JP2007199053A - Horny layer protein detecting method and methods for evaluating epidermis turnover and skin state using same - Google Patents
Horny layer protein detecting method and methods for evaluating epidermis turnover and skin state using same Download PDFInfo
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Abstract
Description
本発明は、角層中に存在する角層蛋白質の検出方法に関するものであり、より詳細には、テープストリッピングにより角層を剥離、採取し、簡便な方法でこれを視覚化し検出する方法並びにこれを利用する表皮ターンオーバーの評価方法及び肌状態評価方法に関するものである。 The present invention relates to a method for detecting a stratum corneum protein present in the stratum corneum, and more specifically, a method for peeling and collecting the stratum corneum by tape stripping, visualizing and detecting the stratum corneum by a simple method, and this The present invention relates to an evaluation method for skin turnover and a skin condition evaluation method using a skin.
表皮は様々な種類の細胞により構成されており、角化細胞（ケラチノサイト）が大部分を占めている。このケラチノサイトが増殖し、上層に移行するに伴い、角化により最外層の角層を形成する。このような表皮の正常な角化や角層の維持には表皮細胞が重要な役割を果たしている。また、ケラチノサイトの細胞接着の異常は、角化症等の皮膚疾患原因であると言われている。更に、ニキビ、フケ、落屑等のスキントラブルは細胞接着性の亢進により角化が亢進することが原因であることが知られている。また、角層には他にも多くの角層蛋白質が存在し、これらが皮膚の老化等に関連しているとされている。 The epidermis is composed of various types of cells, and keratinocytes (keratinocytes) occupy the majority. As this keratinocyte grows and moves to the upper layer, the outermost stratum corneum is formed by keratinization. Epidermal cells play an important role in the normal keratinization and maintenance of the stratum corneum. In addition, it is said that abnormal cell adhesion of keratinocytes is a cause of skin diseases such as keratosis. Furthermore, it is known that skin troubles such as acne, dandruff and desquamation are caused by increased keratinization due to increased cell adhesion. In addition, there are many other stratum corneum proteins in the stratum corneum, and these are considered to be related to skin aging and the like.
上記した角層蛋白質としては、細胞接着蛋白、角層間物質、角層細胞構成成分等が挙げられるが。このうち、角化細胞の接着装置として代表的なものにデスモソームがある。デスモソームは、表皮細胞間及び角層細胞間の接着に関与している構造物であり、デスモソームを介する表皮細胞同士の接着はケラチン線維からなる細胞骨格と連携し、上皮組織における強固な構築が維持されるものであることが知られている（非特許文献１）。このデスモソームの構成タンパクは、デスモグレイン１、２、３、４、デスモコリン１、２、３、デスモプラキン１、２等が知られており、これら細胞接着因子、細胞骨格のいずれの構造あるいは結合能が傷害されても、表皮細胞骨格ネットワークは崩壊し、水疱症を生じるものである（非特許文献２）。 Examples of the stratum corneum protein include cell adhesion proteins, stratum corneum substances, and stratum corneum cell constituents. Of these, desmosomes are typical keratinocyte adhesion devices. Desmosomes are structures that are involved in the adhesion between epidermal cells and stratum corneum cells, and the adhesion between epidermal cells via desmosomes is linked to the cytoskeleton composed of keratin fibers, maintaining a strong structure in epithelial tissues. It is known that this is done (Non-Patent Document 1). The desmosome constituent proteins are known as desmogleins 1, 2, 3, 4, desmocollins 1, 2, 3, desmoplakin 1, 2, etc., and any structure or binding ability of these cell adhesion factor and cytoskeleton is known. Even if injured, the epidermal cytoskeleton network collapses and causes blistering (Non-patent Document 2).
これらデスモソームの構成タンパクの中でも、特にデスモグレイン１が角層デスモソーム成分の蛋白分解と細胞剥離との間に密接な相関関係がある。このため、角層のデスモグレインの存在状態を確認することは、デスモソーム機能を評価することでもあり、皮膚の状態を評価する上で極めて重要である。このため、従来より様々な評価方法が提案されている。具体的には、顕微鏡による組織観察や、角層に存在するプロテアーゼ類の作用からキモトリプシン様酵素およびトリプシン様酵素の活性を評価する方法（特許文献１参照）等が挙げられる。 Among these constituent proteins of desmosome, especially desmoglein 1 has a close correlation between proteolysis of the stratum corneum desmosome component and cell detachment. For this reason, confirming the presence state of desmoglein in the stratum corneum is also an evaluation of the desmosome function, and is extremely important in evaluating the skin state. For this reason, various evaluation methods have been conventionally proposed. Specific examples include a method of evaluating the activity of chymotrypsin-like enzyme and trypsin-like enzyme from the observation of a structure with a microscope and the action of proteases present in the stratum corneum (see Patent Document 1).
しかし、従来の評価方法では、角層細胞の接着機能またはそれらの剥離機能がどのような指標によれば正確に評価できるがについては、明確になっておらず、特許文献１記載の方法は、デスモグレインの残量を測定するため、簡便な方法ではない上、角層でデスモグレインがどのように存在しているのかを示すことはできなかった。同様、他の角層蛋白質についても、簡単にその存在状態を示す方法は知られていなかった。
従って、デスモグレインをはじめとする角層蛋白質の発現ないしは存在状態を簡便に検出する方法を見出し、これに基づいた皮膚評価方法の開発が求められていた。 Accordingly, there has been a demand for the development of a skin evaluation method based on the discovery of a method for simply detecting the expression or presence of stratum corneum proteins such as desmoglein.
上記実情に鑑み、本発明者らは鋭意検討した結果、表皮から角層をテープストリッピングにより剥離し、これを蛍光抗体法により染色することにより角層中の角層蛋白質を視覚化して検出できることを見出した。そして、同一部位の角層をテープストリッピングにより複数剥離し、これを蛍光抗体法により染色することにより角層中のデスモグレインの存在位置を検出することができること、更にはこの方法は、角層中の角層蛋白質の存在位置を指標とする表皮ターンオーバーの評価方法及び肌状態評価方法に利用できることを見出し、本発明を完成した。 In view of the above situation, as a result of intensive studies, the present inventors have found that the horny layer protein in the horny layer can be visualized and detected by peeling the horny layer from the epidermis by tape stripping and staining this with the fluorescent antibody method. I found it. Then, the stratum corneum at the same site is peeled off by tape stripping, and the presence of desmoglein in the stratum corneum can be detected by staining this with a fluorescent antibody method. The present invention was completed by discovering that the present invention can be used in an evaluation method for skin turnover and a skin condition evaluation method using the existing position of the stratum corneum protein as an index.
すなわち本発明は、テープストリッピングにより皮膚から角層を剥離、採取し、採取した角層試料を蛍光抗体法により染色することを特徴とする角層蛋白質の検出方法を提供するものである。 That is, the present invention provides a method for detecting a stratum corneum protein comprising peeling and collecting the stratum corneum from the skin by tape stripping, and staining the collected stratum corneum sample by a fluorescent antibody method.
また本発明は、皮膚同一部位の角層をテープストリッピングにより複数回剥離、採取し、採取した複数の角層試料を蛍光抗体法により染色することを特徴とする角層中の角層蛋白質の存在位置の検出方法を提供するものである。 The present invention also provides the presence of a stratum corneum protein in the stratum corneum characterized in that the stratum corneum at the same site on the skin is peeled off and collected a plurality of times by tape stripping, and the collected stratum corneum samples are stained by a fluorescent antibody method. A position detection method is provided.
更に本発明は、角層における角層蛋白質の存在位置を指標とすることを特徴とする表皮ターンオーバーの評価方法および肌状態評価方法を提供するものである。 Furthermore, the present invention provides an epidermis turnover evaluation method and a skin condition evaluation method characterized by using the position of the stratum corneum protein in the stratum corneum as an index.
本発明によれば、角層中の角層蛋白質を簡便な方法で視覚化し検出することが可能となる。また、簡便に表皮ターンオーバーを評価することができる。更に、簡便に肌荒れ等の肌状態を評価することが可能となる。 According to the present invention, it becomes possible to visualize and detect horny layer proteins in the horny layer by a simple method. In addition, skin turnover can be easily evaluated. Furthermore, it is possible to easily evaluate skin conditions such as rough skin.
本発明において、測定対象となる角層蛋白質としては、細胞接着蛋白、角層間物質、角層細胞構成成分等が挙げられる。 In the present invention, the stratum corneum protein to be measured includes cell adhesion proteins, stratum corneum substances, stratum corneum cell components, and the like.
このうち、細胞接着蛋白としては、デスモグレイン１、２、３、４、デスモコリン１、２、３、コルネオデスモシン、デスモプラキン１、２、プラコグロビン、プラコフィリン等のデスモソーム構成成分、クローディン、オクルディン、ＺＯ−１、２、３、ＭＵＰＰ−１、トリセルリン、Ｅカドヘリン、Ｎカドヘリン、Ｐカドヘリン、α−カテニン、β−カテニン、ｐ１２０等を挙げることができる。 Among these, as cell adhesion protein, desmoglein 1, 2, 3, 4, desmocollin 1, 2, 3, corneodesmosine, desmoplakin 1, 2, plamoglobin, placofilin and other desmosome components, claudin, occludin, ZO-1, 2, 3, MUPP-1, Tricellulin, E cadherin, N cadherin, P cadherin, α-catenin, β-catenin, p120, and the like.
また、角層間物質としては、グルコシルセラミド、セラミド１〜９、コレステロール、遊離脂肪酸、硫酸コレステロール等の脂質類、カリクレイン５、６、７、８、１０、１１、１３、１４、プラスミン、カテプシンＤ、Ｅ、Ｇ、Ｌ、トランスグルタミナーゼ１、２、３、５、６、ペプチジルアルギニンデイミナーゼ、ステロイドスルファターゼ、リパーゼ、カスパーゼ類等の酵素類、インターロイキン（ＩＬ）１ｒａ、１α、１β、２、３、４、５、６、８、１０、トランスフォーミング成長因子（ＴＧＦ） α、β、インターフェロン（ＩＮＦ）α、β、腫瘍壊死因子（Tumor necrosis factor；ＴＮＦ）、顆粒球マクロファージ・コロニー刺激因子（Granulocyte macrophage colony stimulating factor；ＧＭ−ＣＳＦ）等のサイトカイン類や、フィラグリン、プロフィラグリン、各種リン酸化・脱リン酸化酵素、各種アミノ酸等を挙げることができる。 In addition, as the stratum corneum substance, lipids such as glucosylceramide, ceramide 1-9, cholesterol, free fatty acid, cholesterol sulfate, kallikrein 5, 6, 7, 8, 10, 11, 13, 14, plasmin, cathepsin D, E, G, L, transglutaminase 1, 2, 3, 5, 6, peptidyl arginine deiminase, steroid sulfatase, lipase, caspases and other enzymes, interleukin (IL) 1ra, 1α, 1β, 2, 3, 4, 5, 6, 8, 10, transforming growth factor (TGF) α, β, interferon (INF) α, β, tumor necrosis factor (TNF), granulocyte macrophage colony stimulating factor (Granulocyte macrophage) Cytokines such as colony stimulating factor (GM-CSF), filaggrin, pro Iragurin various phosphorylation-dephosphorylation enzymes, mention may be made of various amino acids and the like.
更に、角層細胞構成成分としては、ロリクリン、インボルクリン、スモールプロリンリッチタンパク等を挙げることができる。 Furthermore, examples of the stratum corneum cell component include loricrin, involucrin, and small proline-rich protein.
本発明方法のうち、テープストリッピングにより皮膚から角層を剥離、採取し、採取した角層試料を蛍光抗体法により染色する角層蛋白質の検出方法（以下、「第一態様発明」という）は、例えば次のようにして実施することができる。 Among the methods of the present invention, the stratum corneum is peeled off and collected from the skin by tape stripping, and the stratum corneum protein detection method for staining the collected stratum corneum sample by the fluorescent antibody method (hereinafter referred to as “first aspect invention”) is: For example, it can be carried out as follows.
第一態様発明を実施するには、まず、皮膚の皮表から角層をテープストリッピングにより剥離、採取し、角層試料とすることが必要である。このテープストリッピングは、皮膚に粘着性の透明テープ、例えば、粘着層は天然ゴムあるいはアクリル系粘着剤を主成分とし、透明フィルムがＰＥＴ、セロファン等であるテープを貼付け、テープ全面が皮膚に張り付くようにした後、該テープを皮膚から剥がすことにより、角層をサンプリングする方法である。 In order to implement the first aspect of the invention, it is first necessary to peel and collect the stratum corneum from the skin surface of the skin by tape stripping to obtain a stratum corneum sample. This tape stripping is a transparent tape that is sticky to the skin, for example, the adhesive layer is made of natural rubber or acrylic adhesive, and the transparent film is made of PET, cellophane, etc. Then, the stratum corneum is sampled by peeling the tape from the skin.
このテープストリッピングにおいては、１回の操作でテープの粘着性により剥離される角層細胞は、通常１層となるので、テープストリッピングの回数により剥離、取得できる角層細胞の深さが決まる。すなわち、この操作を複数回繰り返すことにより、角層を上層から内部（深さ）方向にかけて段階的に角層細胞をサンプリングすることができる。 In this tape stripping, the stratum corneum cells that are peeled off due to the adhesiveness of the tape in one operation are usually one layer. Therefore, the depth of the stratum corneum cells that can be peeled off and obtained is determined by the number of times of tape stripping. That is, by repeating this operation a plurality of times, stratum corneum cells can be sampled step by step from the upper layer to the inner (depth) direction.
次に、テープに写し採った角層細胞に存在する角層蛋白質を、周知の蛍光抗体法により染色、検出する。この蛍光抗体法による染色は、例えば、テープストリッピングにより得た、角層が付着した透明テープをリン酸緩衝生理食塩水（ＰＢＳ）等で洗浄し、次いでこれに蛍光標識された目的角層蛋白質に対する抗体を所定時間、室温等適当な温度にて反応させるか、上記透明テープを洗浄後、目的角層蛋白質に対する抗体を反応させ、次いで、標識されたこの抗体に対する抗体を反応させることにより行われる。蛍光抗体法による染色の好ましい例としては、一次抗体として目的とする角層蛋白質（例えば、デスモグレイン）に対するマウスＩｇＧ抗体を用い、二次抗体に蛍光標識抗マウスＩｇＧ抗体を用いることにより、免疫蛍光染色する方法が挙げられる。 Next, stratum corneum proteins present in stratum corneum cells copied on the tape are stained and detected by a well-known fluorescent antibody method. This fluorescent antibody method is performed by, for example, washing a transparent tape with a stratum corneum obtained by tape stripping with phosphate buffered saline (PBS) or the like, and then subjecting this to fluorescently labeled target stratum corneum protein. The reaction is carried out by reacting the antibody for a predetermined time at an appropriate temperature such as room temperature, or by reacting the antibody against the target stratum corneum protein after washing the transparent tape and then reacting the antibody against the labeled antibody. As a preferable example of staining by the fluorescent antibody method, a mouse IgG antibody against a target stratum corneum protein (for example, desmoglein) is used as a primary antibody, and a fluorescent-labeled anti-mouse IgG antibody is used as a secondary antibody. The method of dyeing is mentioned.
なお、上記において使用する抗体は、モノクローナル抗体であっても、ポリクローナル抗体であっても良く、これらの組み合わせであっても良い。またこれらの目的とする角層蛋白質に対する抗体は、いずれも公知であるか成書に記載の周知な方法により、容易に得ることができるものである。また、抗体に対する蛍光標識等も周知な方法により容易に実施可能である。 The antibody used in the above may be a monoclonal antibody, a polyclonal antibody, or a combination thereof. In addition, antibodies against these target stratum corneum proteins are either known or can be easily obtained by a well-known method described in the literature. In addition, fluorescent labeling for antibodies can be easily carried out by a known method.
そして、蛍光染色された試料を蛍光顕微鏡にて観察することにより、角層の形状と同時に角層蛋白質（例えば、デスモグレイン）の存在位置を蛍光により視覚化し、検出することができる。尚、得られた顕微鏡画像を画像処理し、角層蛋白質の蛍光分布を数値化して評価することも可能である。 Then, by observing the fluorescently stained sample with a fluorescence microscope, the location of the stratum corneum protein (eg, desmoglein) can be visualized and detected simultaneously with the shape of the stratum corneum. In addition, it is also possible to perform image processing on the obtained microscopic image and to evaluate the fluorescence distribution of the stratum corneum protein in numerical values.
また本発明方法のうち、皮膚同一部位の角層をテープストリッピングにより複数回剥離、採取し、採取した複数の角層試料を蛍光抗体法により染色する角層中の角層蛋白質の存在位置の検出方法（以下、「第二態様発明」という）は、上記第一態様発明を繰り返すことにより実施できるものである。 Also, in the method of the present invention, the stratum corneum at the same site in the skin is peeled and collected multiple times by tape stripping, and the location of the stratum corneum protein in the stratum corneum is detected by staining the collected plurality of stratum corneum samples by the fluorescent antibody method The method (hereinafter referred to as “second aspect invention”) can be carried out by repeating the first aspect invention.
すなわち前記したように、テープストリッピングにおいては、１回の操作でテープの粘着性により剥離される角層細胞は、通常１層であるから、例えば皮膚の同じ部分で、最初にテープストリッピングして得た角層試料（表面試料）、１０〜１５回程度テープストリッピングして得た角層試料（中間層浅部試料）、２０〜３０回程度テープストリッピングして得た角層試料（中間層深部試料）、３５〜４０回程度テープストリッピングして得た角層試料（深層部試料）の４試料を得、これらについてそれぞれ目的とする角層蛋白質を検出すれば、この角層の存在位置やその分布（例えば、何れの部分に多いかや、どの様な形状で存在しているか）が明らかとなる。無論、この方法は、上記のように４試料で行うことに限定されるものでなく、簡易的には３試料でも、正確に分布を求める場合は４試料以上で実施しても良い。 That is, as described above, in tape stripping, the stratum corneum cells that are peeled off due to the adhesiveness of the tape in a single operation are usually one layer. For example, tape stripping is first performed on the same part of the skin. Corn layer sample (surface sample), stratum corneum sample obtained by tape stripping about 10-15 times (intermediate layer shallow sample), stratum corneum sample obtained by tape stripping about 20-30 times (intermediate layer deep sample) ) If four samples of stratum corneum samples (deep layer samples) obtained by tape stripping about 35 to 40 times are obtained, and the target stratum corneum protein is detected for each of these samples, the location and distribution of the stratum corneum It becomes clear (for example, in which part it is often present and in what form it exists). Needless to say, this method is not limited to using four samples as described above, and may be performed simply with three samples or with four or more samples when the distribution is accurately obtained.
本発明の、角層における角層蛋白質の存在位置を指標とする表皮ターンオーバーの評価方法（以下、「第三態様発明」という）および角層における角層蛋白質の存在位置を指標とする肌状態評価方法（以下、「第四態様発明」という）は、上記第二態様発明を利用することにより容易に実施することができる。 Epidermal turnover evaluation method (hereinafter referred to as “third aspect invention”) using the present position of stratum corneum protein in the stratum corneum as an index, and skin condition using the present position of stratum corneum protein in the stratum corneum as an index The evaluation method (hereinafter referred to as “fourth aspect invention”) can be easily implemented by utilizing the second aspect invention.
すなわち、複数のパネルについて、特定の角層蛋白質の存在位置と表皮ターンオーバーの関係を調べた後、評価すべき対象者の角層蛋白質の存在位置を調べ、これにより表皮ターンオーバーを評価すればよい。例えば、角層蛋白質としてデスモグレインを選択し、この存在位置を指標とすることにより、表皮ターンオーバーを評価する方法について説明すれば次の通りである。 That is, after investigating the relationship between the location of specific horny layer proteins and epidermal turnover for multiple panels, the location of the subject's horny layer proteins to be evaluated is examined, and the epidermal turnover can be evaluated accordingly. Good. For example, a method for evaluating epidermis turnover by selecting desmoglein as a stratum corneum protein and using this location as an index is as follows.
表皮ターンオーバーは、角層を深さ方向に調べることで、これが正常な状態にあるか否かを知ることができる。従って、まず、前記第二態様発明により、角層のどの位置にデスモグレインが存在しているかを調べ、これから角化、ひいては表皮ターンオーバーがどの状態にあるかを評価することができる。そして、例えば、デスモグレインが、主に角層深層部試料においては細胞全面に存在しているが、角層表面試料では細胞辺縁部に存在する場合は、角化が良好な状態にあり、皮膚のターンオーバーが適切に行われていると評価できる。一方、デスモグレインが、角層表面試料においても細胞全面に存在する場合は、角化は良好でない状態にあり、皮膚のターンオーバーが適切に行われていないと評価することができる。また、テープストリッピングする部位を変え、例えば、顔面と上腕内部とで比較することにより、皮膚の部位による表皮ターンオーバーの状態を比較することができる。 Skin turnover can be determined by examining the stratum corneum in the depth direction to determine whether it is in a normal state. Therefore, first, according to the second aspect of the invention, it is possible to examine in which position of the stratum corneum the desmoglein is present, and from this, it is possible to evaluate in which state the keratinization and thus the epidermis turnover is present. And, for example, desmoglein is present on the entire cell surface mainly in the stratum corneum deep layer sample, but in the stratum corneum surface sample, if it is present on the cell edge, the keratinization is in a good state, It can be evaluated that the skin has been turned over properly. On the other hand, when desmoglein is present on the entire cell surface even in the stratum corneum surface sample, it can be evaluated that the keratinization is not good and the skin is not turned over properly. Moreover, the state of the epidermis turnover by the part of the skin can be compared by changing the part where the tape stripping is performed, for example, by comparing the face and the upper arm.
また、肌状態は、角層の最外層の状態で評価できる、すなわち、第四態様発明は第一態様発明により角層蛋白質を検出し、その存在量を検出し、この存在量から肌状態を評価するものである。例えば、経表皮水分蒸散量（ＴＥＷＬ）や角層水分量、油分量は角層の状態あるいは肌状態を表すパラメーターとしてよく知られているものであるが、本発明では、これらのパラメータと、角層のデスモグレインの発現状態との間に相関性が見出された。特にＴＥＷＬと水分量に高い相関性があることが見出された。従って、第一態様発明により、例えば、デスモグレインを染色し、これが、主に角層細胞の辺縁部に存在する場合は、皮膚状態が良好であると評価できるし、逆に、デスモグレインが、主に角層細胞の全面に存在する場合は、皮膚状態が良好ではないと評価することができる。 In addition, the skin condition can be evaluated in the state of the outermost layer of the stratum corneum, that is, the fourth aspect invention detects the stratum corneum protein according to the first aspect invention, detects its abundance, and determines the skin condition from this abundance. It is something to evaluate. For example, transepidermal water transpiration (TEWL), stratum corneum water content, and oil content are well known as parameters representing the stratum corneum state or skin state. A correlation was found between the expression state of the layer's desmoglein. In particular, it has been found that there is a high correlation between TEWL and water content. Therefore, according to the first aspect of the invention, for example, when desmoglein is stained and is present mainly in the peripheral part of the horny layer cells, it can be evaluated that the skin condition is good, and conversely, desmoglein is When it exists mainly on the entire surface of the stratum corneum, it can be evaluated that the skin condition is not good.
すなわち、ニキビ、フケ、日焼けによる落屑等のスキントラブルは、細胞接着性の亢進による角層の重層化が原因とされるのであるから、角層のどの位置にデスモグレインが存在しているかを指標とすることにより、肌の荒れ具合、落屑状態等を評価することができるのである。また、テープストリッピングする部位を変え、例えば、顔面と上腕内部とで比較することにより、皮膚の部位による肌状態が異なることが確認できる。 In other words, skin troubles such as acne, dandruff, and tanning are caused by stratification of the stratum corneum due to increased cell adhesion, so it is an indicator of where in the stratum corneum desmoglein is present. By doing so, it is possible to evaluate the roughness of the skin, the desquamation state, and the like. Moreover, it can confirm that the skin state by a skin site | part differs by changing the site | part which carries out a tape stripping, for example by comparing with a face and the upper arm inside.
以下、実施例を挙げ、本発明をさらに具体的に説明するが、本発明はこれら実施例に何ら制約されるものではない。 EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further more concretely, this invention is not restrict | limited at all by these Examples.
実 施 例 １
ヒト上腕内側を用いたデスモグレインの検出：
（試料調製）
透明両面テープ（ニチバン社製）を１.８ｃｍ×２.０ｃｍの大きさに切り、これをスライドガラスに貼り付ける。そして、このスライドガラスの粘着面をヒト上腕内側に軽く押し当て、テープ全面が皮膚に張り付くようにした後、皮膚から剥がして最外層の試料とした。また、新たなスライドガラスを用いて、同様に同じ部位に塗布し、テープストリッピングを２〜３９回繰り返し、中層、深部、最深部の試料を調製した。
Example 1
Detection of desmoglein using the human inner upper arm:
(Sample preparation)
A transparent double-sided tape (manufactured by Nichiban Co., Ltd.) is cut into a size of 1.8 cm × 2.0 cm, and this is attached to a slide glass. Then, the adhesive surface of this slide glass was lightly pressed against the inner side of the human upper arm so that the entire surface of the tape adhered to the skin, and then peeled off from the skin to obtain an outermost layer sample. Moreover, it applied to the same site | part using the new slide glass similarly, tape stripping was repeated 2 to 39 times, and the sample of the middle layer, the deep part, and the deepest part was prepared.
（一次抗体反応）
前記スライドガラス試料のテープ表面に抗デスモグレイン１ マウスＩｇＧモノクロナール抗体（ＰＲＯＧＥＮ社製）液を適量かけ、室温にて1時間反応させる。その後、リン酸緩衝化生理食塩水（ＰＢＳ）に浸し、未反応の抗体を洗い流す。
(Primary antibody reaction)
An appropriate amount of an anti-desmoglein 1 mouse IgG monoclonal antibody (PROGEN) solution is applied to the tape surface of the slide glass sample and allowed to react at room temperature for 1 hour. Then, it is immersed in phosphate buffered saline (PBS) to wash away unreacted antibodies.
（二次抗体反応）
前記一次抗体反応させたスライドガラス試料のテープ表面に蛍光標識抗マウスＩｇＧ抗体（Ｉｎｖｉｔｒｏｇｅｎ社製）の２００倍ＰＢＳ希釈溶液を適量かけ、室温にて１時間反応させる。その後、ＰＢＳに浸し、未反応の抗体を洗い流す。
(Secondary antibody reaction)
An appropriate amount of a 200-fold PBS diluted solution of fluorescently labeled anti-mouse IgG antibody (manufactured by Invitrogen) is applied to the tape surface of the slide glass sample subjected to the primary antibody reaction, and reacted at room temperature for 1 hour. Then, it is immersed in PBS to wash away unreacted antibodies.
（封入）
前記二次抗体反応させたスライドガラス試料の表面にマウント・クイック・アクオス（Mount Quick Aqueous；SPI SUPPLIES)を滴下し、カバーガラスで覆い観察用試料とした。
(Enclosed)
Mount Quick Aqueous (SPI SUPPLIES) was dropped on the surface of the slide glass sample reacted with the secondary antibody, and the sample was covered with a cover glass to obtain a sample for observation.
（観察）
前記包埋したスライドガラス試料をオリンパス社製の倒立型蛍光顕微鏡を用いて観察した。この観察画面を図１〜４に示した。
(Observation)
The embedded slide glass sample was observed using an inverted fluorescent microscope manufactured by Olympus. This observation screen is shown in FIGS.
（結果）
図１に示すように、角層細胞間付近を中心としたドット上の染色像が得られ、デスモグレインを視覚化し検出することができた。また、図１〜４より、角層の深い部分と表面でのデスモグレインの発現状態が変化していることが確認された。これより、表皮の上層に向かうほどデスモグレインがなくなるので、細胞接着力が弱まって、最終的に角層が剥がれていく過程を示している。このことより、本発明の方法により、表皮ターンオーバーを評価することが可能であることが示された。
(result)
As shown in FIG. 1, a stained image on a dot centered around the stratum corneum cells was obtained, and desmoglein could be visualized and detected. Moreover, it was confirmed from FIGS. 1-4 that the expression state of the desmoglein in the deep part and surface of a stratum corneum has changed. From this, desmoglein disappears toward the upper layer of the epidermis, indicating that cell adhesion is weakened and the stratum corneum is finally peeled off. From this, it was shown that the skin turnover can be evaluated by the method of the present invention.
実 施 例 ２
ヒト顔面と口唇および上腕外側を用いたデスモグレインの検出：
前記実施例１と同様に、サンプリング部位をヒト顔面と口唇、上腕外側に変えて、第１層の表皮を剥離し、一次抗体、二次抗体処理後、顕微鏡観察した。これにより得られた顔面の画像を図５、口唇の画像を図６、上腕外側を図７に示した。
Example 2
Detection of desmoglein using human face, lips and outer upper arm:
In the same manner as in Example 1, the sampling site was changed to the human face, lips, and outer upper arm, and the epidermis of the first layer was peeled off, and after microscopic observation after treatment with the primary antibody and the secondary antibody. The facial image thus obtained is shown in FIG. 5, the lip image in FIG. 6, and the outer upper arm in FIG.
（結果）
図１〜４、図５〜７より、顔面では上腕内側の深い部分のように、角層細胞全体にデスモグレインが検出された。これは、外部環境刺激を受け易い顔面は、刺激を受け難い上腕内側より角化が亢進しており、肌荒れを生じているものと考えられる。また、角層の非常に薄い口唇では、角層細胞全体から上腕内側の深い部分のように、デスモグレインが検出された。逆に、上腕外側の角層は内側に近い結果であった。
(result)
1-4 and FIGS. 5-7, desmoglein was detected in the entire stratum corneum cells like a deep part inside the upper arm on the face. This is considered to be because the face that is susceptible to external environmental stimulation is more keratinized than the inner side of the upper arm, which is difficult to receive stimulation, and the skin is rough. In the lips with a very thin stratum corneum, desmoglein was detected from the entire stratum corneum cells, as in the deep part inside the upper arm. Conversely, the stratum corneum outside the upper arm was close to the inside.
実 施 例 ３
皮膚状態と角層デスモグレインの発現状態との相関性検討：
（１）ＴＥＷＬ・水分量の測定方法（皮膚計測）
インフォームドコンセントを行った健常人ボランティアを被験者とし、次の方法で経表皮水分蒸散量（ＴＥＷＬ）および角層水分量を測定した。まず、被験者の被験部位を洗浄剤にて洗浄後、室温２２℃、湿度５０％に保たれた試験室にて１５分安静にした。次いで、デルフィン・テクノロジイズ（Delfin Technologies）社製バポメーター（Vapometer）
ＳＷＬ２ タイプにてＴＥＷＬを４回測定し、その平均値を使用した。また水分量はアイ・ビイ・エス社製皮表角層水分量測定装置ＳＫＩＣＯＮ−２００ＥＸにて４回測定し、その平均値を使用した。
Example 3
Examination of correlation between skin condition and expression state of stratum corneum desmoglein:
(1) Method for measuring TEWL / water content (skin measurement)
Using healthy volunteers who performed informed consent as subjects, transepidermal water transpiration (TEWL) and stratum corneum water were measured by the following methods. First, the test site of the subject was washed with a cleaning agent, and then rested for 15 minutes in a test room maintained at room temperature 22 ° C. and humidity 50%. Next, Delfin Technologies Vapometer
TEWL was measured 4 times with the SWL2 type, and the average value was used. The water content was measured four times with a skin surface angle layer water content measuring device SKICON-200EX manufactured by IBS, and the average value was used.
（２）画像解析方法
上記（１）にてＴＥＷＬ等を測定した被験者の測定部位から、透明両面テープ（ニチバン社製）を用いて１回テープストリッピングを行い、角層サンプルを採取した。この角層サンプルを、蛍光抗体法にてデスモグレインを染色後、これを蛍光顕微鏡に付属するＣＣＤカメラにて撮影した。この撮影した画像は、三谷商事社製画像解析ソフト、ＷｉｎＲ００Ｆにて解析を行った。具体的には顕微鏡にて撮影を行った画像において１ｍｍ２の正方形のエリアを無作為に３ヶ所抽出し、その範囲に含まれる蛍光のドット数をＷｉｎＲ００Ｆにて測定し、その平均値を使用して数値化した。
(2) Image analysis method From the measurement site | part of the test subject who measured TEWL etc. in said (1), tape stripping was performed once using the transparent double-sided tape (made by Nichiban), and the stratum corneum sample was extract | collected. This stratum corneum sample was stained with desmoglein by a fluorescent antibody method, and then photographed with a CCD camera attached to a fluorescence microscope. This photographed image was analyzed with image analysis software, WinR00F, manufactured by Mitani Corporation. Specifically, three square areas of 1 mm 2 were randomly extracted from the image taken with a microscope, the number of fluorescent dots included in the range was measured with WinR00F, and the average value was used. And quantified.
（３）相関性検討
上記（２）にて数値化したデスモグレインの発現状態と、前記（１）で得た角層水分量、およびＴＥＷＬとの相関性を調べたところ、図８および図９に示すようにデスモグレインの発現量とこれら２つの皮膚パラメーターに高い相関性が見られた。
(3) Correlation study The correlation between the expression state of desmoglein quantified in (2) above, the stratum corneum water content obtained in (1), and TEWL was examined. As shown in Fig. 2, a high correlation was found between the expression level of desmoglein and these two skin parameters.
すなわちデスモグレインが角層細胞全面に見られ、その量が多いほど水分量は低く、ＴＥＷＬは高い（いわゆる荒れ肌）。また逆にデスモグレインが角層細胞辺縁に限局して観察され、その量が少ないほど水分量は高く、ＴＥＷＬは低い（いわゆる理想肌）ことが明らかとなった。この結果より角層内デスモグレインは皮膚のバリア形成に関与していると示唆された。また同時に本方法が角層の状態ならびにバリア状態の評価に有効であることが示された。 That is, desmoglein is found on the entire stratum corneum cells, and the greater the amount, the lower the water content and the higher the TEWL (so-called rough skin). Conversely, desmoglein was observed confined to the stratum corneum cell margin, and it was found that the smaller the amount, the higher the water content and the lower the TEWL (so-called ideal skin). This result suggests that desmoglein in the stratum corneum is involved in skin barrier formation. At the same time, it was shown that this method is effective for the evaluation of the stratum corneum and the barrier state.
実 施 例 ４
口唇からのデスモグレイン３の検出：
インフォームドコンセントを行った何人かのボランティアの口唇について、透明両面テープ（ニチバン社製）を用い、１回ストリッピングを行った後、口唇角層サンプルを得た。この口唇角層サンプルについて、抗デスモグレイン３マウスモノクローナル抗体（Pｒｏｇｅｎ社製）と、ＦＩＴＣ標識抗マウスＩｇＧ抗体を用い、実施例１と同様にデスモグレイン３の検出を行った。
Example 4
Detection of desmoglein 3 from the lips:
The lips of some volunteers who gave informed consent were stripped once using a transparent double-sided tape (manufactured by Nichiban Co., Ltd.), and a lip horny layer sample was obtained. For this lip horny layer sample, desmoglein 3 was detected in the same manner as in Example 1 using an anti-desmoglein 3 mouse monoclonal antibody (Progen) and a FITC-labeled anti-mouse IgG antibody.
そして、口唇の荒れた状態ではデスモグレイン３の発現が多く、デスモグレイン１の発現は口唇の状態には大きく影響されないことから、デスモグレイン１と３を検出し、それらの比を見ることで、口唇の荒れた状態の指標となることを見出した。 And, since the expression of desmoglein 3 is much in the rough lips, and the expression of desmoglein 1 is not greatly influenced by the state of the lips, by detecting desmogleins 1 and 3 and looking at their ratio, It has been found that it is an indicator of the rough state of the lips.
実 施 例 ５
インボルクリン（Involucrin）の検出：
実施例１と同様の方法で角層からテープストリッピングを行い、得られたテープをＰＢＳで洗浄後、これにＰＢＳで５００倍に希釈した抗インボルクリン抗体（ａｂｃａｍ社製）を１時間反応させた。その後ＰＢＳで２００倍に希釈したアレクサ・フルオル（Alexa Fluor）５４６抗マウスＩｇＧ抗体を１時間反応させて、蛍光顕微鏡観察を行った。その結果、角層細胞の全面が染色されるものとほとんど染色されない細胞が観察されることが明らかとなった（図１０）。この結果もデスモグレインの結果と同様に、角層のターンオーバー、あるいは肌状態の評価法として利用できることを示唆するものであった。
Example 5
Involucrin detection:
Tape stripping was performed from the stratum corneum in the same manner as in Example 1. The obtained tape was washed with PBS, and then reacted with an anti-involucrin antibody (abcam manufactured by Abcam) diluted 500 times with PBS for 1 hour. Thereafter, Alexa Fluor 546 anti-mouse IgG antibody diluted 200-fold with PBS was reacted for 1 hour and observed with a fluorescence microscope. As a result, it was revealed that the entire surface of the stratum corneum cells were stained and cells that were hardly stained were observed (FIG. 10). This result also suggests that it can be used as a method for evaluating the turnover of the stratum corneum or the skin condition, similar to the result of desmograin.
本発明の角層中のデスモグレインの検出方法や、表皮ターンオーバーの評価方法及び肌状態評価方法は、医薬品、化粧品の開発に用いられるばかりでなく、顧客の肌の状態を客観的に示すものであるため、顧客に適する化粧品の選択する指標にも用いることができるものである。 The method for detecting desmoglein in the stratum corneum, the method for evaluating skin turnover and the method for evaluating skin condition according to the present invention are not only used for the development of pharmaceuticals and cosmetics but also objectively indicate the condition of the customer's skin. Therefore, it can also be used as an index for selecting a cosmetic suitable for the customer.
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