JP6898720B2 - Skin internal structure evaluation method - Google Patents
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本発明は、皮膚内部構造の評価方法に関する。 The present invention relates to a method for evaluating the internal structure of the skin.
皮膚内部の線維構造は、皮膚弾力性に大きく関わっている。皮膚内部の線維構造を評価する方法として、ｉｎ ｖｉｖｏ共焦点レーザー顕微鏡で評価する方法、免疫染色した皮膚組織の切片を光学顕微鏡で評価する方法、電子顕微鏡で評価する方法が用いられている。しかし、これらの手法では、皮膚内部の線維構造を三次元的かつタンパク特異的に評価することは困難である。 The fibrous structure inside the skin is greatly involved in skin elasticity. As a method for evaluating the fibrous structure inside the skin, a method for evaluating with an in vivo confocal laser scanning microscope, a method for evaluating a section of immunostained skin tissue with an optical microscope, and a method for evaluating with an electron microscope are used. However, with these methods, it is difficult to evaluate the fibrous structure inside the skin three-dimensionally and protein-specifically.
特許文献１には、組織を透明化し、蛍光標識し、蛍光顕微鏡などで観察する方法が記載されている。特許文献１には、皮膚、皮下組織を対象臓器とすることは記載されているが、観察した結果を解析・評価する手法については何ら開示されていない。
本発明は、皮膚内部の線維構造の状態を正確に把握し、皮膚内部構造を正しく評価することを課題とする。 An object of the present invention is to accurately grasp the state of the fibrous structure inside the skin and correctly evaluate the internal structure of the skin.
本発明は、上記課題を解決するために鋭意検討した結果、蛍光標識し、透明化した皮膚組織により皮膚内部の線維構造を三次元的かつタンパク特異的に観察することができ、フィブリリン構造により皮膚内部構造を正確に評価できることを見出してなされたものである。 As a result of diligent studies to solve the above problems, the present invention can observe the fibrous structure inside the skin three-dimensionally and protein-specifically by the fluorescently labeled and transparent skin tissue, and the skin by the fibrillin structure. It was made by finding that the internal structure can be evaluated accurately.
本発明は、具体的には次の事項を要旨とする。
１．皮膚組織透明化と免疫蛍光染色法を組み合わせることにより得られる皮膚内部構造の三次元画像を画像解析し、皮膚組織内のフィブリリン構造を数値化することを特徴とする皮膚内部構造評価方法。
２．前記フィブリリン構造の長さ、径、直線性のいずれか１以上を数値化することを特徴とする１．に記載の皮膚内部構造評価方法。
３．前記数値化された値を、皮膚線維の老化度の指標とすることを特徴とする１．または２．に記載の皮膚内部構造評価方法。
Specifically, the gist of the present invention is as follows.
1. 1. A method for evaluating the internal structure of the skin, which comprises analyzing a three-dimensional image of the internal structure of the skin obtained by combining the clearing of the skin tissue and the immunofluorescence staining method and quantifying the fibrilline structure in the skin tissue.
2. 1. It is characterized in that any one or more of the length, diameter, and linearity of the fibrillin structure is quantified. The method for evaluating the internal structure of the skin according to.
3. 3. 1. The quantified value is used as an index of the degree of aging of skin fibers. Or 2. The method for evaluating the internal structure of the skin according to.
本発明の皮膚内部構造評価方法は、皮膚組織透明化と免疫蛍光染色法を組み合わせることにより、皮膚内部の線維構造を三次元的かつタンパク特異的に観察することができる。皮膚真皮内部の線維構造としては、膠原線維、弾性線維が知られ、膠原線維は、Ｉ型コラーゲンを主成分とし、弾性線維は、骨格線維としてのマイクロフィブリル、フィブリリン、均質物質としてエラスチンから構成され、さらに細胞外基質として、フィブロネクチンなどの糖タンパクやプロテオグリカンを主成分とする基質が存在する。これらの中で、フィブリリン構造を画像処理、数値化して得られる変数により、皮膚内部構造を正しく評価することができる。フィブリリン構造は、長さ、径、直線性について、年齢による差異が認められるため、フィブリリン構造を老化度、肌年齢の指標とすることができる。 In the skin internal structure evaluation method of the present invention, the fibrous structure inside the skin can be observed three-dimensionally and protein-specifically by combining skin tissue clearing and immunofluorescence staining. Collagen fibers and elastic fibers are known as the fibrous structures inside the dermis of the skin. Collagen fibers are mainly composed of type I collagen, and elastic fibers are composed of microfibrils and fibrils as skeletal fibers and elastin as a homogeneous substance. Furthermore, as extracellular matrix, there are substrates containing glycoproteins such as fibrillintin and proteoglycan as main components. Among these, the internal structure of the skin can be correctly evaluated by the variables obtained by image processing and quantifying the fibrillin structure. Since the fibrillin structure has a difference in length, diameter, and linearity depending on the age, the fibrillin structure can be used as an index of aging degree and skin age.
本発明は、皮膚組織透明化と免疫蛍光染色法を組み合わせることにより得られる皮膚内部構造の三次元画像を画像解析し、皮膚組織内のフィブリリン構造を数値化し、この数値に基づいて皮膚内部構造を評価する方法に関する。 The present invention analyzes a three-dimensional image of the internal skin structure obtained by combining skin tissue clearing and immunofluorescence staining, quantifies the fibrilline structure in the skin tissue, and determines the internal skin structure based on this numerical value. Regarding the method of evaluation.
皮膚組織は、表面から順に表皮、真皮、皮下組織により構成される。真皮は、柔軟性と弾性に富んだ組織であり、場所によって異なるが１〜２ｍｍの厚さを有する。真皮は、主に細胞と細胞外マトリックス（線維と基質）とから構成される。細胞外マトリックスの主成分は膠原線維であり、その他に弾性線維、フィブロネクチンなどの糖タンパクやプロテオグリカンを主成分とする基質等が含まれる。 The skin tissue is composed of epidermis, dermis, and subcutaneous tissue in this order from the surface. The dermis is a flexible and elastic tissue with a thickness of 1-2 mm, depending on the location. The dermis is mainly composed of cells and extracellular matrix (fibers and substrates). The main component of the extracellular matrix is collagen fibers, and in addition, elastic fibers, glycoproteins such as fibronectin, and substrates containing proteoglycan as the main component are included.
フィブリリンは、弾性繊維の骨格を形成するタンパク質であり、弾性繊維の構造を形成する。
本発明の皮膚内部構造評価方法は、フィブリリン構造を数値化して得られる変数により、皮膚内部構造を評価することを特徴とする。
Fibrillin is a protein that forms the skeleton of elastic fibers and forms the structure of elastic fibers.
The skin internal structure evaluation method of the present invention is characterized in that the skin internal structure is evaluated by a variable obtained by quantifying the fibrillin structure.
＜タンパク特異的蛍光染色＞
皮膚組織内部の線維構造を特異的に可視化する手法として、皮膚弾力性線維を構成するタンパク質であるフィブリリンを抗原特異的に結合する１次抗体および１次抗体と結合し蛍光ラベル化された２次抗体を用い、抗原抗体反応による免疫蛍光染色法を用いる。
<Protein-specific fluorescent staining>
As a method for specifically visualizing the fibrous structure inside the skin tissue, a primary antibody that specifically binds fibrillin, which is a protein constituting skin elastic fibers, and a secondary antibody that binds to the primary antibody and are fluorescently labeled. An antibody is used, and an immunofluorescent staining method based on an antigen-antibody reaction is used.
＜皮膚組織の透明化＞
通常、皮膚組織は、メラニンや血液中の赤色色素を代表とする色素や脂質による吸光や組織表面および内部での散乱が発生するため、光が深部まで透過せず、皮膚内部構造の三次元的蛍光画像を取得することは困難である。そこで、光を皮膚深部まで透過させるため、透明化試薬を用いて、これらの生体色素の脱色、脂質の除去、および屈折率の均一化を行うことにより光の透過性を向上させ、皮膚組織内部の線維構造の三次元画像を取得する。
<Transparency of skin tissue>
Normally, skin tissue undergoes absorption by pigments and lipids such as melanin and red pigments in blood and scattering on the surface and inside of the tissue, so light does not penetrate deeply and the internal structure of the skin is three-dimensional. It is difficult to obtain a fluorescent image. Therefore, in order to allow light to pass deep into the skin, a clearing reagent is used to decolorize these biopigments, remove lipids, and homogenize the refractive index to improve light transmission and inside the skin tissue. Acquire a three-dimensional image of the fibrous structure of.
＜蛍光染色された組織の評価方法＞
皮膚内部の三次元画像について、画像解析ソフトウェアを用いて、２値化及び細線化処理を行い、フィブリリン構造の長さ、径、直線性を数値化し、得られた変数により皮膚内部構造を評価する。
<Evaluation method for fluorescently stained tissue>
The three-dimensional image inside the skin is binarized and thinned using image analysis software, the length, diameter, and linearity of the fibrilline structure are quantified, and the skin internal structure is evaluated using the obtained variables. ..
＜検体＞
２０代女性３名（２３、２４、２８歳）、６０代女性３名（６１、６１、６４歳）の腹部皮膚を採取し、検体とした。
検体のサイズは表皮面積がタテ約１０ｍｍ、ヨコ約１０ｍｍで、厚さは約１ｍｍである。
<Sample>
The abdominal skin of 3 women in their 20s (23, 24, 28 years old) and 3 women in their 60s (61, 61, 64 years old) was collected and used as samples.
The size of the sample has an epidermis area of about 10 mm in length and about 10 mm in width, and a thickness of about 1 mm.
＜真皮内の蛍光染色＞
エラスチン抗体、フィブリリン−１抗体を含む水溶液に検体を浸漬して、タンパク特異的に標識した後、Ａｌｅｘａ４８８、５９４（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）を２次抗体として蛍光標識した。さらに各種細胞の位置を認識するため、蛍光プローブＤＡＰＩ（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ社）を用いて、細胞核の染色を行った。
<Fluorescent staining in the dermis>
The sample was immersed in an aqueous solution containing an elastin antibody and a fibrillin-1 antibody, labeled in a protein-specific manner, and then fluorescently labeled with Alexa488,594 (Thermo Fisher Scientific) as a secondary antibody. Furthermore, in order to recognize the positions of various cells, cell nuclei were stained using a fluorescent probe DAPI (Thermo Fisher Scientific).
＜皮膚組織の透明化＞
組織抗体染色した後、Ｒａｐｉ ｃｌｅａｒ １．４９（ＳｕｎＪｉｎ Ｌａｂ社）に検体を約３０分間浸漬して組織透明化を行った。
<Transparency of skin tissue>
After staining with tissue antibody, the sample was immersed in Rapi clear 1.49 (SunJin Lab) for about 30 minutes to clear the tissue.
＜画像取得＞
線維構造を構成するタンパク質を蛍光染色し、皮膚組織の透明化をした検体を超解像型共焦点レーザー顕微鏡ＬＳＭ−７００（ＺＥＩＳＳ社製）で観察して、三次元画像を取得した。観察方向は皮膚側面を対物レンズに垂直となるよう設置し、組織側面から、深さ７０μｍ深部まで撮像した。なお、撮像部位を統一するため、画像取得の範囲はタテ３００μｍ、ヨコ３００μｍとし、表皮の上部に約２０μｍの空白の画像取得領域を設けた。すなわち、表皮から真皮方向に向けて約２８０μｍの範囲が画像取得の範囲に含まれるようにした。
励起光は、４０５、４８８、５９４ｎｍのレーザー光を用い、それぞれ４２５〜４７５ｎｍ、５００〜５３０ｎｍ、６０５〜６４５ｎｍの蛍光を検出した。励起光４０５ｎｍ、検出光４２５〜４７５ｎｍで細胞核が検出できる。励起光４８８ｎｍ、検出光５００〜５３０ｎｍでエラスチンが検出できる。励起光５９４ｎｍ、検出光６０５〜６４５ｎｍでフィブリリン−１が検出できる。励起光５９４ｎｍ、検出光６０５〜６４５ｎｍでフィブリリン−１を検出した２３歳女性、６４歳女性の皮膚内部構造の三次元画像を、それぞれ図１、２に示す。
<Image acquisition>
A three-dimensional image was acquired by observing a sample in which the proteins constituting the fibrous structure were fluorescently stained and the skin tissue was made transparent with a super-resolution confocal laser scanning microscope LSM-700 (manufactured by ZEISS). The observation direction was set so that the side surface of the skin was perpendicular to the objective lens, and images were taken from the side surface of the tissue to a depth of 70 μm. In order to unify the imaging sites, the image acquisition range was set to 300 μm in the vertical direction and 300 μm in the horizontal direction, and a blank image acquisition area of about 20 μm was provided in the upper part of the epidermis. That is, the range of about 280 μm from the epidermis to the dermis was included in the image acquisition range.
As the excitation light, laser light of 405, 488, 594 nm was used, and fluorescence of 425 to 475 nm, 500 to 530 nm, and 605 to 645 nm was detected, respectively. The cell nucleus can be detected with an excitation light of 405 nm and a detection light of 425 to 475 nm. Elastin can be detected with an excitation light of 488 nm and a detection light of 500 to 530 nm. Fibrillin-1 can be detected with an excitation light of 594 nm and a detection light of 605 to 645 nm. Three-dimensional images of the internal skin structure of a 23-year-old woman and a 64-year-old woman who detected fibrillin-1 with an excitation light of 594 nm and a detection light of 605 to 645 nm are shown in FIGS. 1 and 2, respectively.
＜画像解析＞
得られた画像に対し、画像解析ソフトウェアＩｍａｒｉｓ（ＺＥＩＳＳ社製）を用いて２値化及び細線化処理を行い以下の解析変数を数値化した。
長さ
定義：１画像内における細線化した線維の長さの総和
径
定義：１画像内における線維の半径の平均値
直線性
定義：線維始点と線維末端との間の直線距離／細線化した線維の長さ
<Image analysis>
The obtained image was binarized and thinned using image analysis software Imaris (manufactured by ZEISS), and the following analysis variables were quantified.
Length definition: Sum of the lengths of thinned fibers in one image
Diameter definition: Average fiber radius in one image
Linear definition: Linear distance between fiber start point and fiber end / length of thinned fiber
＜解析結果＞
フィブリリン構造の長さ、径、直線性に年代差が認められた。結果を図３に示す。２０代の皮膚組織のフィブリリン構造は、６０代と比べて、長く、細く、直線性が高かった。一方、フィブリリン構造の分岐の角度、体積、表面積、数、分岐の数についても画像解析ソフトウェアを用いて数値化したところ、これらについては、年代差は認められなかった。
<Analysis result>
Age differences were observed in the length, diameter, and linearity of the fibrillin structure. The results are shown in FIG. The fibrillin structure of the skin tissue in the 20s was longer, thinner and more linear than in the 60s. On the other hand, when the angle, volume, surface area, number, and number of branches of the fibrillin structure were also quantified using image analysis software, no age difference was observed for these.
＜まとめ＞
フィブリリン構造の長さ、径、直線性は、年齢による差異が認められ、これらの値が皮膚内部構造の評価に有用であることが確かめられた。フィブリリン構造の長さ、径、直線性を数値化して得られた値は、肌年齢、老化度等の指標とすることができる。また、皮膚内部構造を正確に評価することにより、個人ごとに適したスキンケアを提供することができる。
<Summary>
The length, diameter, and linearity of the fibrillin structure differed with age, confirming that these values are useful for evaluating the internal structure of the skin. The values obtained by quantifying the length, diameter, and linearity of the fibrillin structure can be used as an index of skin age, aging degree, and the like. In addition, by accurately evaluating the internal structure of the skin, it is possible to provide skin care suitable for each individual.
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