JP5399726B2 - Method for measuring lipid structure of living tissue and apparatus for evaluating lipid structure of epidermis - Google Patents
Method for measuring lipid structure of living tissue and apparatus for evaluating lipid structure of epidermis Download PDFInfo
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本発明は、角層細胞間脂質構造の評価方法及び装置に関し、具体的には、工学的手法による角層細胞間脂質構造の評価方法及び装置に関する。 The present invention relates to a method and an apparatus for evaluating the stratum corneum intercellular lipid structure, and specifically relates to a method and an apparatus for evaluating a stratum corneum intercellular lipid structure by an engineering technique.
角層は皮膚の最外層にある厚さ１０〜２０μｍの組織である。角層の細胞間脂質は、セラミド、コレステロール、脂肪酸等の化学物質からなるマルチラメラ構造を形成し、皮膚のバリア機能に関与すると考えられている。そこで皮膚のバリア機能を解明するためには、角層細胞間脂質の構造を物理学的に解析することが重要である。 The stratum corneum is a 10-20 μm thick tissue in the outermost layer of the skin. The intercellular lipids in the stratum corneum are thought to form a multilamellar structure composed of chemical substances such as ceramide, cholesterol, and fatty acids, and to be involved in the skin barrier function. Therefore, in order to elucidate the barrier function of the skin, it is important to physically analyze the stratum corneum intercellular lipid structure.
従来、角層細胞間脂質の構造の物理学的な解析方法としては、熱量測定（ＤＳＣ）法（非特許文献１）、Ｘ線回折法（非特許文献２）、赤外吸収法（非特許文献３）、ＥＳＲ法（非特許文献４）等が試みられてきた。しかしこれらの解析方法は、大型の測定機器を運転したり、高価な試薬を用いたり、サンプル調製に時間と手間がかかったりするため、スクリーニングにコストがかかり、スループットが低いという課題があった。 Conventionally, as a physical analysis method of stratum corneum intercellular lipid structure, calorimetry (DSC) method (non-patent document 1), X-ray diffraction method (non-patent document 2), infrared absorption method (non-patent document) Document 3), ESR method (Non-patent Document 4) and the like have been tried. However, these analysis methods have problems that a large measuring instrument is operated, expensive reagents are used, and time and labor are required for sample preparation, so that screening is expensive and throughput is low.
そこで、皮膚バリア機能を改善又は向上させる物質を角層細胞間脂質の構造の変化を指標として多数の候補物質の中から探索するために、安価で簡便なスループットの高い角層細胞間脂質の構造の評価方法及び装置を開発する必要がある。 Therefore, in order to search for substances that improve or improve the skin barrier function from among a large number of candidate substances using changes in the structure of stratum corneum intercellular lipids as an index, the structure of stratum corneum intercellular lipids that is inexpensive and simple and has high throughput is used. It is necessary to develop an evaluation method and apparatus.
本発明は、生体組織サンプルの全透過率を得るステップを含む、生体組織の脂質構造の評価方法を提供する。 The present invention provides a method for evaluating the lipid structure of a biological tissue, including the step of obtaining the total transmittance of the biological tissue sample.
本発明の評価方法において、前記生体組織は表皮角層の場合がある。 In the evaluation method of the present invention, the living tissue may be an epidermal stratum corneum.
本発明の評価方法において、前記生体組織サンプルは培養下で維持された表皮組織に由来する場合がある。 In the evaluation method of the present invention, the biological tissue sample may be derived from epidermal tissue maintained in culture.
本発明の評価方法において、前記生体組織サンプルは生体から採取された新鮮な表皮組織に由来する場合がある。 In the evaluation method of the present invention, the biological tissue sample may be derived from fresh epidermal tissue collected from a living body.
本発明は表皮の脂質構造の評価装置を提供する。本発明の評価装置は、白色光源と、生体組織サンプルの保持具と、集光器と、測光器とを含み、前記白色光源、前記生体組織サンプルの保持具、前記集光器及び前記測光器は、前記白色光源から発して、前記保持具に保持される前記生体組織サンプルを透過した光だけが前記集光器に導かれ、該集光器から導かれた光の強度が前記測光器によって計測されるように配置される。 The present invention provides an apparatus for evaluating the lipid structure of the epidermis. The evaluation apparatus of the present invention includes a white light source, a biological tissue sample holder, a condenser, and a photometer, and the white light source, the biological tissue sample holder, the condenser, and the photometer. Only the light emitted from the white light source and transmitted through the biological tissue sample held by the holder is guided to the condenser, and the intensity of the light guided from the condenser is measured by the photometer. Arranged to be measured.
本発明の評価装置において、前記生体組織は表皮角層の場合がある。 In the evaluation apparatus of the present invention, the living tissue may be an epidermal stratum corneum.
本発明の評価装置において、前記生体組織サンプルは培養下で維持された表皮組織に由来する場合がある。 In the evaluation apparatus of the present invention, the biological tissue sample may be derived from an epidermal tissue maintained in culture.
本発明の評価装置において、前記生体組織サンプルは生体から採取された新鮮な表皮組織に由来する場合がある。 In the evaluation apparatus of the present invention, the biological tissue sample may be derived from a fresh epidermal tissue collected from a living body.
本明細書において「生体組織」とは、いかなる種の生物由来のいかなる組織でもかまわないが、ほ乳類の皮膚組織が好ましく、ヒトの他、マウス、ラット、ブタ等の皮膚科学の研究用の実験動物の皮膚組織がより好ましい。皮膚組織は最外層の角層（角質層）から基底層までの表皮組織を含む場合があるが、角層がバリア機能の主要な担い手であるため、少なくとも角層を含むことが好ましい。 In the present specification, the “biological tissue” may be any tissue derived from any species of organism, but mammalian skin tissue is preferable, and in addition to humans, laboratory animals for dermatological research such as mice, rats, pigs, etc. More preferred is skin tissue. The skin tissue may include an epidermal tissue from the outermost stratum corneum (stratum corneum) to the basal layer. However, since the stratum corneum is a main player of the barrier function, it is preferable to include at least the stratum corneum.
本明細書において「生体組織サンプル」とは、生体から採取された新鮮な組織を調製したサンプルである場合の他、培養下で維持される組織を調製したサンプルである場合も含まれる。生体から皮膚組織、特に皮膚角層を採取するには、例えば、ガラス、樹脂等の光学的に透明な固体支持体に透明な接着剤を付着させて皮膚表面に押し当てて角層を剥離するやり方があるが、他のやり方を用いてもかまわない。培養下で維持された表皮組織から角層シートを単離するには、前記表皮組織をカルシウムイオン及びマグネシウムイオン濃度を制御した生理食塩水中でタンパク質分解酵素で処理する等の手法を用いることができる。前記角層シートは表皮組織から剥離又は単離された直後、あるいは、なんらかの処理後に全透過率を測定される場合もある。代替的には、温度及び湿度が制御可能な恒温恒湿インキュベータを用いて調製した後に全透過率を測定される場合がある。 In this specification, the “biological tissue sample” includes not only a sample prepared from fresh tissue collected from a living body but also a sample prepared from tissue maintained in culture. In order to collect skin tissue, especially the skin stratum corneum from a living body, for example, a transparent adhesive is attached to an optically transparent solid support such as glass or resin and pressed against the skin surface to peel off the stratum corneum. There are ways, but you can use other ways. In order to isolate the stratum corneum sheet from the epidermal tissue maintained in culture, a technique such as treating the epidermal tissue with a proteolytic enzyme in physiological saline with controlled calcium ion and magnesium ion concentrations can be used. . The stratum corneum sheet may be measured for total transmittance immediately after being peeled or isolated from the epidermal tissue, or after any treatment. Alternatively, the total transmittance may be measured after preparation using a constant temperature and humidity incubator with controllable temperature and humidity.
本明細書において「培養下で維持される表皮組織」とは、ヒト又は実験動物の皮膚から採取した皮膚組織か、胚性幹細胞又は成体幹細胞を試験管内で分化させて得られた皮膚組織かを重層構造を維持しながら試験管内で培養した組織をいう。特にヒトについては、ＬａｂＣｙｔｅ ＥＰＩ−ＭＯＤＥＬ（株式会社ジャパン・ティッシュ・エンジニアリング）のような商業的に入手可能なヒト3次元培養表皮モデル製品を用いる場合がある。 As used herein, “epidermal tissue maintained in culture” refers to skin tissue collected from human or laboratory animal skin, or skin tissue obtained by differentiating embryonic stem cells or adult stem cells in vitro. Tissue that has been cultured in a test tube while maintaining a multilayer structure. Particularly for humans, commercially available human three-dimensional cultured epidermis model products such as LabCyte EPI-MODEL (Japan Tissue Engineering Co., Ltd.) may be used.
本発明の表皮の脂質構造の評価装置において、白色光源とは、発光スペクトルの分布が可視領域のほぼ全域にわたっていて肉眼で白色に見えるような光の光源をいう。生体組織サンプルの保持具は、本発明の生体組織サンプルが前記白色光源からの光路を横切る位置に保持することができることを条件として、いかなる形状のものであってもかまわない。集光器は、前記生体組織サンプルを透過した光を集めて均一化して、測光器に導くことができることを条件としていかなる形状及び／又は構造のものであってもかまわない。好ましい集光器は積分球である。前記生体組織サンプルを透過した光だけが前記集光器に入射するように、前記生体組織サンプルと集光器の間に遮光体が設けられる場合がある。好ましい遮光体は暗幕である。測光器は集光器から導かれた可視光の強度を測定することができることを条件としていかなる種類の測光器であってもかまわない。好ましい測光器は光電素子である。 In the apparatus for evaluating the lipid structure of the epidermis of the present invention, the white light source refers to a light source that emits white light with the naked eye and has an emission spectrum distribution over almost the entire visible region. The biological tissue sample holder may have any shape as long as the biological tissue sample of the present invention can be held at a position crossing the optical path from the white light source. The collector may be of any shape and / or structure provided that the light transmitted through the biological tissue sample can be collected and uniformed and guided to the photometer. A preferred collector is an integrating sphere. There is a case where a light-shielding body is provided between the biological tissue sample and the collector so that only light transmitted through the biological tissue sample is incident on the collector. A preferred light shield is a black curtain. The photometer may be any type of photometer provided that it can measure the intensity of visible light guided from the condenser. A preferred photometer is a photoelectric element.
本明細書において「全透過率」とは、本発明の評価方法又は評価装置を用いて得られた生体組織サンプルを透過して測光した白色光の強度の測定値を、該生体組織サンプルのかわりにブランク状態で測光した白色光の強度の測定値で除算した百分率をいう。全透過率で表される透明度が高い角層シートは、乱反射が起こりにくいはずだから、より角層シートを構成する細胞間脂質の配向が揃っていると考えられるので、ＥＳＲ法で測定できる角層細胞間脂質秩序度と、Ｘ線解析法で測定できる脂質パッキング構造のピーク強度と相関性があると考えられる。すなわち、より全透過率の高い角層シートサンプルは角層細胞間脂質秩序度が高く、脂質パッキング構造のピーク強度も高いとの仮説を立てることができる。 In this specification, “total transmittance” refers to a measured value of the intensity of white light measured through a biological tissue sample obtained by using the evaluation method or evaluation apparatus of the present invention, instead of the biological tissue sample. The percentage divided by the measured value of the intensity of white light measured in the blank state. The stratum corneum sheet with high transparency expressed by the total transmittance should be less likely to cause irregular reflection, so the orientation of the intercellular lipids constituting the stratum corneum sheet is considered to be more uniform, so the stratum corneum that can be measured by the ESR method It is considered that there is a correlation between the degree of intercellular lipid order and the peak intensity of the lipid packing structure that can be measured by X-ray analysis. That is, it can be hypothesized that a stratum corneum sheet sample having a higher total transmittance has a high degree of stratum corneum lipid order and a high peak intensity of the lipid packing structure.
以下の実施例によって本発明について詳細な説明を行なうが、本発明はこれらの実施例により何ら制限されるものではない。 The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.
１−１角層シートの調製
１２穴プレートの３次元ヒト表皮培養モデル（ＬａｂＣｙｔｅ ＥＰＩ−ＭＯＤＥＬ、株式会社ジャパン・ティッシュ・エンジニアリング）を、０．１％トリプシン溶液／等張リン酸緩衝液中で３７°Ｃで３０分間インキュベーションすることにより、直径約１０mmの角層シートを調製した。該角層シートを、恒温恒湿槽ＭＴＨ−２２００（三洋電機株式会社）を用いて、温度３４°Ｃ、相対湿度６０％の条件下で最長１８時間乾燥させた。乾燥時間を変えることによって、４種類の異なる水分量を備えた角層シートを調製した。
1-1 Preparation of the stratum corneum sheet A three-dimensional human epidermis culture model (LabCyte EPI-MODEL, Japan Tissue Engineering Co., Ltd.) in a 12-well plate was added in 0.1% trypsin solution / isotonic phosphate buffer solution. By incubating at ° C for 30 minutes, a stratum corneum sheet having a diameter of about 10 mm was prepared. The stratum corneum sheet was dried for a maximum of 18 hours under conditions of a temperature of 34 ° C. and a relative humidity of 60% using a thermo-hygrostat MTH-2200 (Sanyo Electric Co., Ltd.). By changing the drying time, four types of stratum corneum sheets with different moisture contents were prepared.
１−２角層全透過率の測定
図１に本発明の透明度測定装置の模式図を示す。本発明の透明度測定装置は、ＣＩＥ（国際照明委員会）標準光源Ｃ規格の白色光源と、角層サンプルの保持具（図示されない）と、角層サンプルを透過しない光の遮光体としての暗幕と、角層サンプルを透過した光を均一化する積分球と、積分球から導かれた光の強度を測定する測光器（図示されない）とを含む。１−１で説明したとおり調製された角層シートを本発明の透明度測定装置に装着し、前記光源からの白色光を照射して前記角層シートを透過した光を積分球に導いて均一化された光の強度を測定した。結果は、全透過率、すなわち、前記角層シートを透過した光の強度の測定値を、角層シートを装着しないブランク状態で前記光源からの白色光を直接積分球に導いた光の強度の測定値で除算した百分率で表した。
1-2. Measurement of total transmittance of stratum corneum FIG. 1 shows a schematic diagram of the transparency measuring apparatus of the present invention. The transparency measuring apparatus of the present invention includes a CIE (International Commission on Illumination) standard light source C standard white light source, a stratum corneum sample holder (not shown), and a black curtain as a light shield that does not transmit the stratum corneum sample. An integrating sphere for uniformizing the light transmitted through the stratum corneum sample, and a photometer (not shown) for measuring the intensity of the light guided from the integrating sphere. The stratum corneum sheet prepared as described in 1-1 is mounted on the transparency measuring apparatus of the present invention, and the white light from the light source is irradiated and the light transmitted through the stratum corneum is guided to an integrating sphere for uniformization. The intensity of the emitted light was measured. The result is the total transmittance, that is, the measured value of the intensity of the light transmitted through the stratum corneum sheet. Expressed as a percentage divided by the measured value.
１−３角層細胞間脂質秩序度の測定
１−１で説明したとおり調製された角層シートを８ｍｍ ｘ ７０ｍｍのガラスに乗せた後、スピンプローブである５−ドキシルステアリン酸（５−ＤＳＡ、Ａｌｄｒｉｃｈ）を０．０１％含む水溶液で３７°Ｃ、１時間処理した。その後角層シートを蒸留水で洗浄して過剰なスピンプローブを除去した後、シアノアクリレート系接着剤で前記ガラス板に接着させた状態で、電子スピン共鳴（ＥＳＲ）の測定を実施した。ＥＳＲの測定は、ＸバンドＥＳＲ装置ＪＥＳ−ＲＥ１Ｘ（日本電子株式会社）を用いて室温で行なった。マイクロ波出力は１０ｍＷ、磁場変調幅は０．２ｍＴ、時定数は１秒、掃引速度は８分であった。
1-3 Measurement of degree of lipid order between stratum corneum cells After placing the stratum corneum sheet prepared as described in 1-1 on a glass of 8 mm x 70 mm, 5-doxyl stearic acid (5-DSA, which is a spin probe) (Aldrich) was treated with an aqueous solution containing 0.01% at 37 ° C for 1 hour. Thereafter, the stratum corneum sheet was washed with distilled water to remove excess spin probe, and then measured for electron spin resonance (ESR) in a state where it was adhered to the glass plate with a cyanoacrylate adhesive. The measurement of ESR was performed at room temperature using an X-band ESR device JES-RE1X (JEOL Ltd.). The microwave output was 10 mW, the magnetic field modulation width was 0.2 mT, the time constant was 1 second, and the sweep speed was 8 minutes.
得られたシグナルに対して幾何学法による秩序度解析を実施した。Ｓｈｉｍｓｈｉｃｋ，Ｅ．Ｊ．ら、（Ｂｉｏｃｈｅｍｉｓｔｒｙ、１２：２３５１−２３６０、１９７３）にしたがって、角層シートに取り込まれた５−ＤＳＡのＥＳＲスペクトルの波形図から５−ＤＳＡの回転対称軸に対して平行方向及び垂直方向の超微細結合定数それぞれＡＰａｒａ及びＡＰｅｒｐを求め、これらに基づいて以下の式（１）ないし（３）からＳ値を算出した。ここで主値は、（ＡＸＸ，ＡＹＹ，ＡＺＺ）＝（０．６６，０．５５，３．４５）とした（Ｇｅ Ｍ．ら、Ｂｉｏｃｈｅｍ． Ｂｉｏｐｈｙｓ． Ａｃｔａ １０３６：２２８−３６、１９９０）。 The degree of order was analyzed by geometric method for the obtained signal. Shimick, E .; J. et al. (Biochemistry, 12: 2351-1360, 1973), the waveform diagram of the ESR spectrum of 5-DSA incorporated in the stratum corneum is shown in the direction parallel to and perpendicular to the rotational symmetry axis of 5-DSA. The fine coupling constants A Para and A Perp were obtained, and based on these, the S value was calculated from the following formulas (1) to (3). Here, the main values were (A XX , A YY , A ZZ ) = (0.66, 0.55, 3.45) (Ge M. et al., Biochem. Biophys. Acta 1036: 228-36, 1990). ).
１−４結果
図２は、水分含有量の異なる４種類の角層シートの全透過率と角層細胞間脂質秩序度との関係を示すグラフである。図２の横軸は角層全透過率（単位：％）で、縦軸は角層細胞間脂質秩序度であり、各プロットからの横軸及び縦軸方向の誤差棒の長さは、それぞれの角層シートの全透過率及び角層細胞間脂質秩序度の標準偏差を示す。図２に示すとおり、角層シートの全透過率が約６８％から約８８％まで増加するのに伴い、角層細胞間脂質秩序度は約０．５１から約０．５７まで増加し、角層シートの全透過率とＳ値との間には正の相関が見出された。
1-4 Results FIG. 2 is a graph showing the relationship between the total transmittance of four types of stratum corneum sheets with different water contents and the degree of stratum corneum lipid order. The horizontal axis in FIG. 2 is the stratum corneum total transmittance (unit:%), the vertical axis is the stratum corneum lipid order, and the length of error bars in the horizontal and vertical directions from each plot is The standard deviation of the total transmittance | permeability and the stratum corneum lipid order degree of a stratum corneum is shown. As shown in FIG. 2, as the total transmittance of the stratum corneum increases from about 68% to about 88%, the degree of stratum corneum intercellular lipid increases from about 0.51 to about 0.57. A positive correlation was found between the total transmittance of the layer sheet and the S value.
２−１角層細胞間脂質パッキング構造の解析（Ｘ線解析）
調製した角層シートについて、角層細胞間脂質パッキング構造の解析を行なった。Ｘ線光源としてフラックスが１秒間当たり光子数１０１１個で波長が１オングストロームの放射光ビームを用い、カメラ長４０ｃｍにセットした３０ｃｍ ｘ ３０ｃｍのイメージングプレートでＸ線散乱像を取得した。得られたＸ線散乱像からＯｈｔａ、Ｎ．ら（Ｃｈｅｍｉｓｔｒｙ ａｎｄ Ｐｈｙｓｉｃｓ ｏｆ Ｌｉｐｉｄｓ、１２３：１−８、２００３）に従って散乱角に対するＸ線散乱強度を計測し、脂質パッキング構造に対応する散乱ピーク強度を算出した。
2-1 Analysis of lipid packing structure between stratum corneum cells (X-ray analysis)
The prepared stratum corneum sheet was analyzed for the lipid packing structure between stratum corneum cells. An X-ray scattering image was obtained with a 30 cm × 30 cm imaging plate set at a camera length of 40 cm using a synchrotron radiation beam having a flux of 10 11 photons per second and a wavelength of 1 angstrom as an X-ray light source. From the obtained X-ray scattering image, Ohta, N. et al. (Chemistry and Physics of Lipids, 123: 1-8, 2003), the X-ray scattering intensity with respect to the scattering angle was measured, and the scattering peak intensity corresponding to the lipid packing structure was calculated.
図３は、水分含有量の異なる４種類の角層シートの全透過率と脂質パッキング構造ピーク強度との関係を示すグラフである。図３の横軸は角層全透過率（単位：％）で、縦軸は脂質パッキング構造ピーク強度であり、各プロットからの誤差棒の長さは、それぞれの角層シートの全透過率及び脂質パッキング構造ピーク強度の標準偏差の大きさを示す。図３に示すとおり、角層シートの全透過率が約６８％から約８８％まで増加するのに伴い、散乱ピーク強度は約２０から約１４５まで増加し、角層シートの全透過率と散乱ピーク強度との間には正の相関が見出された。 FIG. 3 is a graph showing the relationship between the total transmittance of four types of stratum corneum sheets having different water contents and the peak intensity of the lipid packing structure. The horizontal axis of FIG. 3 is the stratum corneum total transmittance (unit:%), the vertical axis is the lipid packing structure peak intensity, and the length of the error bar from each plot is the total transmittance of each stratum corneum sheet and The magnitude of the standard deviation of the lipid packing structure peak intensity is shown. As shown in FIG. 3, as the total transmittance of the stratum corneum sheet increases from about 68% to about 88%, the scattering peak intensity increases from about 20 to about 145, and the total transmittance and scattering of the stratum corneum sheet increase. A positive correlation was found with the peak intensity.
図２及び３に示す結果から、全透過率は角層細胞間脂質秩序度及び散乱ピーク強度と一定の対応関係があり、より全透過率の高い角層シートサンプルは角層細胞間脂質秩序度が高く、脂質パッキング構造のピーク強度も高いとの仮説が正しいことを証明することができた。したがって、全透過率の測定によって、角層細胞間脂質構造性に関する情報を与えることが明らかになった。全透過率の測定は、角層細胞間脂質秩序度及び散乱ピーク強度の測定に比べてサンプル調製が簡単で測定機器も安価であるため、角層細胞間脂質構造についての性質をより簡便に評価することができる。 From the results shown in FIGS. 2 and 3, the total transmittance has a certain correspondence relationship with the degree of stratum corneum lipid order and the scattering peak intensity, and the stratum corneum sheet sample with higher total transmittance has a stratum corneum lipid order degree. And the hypothesis that the peak intensity of the lipid packing structure is high was proved to be correct. Therefore, it was revealed that measurement of total permeability gives information on stratum corneum lipid structure. Measurement of total transmittance is simpler than sample measurement of stratum corneum intercellular lipid order and scattering peak intensity, and measurement equipment is less expensive. can do.
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