動經驗之問卷調查結果與HSP27量之關係 室外活動經驗之問卷調查結果與HSP27量之關係係示 於表7、第7圖。 24 323933 201235662 [表7] 問卷調查之回答 HSP27量平均值 (pg//zg總蛋白質） 回答人數 (人） 無 8. 4 103 有 16. 7 79 以HSP27量10至15pg///g總蛋白質之範圍作為基 準，若判定為較基準值低者之人類皮膚的壓力蓄積度為 低，較基準值高者之人類皮膚的壓力蓄積度為高，則有對 應於被驗者之因室外紫外線所致之壓力蓄積度的傾向。 【圖式簡單說明】 第1圖係表示紫外線照射所致之壓力負荷之蓄積會反 映於角質層之HSP27表現量增加的圖。 第2圖係表示SDS所致之低刺激之壓力負荷會反映於 人類角質層之HSP27表現量增加的圖。 第3圖係對屬於敏感性肌膚之異位性皮膚炎患者的正 常皮膚與炎症皮膚的角質層的HSP27存在量進行測定之 圖。並且，此圖係確認異位性皮膚炎患者即使於正常皮膚 亦蓄積有壓力之圖。 第4圖係表示藉由ELISA法測定健康者之各種部位皮 膚中之HSP27之結果的圖。越是在曰常中暴露於壓力下之 部位5 HSP27之測定值為越高。 第5圖係標本群組之HSP27表現量的直方圖。 第6圖係肌膚敏感性之問卷調查結果與HSP27量的關 25 323933 201235662 係。 第7圖係室外活動經驗之問卷調查結果與HSP27量的 關係。 【主要元件符號說明】 無0 26 323933Pg/V g protein As shown in the results of Tables 4 and 4, the results of HSP27 were particularly high on the face (cheek) where the pressure was high, and the hips which were less exposed were significantly lower. From the above results, it can be seen that the HSP27 system can be used as an index for evaluating the skin pressure of 19 323933 201235662. [Example 4] (Experiment 4) Hsp27 of the stratum corneum of the specimen group was measured to prepare a histogram. Test method (1) Test sample taken The 445 women who were selected by the Ik machine were used as the object, and the stratum corneum sample was taken from the cheek by the tape peeling method. The sample was taken from one site. (2) Extraction method of protein in the stratum corneum of the skin The RIPA buffer solution 5 ul was placed in the homogenization tube, and the above-mentioned tape-peeled stratum corneum test piece was placed therein, and the keratin was obtained from the tape by homogenization. The protein is extracted from the layer (Extract A). In addition, the extracted stratum corneum test piece was transferred to another homogenization tube, and filled with 1% SDS25 〇 mM Tris-HCL buffer 500 yl, and the protein was extracted from the stratum corneum remaining in the tape by homogenization. (Extract B). Extract A was used for the amount of HSP27 and the amount of stratum corneum protein, and extract B was used for the quantification of stratum corneum proteins. (3) Quantification of skin stratum corneum protein The amount of protein contained in the stratum corneum extract was quantified by BCA protein assay (manufactured by Pierce). The total amount of protein of extract A and extract B was used as the total protein amount. (4) Quantification of HSP27 by ELISA The amount of HSP27 contained in the stratum corneum extract was measured by Total Human HSP27 DuoSet ELISA kit (R&D systems 323933 20 201235662). The results were analyzed for the amount of HSP27 per unit protein mass. The frequency distribution table of the Hsp27 expression of the specimen group is shown in Table 5. The histogram shows [Table 5] The frequency distribution table data area of the HSP27 of the specimen group (5) Frequency 0. 4 0 2. 5 22 5. 0 64 7. 5 79 10. 0 51 12. 5 34 15. 0 27 17. 5 26 20. 0 25 22. 5 17 25. 0 19 27. 5 11 30. 0 15 32. 5 11 35. 0 11 37. 5 12 40. 0 4 42. 5 3 45. 0 〇47. 5 2 50.0 3 More than 50 9 Accumulated 0% 5% 19% 37% 49% 56% 62% 68% 74% 78% 82% 84% 88% 90% 93% 95% 96% 97% 97% 97% 98% 100% 323933 21 201235662 As shown in Table 5 and Figure 5, the performance of HSP27 is distributed from 0. 4pg / #g total protein to more than 50pg / / zg A wide range of total protein values. The average is 15. Opg/#g total protein. The basis for determining the amount of HSP27 expression to be large compared to the frequency distribution can be freely set. For example, the value of the average value of 15.0 pg///g or more of the total protein can be judged to be a large amount of HSP27 expression, and The cumulative frequency of the total protein of Opg//zg is about 50%, so that the value of the total protein exceeding 10.1 pg/#g can be judged as the amount of HSP27 being large. In any case, it can be judged that the greater the amount of expression of HSP27, the greater the pressure accumulation of human skin. Further, the amount of HSP27 expression in Experiment 4 was 1/10 of the amount of HSP27 expression in the cheek portion of Experiment 3, because in the protein extraction method of Experiment 4, after extracting the protein containing HSP27, 50 mM containing 1% SDS was used. Tris-HCL buffer was used to extract the protein of the stratum corneum, and compared with Experiment 3, the amount of total protein detected in Experiment 4 was increased. Experiment 3 and Experiment 4 showed no difference in the HSP27 extraction step, and the solubility of HSP27 was high, so that the step was sufficiently extracted. On the other hand, almost all of the protein was extracted by the SDS extraction method which was added in Experiment 4, but the HSP27 in the extract could not be measured. Since the extraction steps of Experiment 3 and Experiment 4 are different, the data of Experiment 3 and Experiment 4 cannot be compared, but the comparison between the data of Experiment 3 and the data of Experiment 4 can be performed. [Example 5] (Experiment 5) Comparison of the results of the questionnaire on the pressure accumulation of human skin and the amount of HSP27 22 323933 201235662 A total of 189 people were randomly selected from the specimen group of Experiment 4, and the pressure accumulation degree of human skin was implemented. Questionnaires were compared to the HSP27 performance. (1) Questionnaire on Skin Sensitivity The subject was asked to select the skin of the subject as "non-sensitive", "slightly sensitive" and "sensitive". The subject's suggestion of the following 10 sensitivities is such that the subject is judged to be non-sensitive (insensitive), slightly sensitive, and sensitive. 1 Cosmetics will be reddish or itchy 2 The cosmetics with cool blue (cool) will not be in contact with the skin 3 Cosmetics containing ethanol will not match the skin 4 Cosmetics containing perfume or fragrance will not fit with the skin 5 Care for the skin due to fragility 6 The skin condition deteriorates due to physical stimuli such as massage. 7 Even in normal life, it is easy to redden and sting when exposed to UV rays. 8 Itching after sneezing. 9 Cosmetics or detergents will not fit. It causes redness or itching. 10 There is a skin problem caused by cosmetics. The experience of medical treatment predicts that the pressure accumulation of human skin that selects "sensitive" is high, and the pressure accumulation of human skin that selects "non-sensitive" is low. (2) Relationship between the results of the questionnaire on the sensitivity of the skin and the amount of HSP27 The relationship between the results of the questionnaire on the sensitivity of the skin and the amount of HSP27 is shown in Tables 6 and 6. 23 323933 201235662 [Table 6] Questionnaire response HSP27 volume average (pg///g total protein) Number of respondents (person) Non-sensitive 7.0 70 Slightly sensitive 15. 7 94 Sensitive 18.8 18 with HSP27 amount 10.0pg/;ag When the total protein is used as a standard, if the pressure accumulation of the human skin lower than the reference value is determined to be low, and the pressure accumulation degree of the human skin higher than the reference value is high, there is a tendency corresponding to the subject's self-feeling. . (3) Questionnaire for outdoor activity experience Let the subject select either "Yes" or "None" for the outdoor activity experience. Outdoor activities are exemplified by sports or gardening. It is predicted that the pressure accumulation of human skin that is answered as "Yes" for outdoor activity experience is high, and the pressure accumulation of human skin with the answer of "None" is low. (4) Relationship between questionnaire results of outdoor activity experience and HSP27 volume The relationship between the questionnaire results of outdoor activity experience and the amount of HSP27 is shown in Tables 7 and 7. 24 323933 201235662 [Table 7] Questionnaire response HSP27 average value (pg//zg total protein) Number of respondents (person) No 8. 4 103 Yes 16. 7 79 Total amount of protein from HSP27 10 to 15 pg//g In the range of the human skin, the pressure accumulation of the human skin is lower than the reference value, and the pressure accumulation of the human skin is higher than the reference value. The tendency to accumulate pressure. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing that the accumulation of pressure load due to ultraviolet irradiation reflects an increase in the amount of HSP27 expression in the stratum corneum. Figure 2 is a graph showing the increase in the HSP27 performance of the human stratum corneum due to the low stimulation stress caused by SDS. Fig. 3 is a graph showing the amount of HSP27 present in the stratum corneum of normal skin and inflammatory skin of a patient with atopic dermatitis belonging to sensitive skin. Moreover, this figure confirms that a patient with atopic dermatitis accumulates a pressure map even in normal skin. Fig. 4 is a graph showing the results of measuring HSP27 in skins of various parts of healthy persons by ELISA. The more the site is exposed to pressure, the higher the measured value of HSP27. Figure 5 is a histogram of the HSP27 performance of the specimen group. Figure 6 is a comparison of the results of the questionnaire on skin sensitivity with the amount of HSP27 25 323933 201235662. Figure 7 shows the relationship between the results of the questionnaire survey on outdoor activities and the amount of HSP27. [Main component symbol description] None 0 26 323933