JP5100481B2 - Skin stratum corneum moisture content, cholesterol content measurement method - Google Patents
Skin stratum corneum moisture content, cholesterol content measurement method Download PDFInfo
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本発明は、皮膚角層水分量、コレステロール量測定技術に関する。 The present invention relates to a technique for measuring skin stratum corneum moisture content and cholesterol content.
皮膚の状態を知る上で重要な要素として、角層の水分量、コレステロール量があり、皮膚状態評価のための指標とされている。
特許文献１（特開２００４−９７４３６号公報）には、皮膚の角層水分量が少ない場合は表皮のバリア機能が低下していると判断することができることが開示されている。角層水分量の測定方法として、「皮膚の角層に存在する水分量である角層水分量の測定方法としては、高周波電流を用いて皮膚の電気インピータンスを測定する方法、例えば３．５ＭＨｚの高周波電流を直径２ｍｍのカードリングからなる電極を通して皮膚に流してコンダクタンスを測るＳｋｉｃｏｎ−２００（ＩＢＳ社製）や電気容量から水分量を推定するＣｏｒｎｅｏｍｅｔｅｒ（Ｃｏｕｒａｇｅ＋Ｋｈａｚａｋａ Ｇｅｒｍａｎｙ社製）等を用い測定する方法、赤外線スペクトルを用いる方法、マイクロ波を用いる方法、光音響による方法等が挙げられるが、その中でも高周波電流皮表コンダクタンスを測定する方法が好ましい。[００１３]」と紹介されている。その他、皮膚の状態を把握する因子の測定として、この特許文献には、スティンギングテストにより、被験物質である皮膚刺激性化学物質の感覚刺激性を判定する試験を行い敏感肌と非敏感肌を判断する方法、経皮水分蒸散量（ＴＥＷＬ）の測定方法、重層剥離量の測定方法、共焦点レーザー顕微鏡による表皮の厚さと真皮乳頭密度の測定方法、電気刺激感受性（知覚閾値）の測定方法、神経成長因子（ＮＧＦ）産生量の測定方法などが紹介されている。
As important factors for knowing the skin condition, there are the water content of the stratum corneum and the cholesterol content, which are used as indices for evaluating the skin condition.
Patent Document 1 (Japanese Patent Application Laid-Open No. 2004-97436) discloses that it can be determined that the barrier function of the epidermis is lowered when the skin stratum corneum moisture is small. As a measuring method of the stratum corneum water content, “as a measuring method of the stratum corneum water content that is the amount of water existing in the stratum corneum of the skin, a method of measuring the electrical impedance of the skin using a high frequency current, for example, 3.5 MHz A method using Skicon-200 (manufactured by IBS) for measuring conductance by flowing a high-frequency current of 2 mm into the skin through an electrode made of a card ring having a diameter of 2 mm, Corneometer (manufactured by Courtage + Khazaka Germany) for estimating water content from electric capacity, etc. , A method using an infrared spectrum, a method using a microwave, a method using photoacoustics, and the like, among which a method of measuring high-frequency current skin conductance is preferable. [0013] In addition, as a measurement of factors that grasp the skin condition, this patent document describes a test for determining the sensory irritation of a skin-irritating chemical substance, which is a test substance, by a sting test. Method of judging, method of measuring transdermal moisture transpiration (TEWL), method of measuring amount of delamination, method of measuring epidermal thickness and dermal papillary density by confocal laser microscope, method of measuring electrical stimulation sensitivity (perception threshold), Methods for measuring the amount of nerve growth factor (NGF) production are introduced.
特許文献２（特開２００２−２６５３４７号公報）には、角層には角質細胞と細胞間脂質があって、細胞間脂質はセラミド、コレステロール 、脂肪酸などを成分としてラメラ構造を形成し、角層 バリアー機能において重要な役割を演じていることが明らかになっており、角層 バリアー機能が低下する種々の皮膚疾患や、肌荒れなどの皮膚トラブルにおいて、細胞間脂質が形態的にまた組成的にも乱れていることにより裏付けられていると、紹介されている。細胞間脂質に存在するコレステロール量が多いと肌のバリア機能に優れ、角質水分量を維持すると考えられている。 In Patent Document 2 (Japanese Patent Application Laid-Open No. 2002-265347), the stratum corneum has keratinocytes and intercellular lipids, and the intercellular lipids form a lamella structure with ceramide, cholesterol, fatty acid and the like as components, and It has been revealed that it plays an important role in the barrier function, and the stratum corneum has various forms of skin diseases such as rough skin and skin troubles such as rough skin. It is introduced that it is supported by being disturbed. It is thought that when the amount of cholesterol present in intercellular lipids is large, the skin barrier function is excellent and the amount of keratin moisture is maintained.
角層を光学顕微鏡で観察すると、角層は半透明であり、視野がぼやけるので角層剥離の重層度を評価することは困難である。そこで、一般的に角層をゲンチアナバイオレット、ブリリアントグリーンで染色した後に光学顕微鏡で観察し、角層が重なっている色の濃い部分を目視評価するか、画像解析することが行われている。しかし、染色ムラや角層ごとの染色され易さの違いにより、正確な重層度を評価することが困難であった。また、染色操作が煩雑であり、染色操作によって角層が剥離する危険性もあった。 When the stratum corneum is observed with an optical microscope, the stratum corneum is translucent and the field of view is blurred, making it difficult to evaluate the degree of stratum corneum peeling. Therefore, in general, the stratum corneum is stained with gentian violet and brilliant green, and then observed with an optical microscope, and a dark portion where the stratum corneum overlaps is visually evaluated or image analysis is performed. However, it was difficult to accurately evaluate the degree of layering due to uneven dyeing and differences in the ease of dyeing for each stratum corneum. Further, the dyeing operation is complicated, and there is a risk that the stratum corneum is peeled off by the dyeing operation.
染色せずにそのまま角質細胞を観察する方法として、特許文献３（特許第３７０９３８２号公報）には、紫外線下で顕微鏡やビデオマイクロスコープを介して撮影し、角質細胞の形状認識、及び面積測定を行った技術が開示されている。
特許文献４（特開２００６−１７６８８号公報）には、顕微鏡下、乃至は、拡大ビデオでの観察下、油浸オイルで封入されたスライドグラス上の角層細胞に、紫外線光源より紫外線を照射し、紫外線によって励起される、角層細胞の蛍光強度を指標として皮膚より採取した角層細胞のケラチンの立体構造を鑑別する方法が開示されている。具体的には、（１）粘着テープを使用し、テープストリップにより皮膚より角層細胞を採取する、（２）（１）の角層細胞の付着した粘着テープを、そのままスライドグラスに貼り付け、有機溶剤（キシレン等）中で一夜浸漬し、テープの粘着剤を軟化及び／又は溶解させ、スライドグラス上に角層細胞標本を転移させる、（３）の標本の角層細胞数を減少させ、スライドグラス上に角層細胞数１個の標本を作成し、油浸オイルを滴下し、カバーグラスをのせて封入する、（４）（３）の標本を、拡大観察手段の元にセットし、メタルハライド光源或いはＬＥＤ光源の絞りを調節しながら紫外線を照射し、角層細胞の自家蛍光を観察し、その画像を取り込む、（５）（４）の取り込んだ画像の輝度を解析し、その代表値を算出し、角層細胞固有の蛍光強度と推定する、（６）条件の異なる角層細胞の蛍光強度を比較し、角層細胞のケラチンの立体構造の差異を鑑別する、とされている。
特許文献５（特開２００５−３４８９９１号公報）には、皮膚より採取した角層細胞に紫外線を照射し、該紫外線によって発光する蛍光強度によって、角層細胞におけるβシート型のケラチンの存在割合の推定及び／又は皮膚柔軟性を鑑別する方法が、開示されている。
特許文献６（特許３９８０７７４号公報）には、非医療的使用を目的とする敏感肌鑑別法として、（１）年齢の異なるパネラーの角質細胞を各季節毎に採取し、これら採取した角質細胞の面積を測定し、これら角質細胞の面積の結果を回帰分析することにより、年齢を説明変数とする標準角質細胞面積を求める回帰式を各季節毎に予め用意し、（２）敏感肌か否かを鑑別する被験者に対し、その被験者の角質細胞を採取し、その角質細胞の面積を測定し、（３）上記（２）で被験者から角質細胞を採取した季節及び被験者の年齢を考慮することにより、上記（１）で得られた回帰式を用いて、被験者の年齢に対応した標準角質細胞面積を得て、（４）上記（３）で得られた標準角質細胞面積と上記（２）で得られた被験者の角質細胞面積とを比較し、上記（２）で得られた被験者の角質細胞面積が上記（３）で得られた標準角質細胞面積より５０〜２００μｍ2小さい場合に、敏感肌であると鑑別する、非医療目的の敏感肌の鑑別法が、開示されている。この角質細胞の面積測定法としては、角質細胞を粘着剤つきのディスクを用い、角質細胞を採取し、これを粘着テープに転写し、ブリリアントグリーンで染色した後、顕微鏡下角質細胞面積を測定する方法が採用されている。
As a method for observing keratinocytes as they are without staining, Patent Document 3 (Patent No. 3709382) takes photographs through a microscope or a video microscope under ultraviolet light, and recognizes the shape and area measurement of keratinocytes. The technology performed is disclosed.
Patent Document 4 (Japanese Patent Application Laid-Open No. 2006-17688) irradiates a stratum corneum cell on a slide glass sealed with oil-immersed ultraviolet light from an ultraviolet light source under observation with a microscope or enlarged video. However, a method for differentiating the three-dimensional structure of keratin in stratum corneum cells collected from the skin using the fluorescence intensity of stratum corneum cells as an index, which is excited by ultraviolet rays, is disclosed. Specifically, (1) Using an adhesive tape, the stratum corneum cells are collected from the skin with a tape strip. (2) The adhesive tape to which the stratum corneum cells (1) are attached is directly attached to a slide glass. Immerse in an organic solvent (such as xylene) overnight, soften and / or dissolve the adhesive on the tape, transfer the stratum corneum cell specimen onto the slide glass, reduce the number of stratum corneum cells in the specimen of (3), Prepare a specimen with one stratum corneum cell on a slide glass, drop oil immersion oil, put a cover glass and enclose it, set the specimen of (4) (3) under the magnification observation means, Irradiate ultraviolet light while adjusting the aperture of the metal halide light source or LED light source, observe the autofluorescence of the stratum corneum cells, capture the image, analyze the brightness of the captured image in (5) and (4), and representative values Calculate the stratum corneum cell fixation The fluorescence intensity and estimating, there is a, to distinguish the difference of (6) comparing the fluorescence intensity of different corneocytes of conditions, three-dimensional structure of the keratin of the stratum corneum cells.
Patent Document 5 (Japanese Patent Application Laid-Open No. 2005-348991) discloses that the horny layer cells collected from the skin are irradiated with ultraviolet rays, and the β-sheet type keratin is present in the horny layer cells by the fluorescence intensity emitted by the ultraviolet rays. A method for differentiating estimation and / or skin softness is disclosed.
In Patent Document 6 (Patent No. 3980774), as a sensitive skin discrimination method for non-medical use, (1) corneocytes of panelists of different ages are collected every season, and the collected corneocytes are collected. Prepare a regression equation for each season to determine the standard keratinocyte area with age as an explanatory variable by measuring the area and performing regression analysis of the results of these keratinocyte areas. (2) Whether the skin is sensitive By collecting the corneocytes of the subject, measuring the area of the corneocytes, and (3) considering the season in which the corneocytes were collected from the subject in (2) above and the age of the subject Using the regression equation obtained in (1) above, the standard keratinocyte area corresponding to the age of the subject was obtained. (4) The standard keratinocyte area obtained in (3) above and (2) above. The obtained subject's corneocyte area And compare, if keratinocyte area of a subject obtained in the above (2) is 50 to 200 [mu] m 2 less than the standard keratinocyte area obtained in the above (3), is distinguished from sensitive skin, non-medical purposes A method for distinguishing sensitive skin is disclosed. As a method for measuring the area of keratinocytes, a method of measuring keratinocyte area under a microscope after collecting the keratinocytes using a disc with an adhesive, transferring the keratinocytes to an adhesive tape, and staining with brilliant green Is adopted.
本願発明は、染色や転写などの工程を必要とすることなく簡便且つ正確に角質層の状態を知る技術を開発することを目的とする。 The object of the present invention is to develop a technique for knowing the state of the stratum corneum easily and accurately without requiring steps such as dyeing and transfer.
（１）共焦点レーザー顕微鏡を用いて、採取した角質層試料が発する自家蛍光強度を測定し、該測定データに基づいて、角質層のコレステロール量を測定する方法。
（２）波長４５０〜６００ｎｍの自家蛍光を観察することを特徴とする（１）記載の角質層のコレステロール量を測定する方法。
（３）（１）又は（２）に記載の測定方法によって得られた角質層のコレステロール量を基礎として非医療目的として皮膚状態を評価する方法。
(1) A method for measuring the autofluorescence intensity emitted from a collected stratum corneum sample using a confocal laser microscope and measuring the cholesterol level in the stratum corneum based on the measurement data.
(2), wherein the observing autofluorescence wavelengths 450 to 600 nm (1) method for measuring the cholesterol content in the stratum corneum according.
(3) (1) or (2) a method of evaluating the skin condition as a non-medical purposes on the basis of the cholesterol content of the resulting stratum corneum by the measuring method described in.
共焦点レーザー顕微鏡をもちいて剥離した角質試料を直接的に、試料が発する自家蛍光の強度を観察することにより、角質水分量及びコレステロール量を測定することができる。
スライドグラスを肌に押しつけて剥がして得られた試料を用いるので、試料の採取が障害を与えることなく容易に行うことができ、電極や溶解分析等の作業を行うこともなくそのまま共焦点レーザー顕微鏡観察によって、データが得られるので、簡便性と即時性がある。
簡便且つ客観的に正確にストリッピング試料を用いた角層の水分量やコレステロール量の状態からその人の顔や手などの皮膚の状態を知ることができるので、皮膚の状態を勘案した非医療目的とする客観的な化粧品の選定、化粧方法を選択、アドバイスすることができる。
The stratum corneum moisture content and cholesterol content can be measured by directly observing the intensity of autofluorescence emitted from the stratum corneum sample peeled off using a confocal laser microscope.
Because the sample obtained by pressing the slide glass against the skin and peeling off is used, the sample can be collected easily without causing any obstacles, and the confocal laser microscope can be used without any work such as electrodes or dissolution analysis. Since data can be obtained by observation, there is convenience and immediacy.
Non-medical taking into account the skin condition because the skin state of the person's face and hands can be known from the state of water content and cholesterol content of the stratum corneum using the stripping sample simply and objectively and accurately The objective objective cosmetics can be selected and makeup methods can be selected and advised.
被験者の頬や手、腕などの対象部位に対して、スライドグラスに樹脂製粘着剤を薄く塗布し、適当な粘着力を持った半渇き状態で皮膚に圧着し、角層を粘着採取して、共焦点レーザー顕微鏡観察用角層試料とする。共焦点レーザー顕微鏡により、自家蛍光強度データを得て、別途従来法で得た角層の水分量とコレステロール量と比較した。本発明では、自家蛍光とは蛍光誘導体化せずに角層自体が発する蛍光を意味する。自家蛍光として波長４５０〜６００ｎｍを用いることが好ましい。
共焦点レーザー顕微鏡に用いる光源としてレーザー光（固体レーザー、液体レーザー、ガスレーザー、半導体レーザー）を用いることができる。レーザーは光の収束に優れているので、レーザー光を走査して平面分解能の高い観察、測定が可能となる。従来の角層の蛍光観察、測定では隣接する角層からの反射光、蛍光が妨害するため、角層細胞が隣接しないようにばらばらにする前処理が必要であったが、本発明の共焦点レーザー顕微鏡を用いた測定では、剥離角層をそのまま観察、測定することが可能である。
本発明に用いるレーザー光の波長は４００〜４６０ｎｍである。レーザー光による照射波長を４００ｎｍ以上とすることで、蛍光の退色を防ぎ、接着剤などの剥離角層以外の物質の励起を防ぐことができる。４６０ｎｍを超えると、蛍光強度が弱まり、観察が困難となる。
検出波長範囲は、４５０〜６００ｎｍである。４５０ｎｍ未満であると、直接反射光の影響を受ける。６００ｎｍを超えると蛍光強度が弱まり、観察が困難となる。
光学断層スライス像を得るために、検体を乗せるステージが０．１〜５μｍ刻みで、垂直方向に０．１〜１０μｍ移動できる装置を用いることが好ましい。
Apply a thin resin adhesive to the slide glass on the subject's cheeks, hands, arms, etc., and apply pressure to the skin in a half-thirsty state with appropriate adhesive strength. A stratum corneum sample for confocal laser microscope observation is used. Autofluorescence intensity data was obtained with a confocal laser microscope and compared with the water content and cholesterol content of the stratum corneum obtained separately by the conventional method. In the present invention, autofluorescence means fluorescence emitted from the stratum corneum itself without being fluorescently derivatized. It is preferable to use a wavelength of 450 to 600 nm as autofluorescence.
Laser light (solid laser, liquid laser, gas laser, semiconductor laser) can be used as a light source used in the confocal laser microscope. Since the laser is excellent in light convergence, it is possible to observe and measure with high planar resolution by scanning the laser light. In conventional fluorescence observation and measurement of the stratum corneum, reflected light and fluorescence from the adjacent stratum corneum interfere with each other. Therefore, pretreatment to separate the stratum corneum cells so as not to be adjacent was necessary. In measurement using a laser microscope, it is possible to observe and measure the exfoliated stratum corneum as it is.
The wavelength of the laser beam used in the present invention is 400 to 460 nm. By setting the wavelength of irradiation with laser light to 400 nm or more, fading of fluorescence can be prevented, and excitation of substances other than the peeling angle layer such as an adhesive can be prevented. If it exceeds 460 nm, the fluorescence intensity becomes weak and observation becomes difficult.
The detection wavelength range is 450 to 600 nm. If it is less than 450 nm, it is affected by the directly reflected light. If it exceeds 600 nm, the fluorescence intensity becomes weak and observation becomes difficult.
In order to obtain an optical tomographic slice image, it is preferable to use an apparatus in which the stage on which the specimen is placed can move by 0.1 to 10 μm in the vertical direction in steps of 0.1 to 5 μm.
共焦点レーザー顕微鏡にて、角層面積が最大となる高さに共焦点を合わて、角層の画像１（図１参照）を採取した。Scion Corporation製画像解析ソフトScion Imageを用いて画像１の角層の有無を輝度の閾値により区別する。角層が存在しているとみなす閾値以上の自家蛍光（470-475nm）強度を角層が存在しているとみなした画素で平均して、角層の自家蛍光強度の測定値とする。 With a confocal laser microscope, confocal light was focused on the height at which the horny layer area was maximum, and a stratum corneum image 1 (see FIG. 1) was collected. The presence or absence of the stratum corneum in image 1 is discriminated based on the threshold of brightness using image analysis software Scion Image manufactured by Scion Corporation. The autofluorescence (470-475 nm) intensity equal to or higher than the threshold value at which the stratum corneum is present is averaged at the pixels that are regarded as having the stratum corneum, and is used as a measurement value of the autofluorescence intensity of the stratum corneum.
別途採取した角層を、従来手法を用いて角層水分量とコレステロール量を測定し、本願発明の共焦点レーザー顕微鏡を用いた測定値と比較した結果、従来手法と相関関係が認められた。共焦点レーザー顕微鏡で測定した蛍光強度値をもって、信頼性が高い角層の水分量及びコレステロール量の指標とすることができる。
コレステロールは角層細胞間脂質の構成成分の一種であり、コレステロール量が多いと皮膚のバリア機能が優れると予測できる。
The stratum corneum collected separately was measured for the moisture content and cholesterol content of the stratum corneum using the conventional method, and compared with the measured values using the confocal laser microscope of the present invention, and a correlation with the conventional method was recognized. The fluorescence intensity value measured with a confocal laser microscope can be used as a highly reliable index of the water content and cholesterol content of the stratum corneum.
Cholesterol is one of the components of stratum corneum intercellular lipids, and it can be predicted that the skin barrier function is excellent when the amount of cholesterol is large.
前腕内側部の皮膚から角層を採取して、470-475nmの自家蛍光に着目して蛍光強度を測定し、角質水分量とコレステロール量との比較検討を行った。
[共焦点レーザー顕微鏡による角層自家蛍光強度の測定 ]
１．共焦点レーザー顕微鏡観察用角層採取
スライドグラスに樹脂製粘着剤を薄く塗布し、適当な粘着力を持った半渇き状態で皮膚に圧着し、角層を粘着採取した。採取試料は、それぞれ表２、表３に示す者から提供を受けた。なお、従来例も同様である。
The stratum corneum was collected from the skin on the inner side of the forearm, and the fluorescence intensity was measured by focusing on autofluorescence at 470-475 nm, and a comparative study was made between the amount of horny water and the amount of cholesterol.
[Measurement of stratum corneum autofluorescence intensity with confocal laser microscope]
1. Extraction of stratum corneum for confocal laser microscope observation A resin adhesive was thinly applied to a slide glass, and it was pressed against the skin in a half-thirsty state with an appropriate adhesive force, and the stratum corneum was collected. The collected samples were provided by the persons shown in Table 2 and Table 3, respectively. The same applies to the conventional example.
２．共焦点レーザー顕微鏡による測定
共焦点レーザー顕微鏡（OLYMPUS FV-1000）による自家蛍光像（ここで自家蛍光とは蛍光誘導体化せずに角層自体が発する蛍光を意味する）を用いて、角層の剥離状態を観察し、データを採取した。
2. Measurement with confocal laser microscope Using autofluorescence image with confocal laser microscope (OLYMPUS FV-1000) (where autofluorescence means fluorescence emitted from the stratum corneum without being derivatized) The peeled state was observed and data was collected.
２．１ 装置
共焦点レーザー顕微鏡（OLYMPUS FV-1000）
2.1 Equipment Confocal laser microscope (OLYMPUS FV-1000)
２．２ 重層剥離度の測定
共焦点レーザー顕微鏡の条件設定を表１に示す。重層剥離となっているので、最大面積となる角層の高さ位置の画像を画像１として得る。なお、角層は重層剥離を起こしていることが多く、特に、皮膚バリア機能が低下している場合は重層剥離度が大きいと考えられているので、共焦点の高さ方向のレベルを角層の最大データが得られる位置を選択した（図１参照）。重層剥離状態を参考に画像２に示す。画像２は画像１から共焦点の位置を５μｍ高くした角層の画像である。
なお、×２０レンズの焦点深度は５．１μｍなので、画像１と画像２の画像情報はほぼ分離される。画像解析ソフトScion Imageを用いて画像１、２の角層の有無を輝度の閾値により区別した。画像１と画像２の角層面積を求め、以下の式により重層剥離度（%）を算出した。尚、1枚のスライドグラスから4箇所撮影し、測定値を平均した。画像１，２の例を図２、３に示す。
2.2 Measurement of delamination degree Table 1 shows the setting conditions of the confocal laser microscope. Since it is delamination, an image at the height position of the stratum corneum having the maximum area is obtained as image 1. In addition, the stratum corneum often causes delamination, and in particular, when the skin barrier function is deteriorated, it is considered that the delamination degree is large. The position at which the maximum data was obtained was selected (see FIG. 1). Image 2 shows the state of delamination as a reference. Image 2 is a stratum corneum image in which the confocal position is 5 μm higher than image 1.
Since the focal depth of the × 20 lens is 5.1 μm, the image information of the image 1 and the image 2 is almost separated. Using the image analysis software Scion Image, the presence or absence of the stratum corneum in the images 1 and 2 was distinguished by the threshold value of luminance. The stratum corneum areas of image 1 and image 2 were determined, and the degree of delamination (%) was calculated by the following formula. In addition, four places were photographed from one slide glass, and the measured values were averaged. Examples of images 1 and 2 are shown in FIGS.
〔数１〕
重層剥離度（％）＝（画像２の角層面積／画像１の角層面積）×１００ （式１）
[Equation 1]
Delamination degree (%) = (Area of stratum corneum of image 2 / Area of stratum corneum of image 1) × 100 (Formula 1)
[３．従来手法による角質層水分量測定とコレステロール量の測定]
３．１ 角層水分量の測定
前腕内側部を水洗し、１０分後にＩＢＳ社製ｓｋｉｃｏｎ−２００（電気伝導度測定）を用いて角層水分量を測定した。被験者は、表２に示す１２名である。
[3. Measurement of stratum corneum water content and cholesterol content by conventional methods]
3.1 Measurement of stratum corneum moisture content The inner part of the forearm was washed with water, and after 10 minutes, stratum corneum moisture content was measured using skicon-200 (electric conductivity measurement) manufactured by IBS. There are 12 subjects as shown in Table 2.
３．２ コレステロール測定用角層の採取
前腕内側部洗浄１５分後に、次のように角層の採取を行った。被験者は、表３に示す９名である。
幅１．５ｃｍ、長さ５ｃｍのセロハンテープ（ニチバン）を用いて２箇所、２回ずつ（合計４枚）角層を粘着採取した。
3.2 Collecting stratum corneum for cholesterol measurement The stratum corneum was collected as follows 15 minutes after washing the inner forearm. The test subjects are nine persons shown in Table 3.
Using a cellophane tape (Nichiban) with a width of 1.5 cm and a length of 5 cm, the stratum corneum was collected by sticking at two places twice (total of 4 sheets).
３．２．１ コレステロール量の測定
角層を採取したセロハンテープを遠沈管に入れ、酢酸エチル：メタノール＝１：４混液６ｍＬを加え、５分超音波処理した。その後、テープを取り除き、乾固させた後にクロロホルム：メタノール＝２：１混液４００μＬに溶解した。角層などを遠沈し、上清をガスクロマトグラフィーで分析し、コレステロール量を測定した。
カラム：DB-1 0.15μm×0.53mmI.D. 15m
温度 ：100℃→200℃（15℃／分）→270℃（5℃／分）
検出器：FID
キャリャーガス：He 18mL／分
3.2.1 Measurement of Cholesterol Cellophane tape from which the stratum corneum was collected was placed in a centrifuge tube, and 6 mL of a mixture of ethyl acetate: methanol = 1: 4 was added and sonicated for 5 minutes. Thereafter, the tape was removed and dried, and then dissolved in 400 μL of a chloroform: methanol = 2: 1 mixture. The stratum corneum and the like were spun down, and the supernatant was analyzed by gas chromatography to measure the amount of cholesterol.
Column: DB-1 0.15μm × 0.53mmI.D. 15m
Temperature: 100 ° C → 200 ° C (15 ° C / min) → 270 ° C (5 ° C / min)
Detector: FID
Carrier gas: He 18mL / min
[４．自家蛍光強度と角質水分量、重層剥離度の比較]
自家蛍光強度、角質水分量、重層剥離度の測定結果を表２に示す。自家蛍光強度と角質水分量のグラフを図４に示す。
自家蛍光強度と角質水分量との相関係数は０．７６６であり、高い相関関係を示した。従って、自家蛍光強度を測定することにより、肌の乾燥度を評価することができる。
自家蛍光強度と重層剥離度との相関係数は−０．０５１であり、全く相関関係が認められなかった。従って、自家蛍光強度は重層剥離度に影響されない。
[4. Comparison of autofluorescence intensity, stratum corneum moisture content, and degree of delamination]
Table 2 shows the measurement results of the autofluorescence intensity, the amount of stratum corneum, and the degree of delamination. A graph of autofluorescence intensity and keratin water content is shown in FIG.
The correlation coefficient between autofluorescence intensity and keratin water content was 0.766, indicating a high correlation. Therefore, the dryness of the skin can be evaluated by measuring the autofluorescence intensity.
The correlation coefficient between the autofluorescence intensity and the degree of delamination was -0.051, and no correlation was observed. Therefore, the autofluorescence intensity is not affected by the degree of delamination.
[５．自家蛍光強度とコレステロール量、重層剥離度の比較]
自家蛍光強度、コレステロール量、重層剥離度の測定結果を表３に示す。自家蛍光強度とコレステロール量のグラフを図５に示す。
自家蛍光強度とコレステロール量との相関係数は０．５１３であり、相関関係を示した。従って、自家蛍光強度を測定することにより、肌のコレステロール量を評価することができる。コレステロールは細胞間脂質であり、コレステロール量が多いと肌のバリア機能に優れ、角質水分量を維持すると考えられる。
自家蛍光強度と重層剥離度との相関係数は０．３９であり、相関関係が認められなかった。自家蛍光強度が重層剥離度に影響されないことを確認できた。
尚、本測定結果の自家蛍光強度の検出幅と、表２の自家蛍光強度の検出幅は大きく異なる。これは、本測定の自家蛍光強度のノイズ幅が大きく、角層の存在を識別する閾値を表２と比べて大きくし、その結果、自家蛍光強度の低い角層部分がデータから除かれたためである。従って、本測定結果を示す表３と表２の結果を直接比較することはできない。
[5. Comparison of autofluorescence intensity, cholesterol level, and degree of delamination]
Table 3 shows the measurement results of autofluorescence intensity, cholesterol content, and delamination level. A graph of autofluorescence intensity and cholesterol content is shown in FIG.
The correlation coefficient between autofluorescence intensity and cholesterol level was 0.513, indicating a correlation. Therefore, the amount of cholesterol in the skin can be evaluated by measuring the autofluorescence intensity. Cholesterol is an intercellular lipid, and if the amount of cholesterol is large, it is considered that the skin barrier function is excellent and the keratin water content is maintained.
The correlation coefficient between the autofluorescence intensity and the degree of delamination was 0.39, and no correlation was observed. It was confirmed that the autofluorescence intensity was not affected by the degree of delamination.
In addition, the detection range of the autofluorescence intensity of this measurement result and the detection range of the autofluorescence intensity of Table 2 are greatly different. This is because the noise width of the autofluorescence intensity of this measurement is large, and the threshold value for identifying the presence of the stratum corneum is increased compared to Table 2, and as a result, the stratum corneum part having low autofluorescence intensity is excluded from the data. is there. Therefore, the results of Table 3 and Table 2 showing the measurement results cannot be directly compared.
したがって、スライドグラスに付着させた剥離角層試料を直接共焦点レーザー顕微鏡を利用して、自家蛍光強度を測定することにより、角質層に含まれる水分量及びコレステロール量を測定することができる。 Therefore, the moisture content and cholesterol content contained in the stratum corneum can be measured by measuring the autofluorescence intensity of the peeled stratum corneum attached to the slide glass directly using a confocal laser microscope.
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