JP2009294202A - Differentiation method for involvement of tight junction in material transport - Google Patents
Differentiation method for involvement of tight junction in material transport Download PDFInfo
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- Medicines Containing Plant Substances (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
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Abstract
Description
本発明は、化粧料などの皮膚外用剤の生理学的作用の探求に有用な、皮膚内における物質輸送、物質分布の鑑別法に関し、更に詳細には、タイトジャンクションがどの程度に特定の物質の皮膚内における物質輸送、物質分布に関与するかを鑑別する技術に関する。 The present invention relates to a method for distinguishing substance transport and substance distribution in the skin, which is useful for exploring the physiological effects of external preparations for skin such as cosmetics, and more particularly, to what extent a tight junction is a specific substance skin. The present invention relates to a technique for discriminating whether a substance is involved in substance transport or substance distribution.
皮膚におけるバリア機能は、生体にとっての異物の生体内への侵入を防ぐ意味で重要な機能の一つとなっている。かかるバリア機能の不全は肌荒れ、炎症などの原因ともなり、その保全策は化粧料分野においては一大課題となっている。この様な皮膚バリア機能の因子としては、角層細胞の形状や接合状態に起因する角層のバリア機能、表皮・真皮の含水量などの皮膚保湿性などが挙げられ、単純なものではなく、多くの因子が絡み合っていることが既に知られている。これらの因子で近年特に注目されているのは、表皮顆粒層に存在するタイトジャンクション蛋白であり、かかるタイトジャンクション蛋白を介した細胞接合の不全に皮膚バリア機能の低下が起因すると言う説である。（例えば、特許文献１、特許文献２を参照）このタイトジャンクションの機能に注目した化粧料素材のスクリーニング法も前記の特許文献に開示されている。この方法では、タイトジャンクション機能の不全による皮膚バリア機能の低下を改善する成分を、数値として評価でき、タイトジャンクションが皮膚内の物質の移動に対して、何らかの機能を担っていることは推定させているが、皮膚機能としての物質の輸送、分布に対しての働きは明らかにしていない。この様なタイトジャンクションの物質の輸送、分布に対して担っている働きを明らかにすることは、皮膚生理において、タイトジャンクションの果たすべき役割を明確にすることにつながり、非常に有益なことと言える。
The barrier function in the skin is one of the important functions in terms of preventing foreign bodies from entering the living body. Such insufficiency of the barrier function causes rough skin, inflammation, and the like, and its conservation measures are a major issue in the cosmetics field. Such skin barrier function factors include the stratum corneum barrier function due to the shape and bonding state of the stratum corneum cells, skin moisture retention such as the water content of the epidermis and dermis, and is not simple, It is already known that many factors are intertwined. In recent years, these factors have attracted particular attention as a tight junction protein present in the epidermal granule layer, and the theory is that a decrease in skin barrier function is caused by the failure of cell junction via the tight junction protein. (See, for example,
タイトジャンクションの障害因子として、紫外線（例えば、特許文献３を参照）、カプリン酸などが存することは既に知られている（例えば、非特許文献１を参照）が、これらを用いて、培養皮膚三次元モデルのタイトジャンクションを傷害し、それが皮膚内の物質の輸送、分布にどの様な影響を与えるかについては何らの検討も存しない。 It is already known that ultraviolet rays (for example, see Patent Document 3), capric acid, and the like exist as obstacle factors for tight junctions (for example, see Non-Patent Document 1). There is no examination of how the original tight junction is damaged and how it affects the transport and distribution of substances in the skin.
本発明は、この様な状況下為されたものであり、皮膚生理学的に有用な、皮膚内の物質の輸送と分布に対してのタイトジャンクションの関与の程度を明らかにせしめる技術を提供することを課題とする。 The present invention has been made under such circumstances, and provides a technique for clarifying the degree of involvement of tight junctions in the transport and distribution of substances in the skin, which is useful in skin physiology. Is an issue.
この様な状況に鑑みて、本発明者らは、皮膚生理学的に有用な、皮膚内の物質の輸送と分布に対してのタイトジャンクションの関与の程度を明らかにせしめる技術を求めて、鋭意研究努力を重ねた結果、皮膚乃至は培養皮膚三次元モデルのタイトジャンクションを傷害処理して、タイトジャンクション傷害皮膚モデルを作製し、標識してなる皮膚関連成分を含む培地中で培養し、前記標識の分布状況を指標とすることにより、この様な課題が解決できることを見出し、発明を完成させるに至った。即ち、本発明は、以下に示す通りである。
（１）皮膚乃至は培養皮膚三次元モデルのタイトジャンクションを傷害処理して、タイトジャンクション傷害皮膚モデルを作成し、標識してなる皮膚関連成分を含む培地中で培養し、前記標識の分布状況を指標とすることを特徴とする、皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法。
（２）対照として、タイトジャンクションの傷害処理を施さない皮膚乃至は培養皮膚三次元モデルを用いることを特徴とする、（１）に記載の皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法。
（３）前記鑑別法が、角層側の培地中に放出された、標識してなる皮膚関連成分を計測することを特徴とする、請求項１又は２に記載の皮膚関連成分の輸送へのタイトジャンクションの関与の程度の鑑別法。
（４）前記皮膚乃至は皮膚三次元モデルが、培養ヒト皮膚三次元モデルであることを特徴とする、（１）〜（３）の何れか1項に記載の皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法。
（５）前記タイトジャンクションの傷害処理が、中鎖飽和直鎖脂肪酸処理であることを特徴とする、（１）〜（４）何れか１項に記載の皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法。
（６）標識してなる皮膚関連成分は、蛍光標識されたセラミドであることを特徴とする、（１）〜（５）何れか１項に記載の皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法。
In view of such a situation, the present inventors have sought for a technique for clarifying the degree of involvement of tight junctions in the transport and distribution of substances in the skin, which are useful for skin physiology. As a result of repeated efforts, the tight junction of the skin or the cultured skin three-dimensional model is injured to prepare a tight junction injured skin model, which is cultured in a medium containing a labeled skin-related component. The inventors have found that such a problem can be solved by using the distribution status as an index, and have completed the invention. That is, the present invention is as follows.
(1) Tight junction of skin or cultured skin three-dimensional model is injured, tight junction injury skin model is prepared, cultured in a medium containing skin-related components formed by labeling, and the distribution status of the label is determined. A method of distinguishing the degree of involvement of tight junctions in the distribution or transport of skin-related components, characterized by being an index.
(2) As a control, a skin or a cultured skin three-dimensional model not subjected to tight junction injury treatment is used, and the involvement of tight junctions in the distribution or transport of skin-related components according to (1) How to differentiate degree.
(3) The said discrimination method measures the skin-related component formed by labeling, which is released into the medium on the stratum corneum side, and the transport of the skin-related component according to
(4) To the distribution or transport of skin-related components according to any one of (1) to (3), wherein the skin or skin three-dimensional model is a cultured human skin three-dimensional model A method to differentiate the degree of involvement of tight junctions.
(5) The tight treatment to distribution or transport of skin-related components according to any one of (1) to (4), wherein the injury treatment of the tight junction is a medium-chain saturated linear fatty acid treatment A method of distinguishing the degree of junction involvement.
(6) The skin-related component formed by labeling is a ceramide that is fluorescently labeled, and the tight junction to the distribution or transport of the skin-related component according to any one of (1) to (5) The method of differentiation of the degree of involvement.
本発明によれば、皮膚生理学的に有用な、皮膚内の物質の輸送と分布に対してのタイトジャンクションの関与の程度を明らかにせしめる技術を提供することができる。 ADVANTAGE OF THE INVENTION According to this invention, the technique which makes clear the degree of participation of the tight junction with respect to the transport and distribution of the substance in skin useful for skin physiology can be provided.
本発明の皮膚関連成分の分布又は輸送へのタイトジャンクションの関与の程度の鑑別法は、皮膚乃至は培養皮膚三次元モデルのタイトジャンクションを傷害処理して、タイトジャンクション傷害皮膚モデルを作成し、標識してなる皮膚関連成分を含む培地中で培養し、前記標識の分布状況を指標とすることを特徴とする。前記の皮膚としては、所望により体毛を除去し、生体より採取した皮膚断片であって、角層などを除いた、表皮顆粒層、表皮基底層及び真皮部分からなるものが好ましく、マウス、ラット、モルモット、ブタ、ウサギの皮膚断片が好ましい。又、前記培養皮膚三次元モデルとしては、ヒト乃至はヒトを除く動物の皮膚より採取した、正常な（癌化していない）ケラチノサイト、フィブロブラストなどの皮膚細胞を培養し、三次元構造を構築し、皮膚の皮膚の構造に疑似させたものが好ましく、この様な形態の市販品を購入して使用することも出来る。好ましい市販品としては、例えば、倉敷紡績株式会社から販売されている「EFT-400」（正常培養ヒト三次元皮膚モデル）などが好適に例示できる。特に表皮成分のみで構成される「EPI-200」や、USA MaTek社から販売されている「EpiDerm」などがさらに好適に例示できる。かかる皮膚乃至は培養皮膚三次元モデルは、顆粒層側から傷害手段を講じてタイトジャンクション部分を傷害する。傷害手段としては、例えば、紫外線や化学物質などが好ましく例示でき、紫外線であれば、波長２８０〜３２０ｎｍの紫外線を７．５〜２００ｍJ／ｃｍ２、さらに好ましくは５０〜１６０ｍJ／ｃｍ２の単位あたりのエネルギー量で照射すれば良く、化学的な処置であれば、カプリン酸、カプリル酸などの中鎖長（炭素数８〜１２）の脂肪族飽和直鎖脂肪酸またはその一価金属塩の０．１〜１０ｍM、さらに好ましくは０．５〜２ｍMの溶液を真皮側乃至は表皮基底層側から、５〜２４時間、さらに好ましくは１０〜１５時間作用させれば良い。また、オクルディンやクローディンの細胞外ドメインを認識する中和抗体を使用することもできる。これらの傷害処置の内、特に好ましいものは化学的傷害処置であり、なかでもカプリン酸ナトリウムによる処理が特に好ましい。これはタイトジャンクションに対して均質な損傷がなしうるからである。この様な均質な傷害は、後記の傷害タイトジャンクションの回復促進剤のスクリーニングにおいては、そのメカニズムを的確に鑑別する上で非常に重要な因子となる。処置後傷害手段は直ちに皮膚乃至は培養皮膚三次元モデルより離脱させる。離脱は、紫外線照射であれば照射を終了することによりできるし、化学的傷害手段であれば、培地乃至はPBS（リン酸緩衝生理食塩水）などで洗浄することによりなしうる。斯くして得られたタイトジャンクション傷害皮膚モデルは、標識してなる皮膚関連成分を含む培地中で培養し、しかる後に、タイトジャンクション傷害皮膚モデルに取り込まれなかった標識された皮膚関連成分を洗浄などによって除去した後に、暫く培養を続け、培養上清中に放出される、標識してなる皮膚関連成分を測定したり、組織片を切り出し、標本に加工し、標識を認識しうる観察手段によって標識の分布状況を観察される。 The method for distinguishing the degree of involvement of tight junctions in the distribution or transport of skin-related components according to the present invention is to treat a tight junction of a skin or a cultured skin three-dimensional model, create a tight junction injury skin model, It is cultured in a medium containing a skin-related component, and the distribution state of the label is used as an index. The skin is preferably a skin fragment collected from a living body by removing body hair as desired, and excluding the stratum corneum, and composed of an epidermal granule layer, an epidermal basal layer, and a dermis portion, such as a mouse, rat, Guinea pig, pig and rabbit skin fragments are preferred. In addition, as the cultured skin three-dimensional model, normal (non-cancerous) keratinocytes, fibroblasts and other skin cells collected from human or non-human animal skin are cultured to construct a three-dimensional structure. Those which simulate the skin structure of the skin are preferable, and commercially available products of such a form can also be purchased and used. Preferable examples of commercially available products include “EFT-400” (normally cultured human three-dimensional skin model) sold by Kurashiki Boseki Co., Ltd. In particular, “EPI-200” composed only of an epidermis component, “EpiDerm” sold by USA MaTek, and the like can be more suitably exemplified. Such skin or cultured skin three-dimensional models injure tight junctions by taking injury means from the granule layer side. As the injury means, for example, ultraviolet rays and chemical substances can be preferably exemplified. In the case of ultraviolet rays, ultraviolet rays having a wavelength of 280 to 320 nm are per unit of 7.5 to 200 mJ / cm 2 , more preferably 50 to 160 mJ / cm 2 . In the case of chemical treatment, the saturated saturated linear fatty acid having a medium chain length (carbon number of 8 to 12) such as capric acid or caprylic acid or a monovalent metal salt thereof may be used. A solution of 1 to 10 mM, more preferably 0.5 to 2 mM, may be allowed to act for 5 to 24 hours, more preferably 10 to 15 hours from the dermis side or epidermal basal layer side. A neutralizing antibody that recognizes the extracellular domain of occludin or claudin can also be used. Among these treatments for injury, particularly preferred is a treatment for chemical injury, and treatment with sodium caprate is particularly preferred. This is because homogeneous damage can occur to tight junctions. Such homogeneous injury is a very important factor in accurately identifying the mechanism in screening for a recovery accelerator for injury tight junction described later. The post-treatment injury means is immediately removed from the skin or cultured skin three-dimensional model. Detachment can be achieved by terminating the irradiation if ultraviolet irradiation is performed, and can be performed by washing with a medium or PBS (phosphate buffered saline) or the like if chemical injury means. The tight junction injury skin model thus obtained is cultured in a medium containing a labeled skin-related component, and then the labeled skin-related component that has not been incorporated into the tight junction injury skin model is washed. After removing by the above, continue culturing for a while, measure the labeled skin-related components released into the culture supernatant, cut out the tissue piece, process it into a specimen, and label it with observation means that can recognize the label The distribution situation is observed.
ここで、前記標識してなる皮膚関連成分を構成する皮膚関連成分としては、皮膚生理学上、皮膚の生理に関与する蓋然性の高い成分であり、例えば、タイプ１〜７のセラミド、スフィンゴシン、スフィンゴミエリン、スフィンゴ糖脂質、スフィンゴ燐脂質、ホスファチジルコリン、ホスファチジルイノシトール、ホスファチジルグリセロール、ホスファチジルエタノールアミンなどの燐脂質類、コラーゲン、エラスチンなどの繊維素、αーＭＳＨ、コラゲナーゼ、エラスターゼ、グルタチオントランスフェラーゼ、グルタチオンレダクターゼ、プロテアーゼなどの酵素類が好適に例示できる。標識としては、例えば、フルオレセイン等の蛍光発色基をエステル結合やアミド結合で導入したり、アミノ基にヨウ素、テクネチウムなどの放射性同位体を導入したり、水酸基を放射性のフッ素で置換したりした放射性同位体に誘導する方法、ルシェフェラーゼやペルオキシダーゼなどの酵素とコンジュゲートを形成せしめ、基質を反応させて発色や発光をさせる方法などが好ましく例示できる。これらの標識の内、好ましいものは、検知感度及び安全性がともに高い蛍光標識である。この様な蛍光標識は、例えば、対象となる成分と蛍光標識基を有するカルボン酸とをＤＣＣ等のペプチド合成試薬の存在下反応させることにより得ることができる。又、この様な標識皮膚関連成分には市販されているものも存し、この様な市販品を購入し利用することもできる。この様な市販品としては例えば、セラミドの蛍光標識体である「BODIPY（登録商標）FL-C5-Ceramide-BSA」（Ｍｏｌｅｃｕｌａｒ Ｐｒｏｂｅ社製）が特に好ましく例示できる。 Here, the skin-related component constituting the labeled skin-related component is a component highly likely to be involved in skin physiology in terms of skin physiology. For example, type 1-7 ceramide, sphingosine, sphingomyelin , Glycosphingolipid, sphingophospholipid, phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol, phosphatidylethanolamine and other phospholipids, collagen, elastin and other fibrin, α-MSH, collagenase, elastase, glutathione transferase, glutathione reductase, protease, etc. These enzymes can be preferably exemplified. Examples of the label include a radioactive chromophore such as fluorescein introduced by an ester bond or an amide bond, a radioactive isotope such as iodine or technetium introduced into an amino group, or a hydroxyl group substituted with radioactive fluorine. Preferred examples include an isotope induction method, a method in which a conjugate is formed with an enzyme such as luciferase or peroxidase, and a substrate is reacted to develop color or emit light. Among these labels, preferred are fluorescent labels having high detection sensitivity and safety. Such a fluorescent label can be obtained, for example, by reacting a target component with a carboxylic acid having a fluorescent labeling group in the presence of a peptide synthesis reagent such as DCC. In addition, some of such labeled skin-related components are commercially available, and such commercially available products can be purchased and used. As such a commercially available product, for example, “BODIPY (registered trademark) FL-C5-Ceramide-BSA” (manufactured by Molecular Probe), which is a fluorescent label of ceramide, can be particularly preferably exemplified.
この様な標識された皮膚生理関連成分は、例えば市販の培地、「EPI-100」（倉敷紡績株式会社）などの液体培地中に１〜１０μM、好ましくは２．５〜７．５μM溶解させ、かかる培地で前記タイトジャンクション傷害皮膚モデルを０．５〜１０時間、好ましくは１〜５時間培養し、皮膚モデル中に取り込ませる。この時、培養時間のドーズを振り、輸送・分布速度の指標としても良い。斯くして、標識してなる皮膚生理関連成分をチャージしたタイトジャンクション傷害皮膚モデルは、そのまま培養を継続して培養上清中に放出される標識してなる皮膚生理関連成分を測定することができるし、所望により組織固定をして、切片などの観察標本に切り出す。観察標本は、標識検知に適した観察手段で観察される。蛍光標識であれば、顕微鏡標本に切り出し、蛍光顕微鏡下観察される。 Such a labeled skin physiology-related component is dissolved in a liquid medium such as a commercially available medium, “EPI-100” (Kurashiki Boseki Co., Ltd.), for example, 1 to 10 μM, preferably 2.5 to 7.5 μM, The tight junction injury skin model is cultured in such a medium for 0.5 to 10 hours, preferably 1 to 5 hours, and incorporated into the skin model. At this time, the dose of the culture time may be varied to serve as an index for transportation / distribution speed. Thus, the tight junction injury skin model charged with the labeled skin physiology-related component can measure the labeled skin physiology-related component released into the culture supernatant by continuing the culture as it is. Then, if desired, the tissue is fixed and cut into an observation specimen such as a section. The observation specimen is observed by an observation means suitable for detecting a sign. If it is a fluorescent label, it is cut out into a microscope specimen and observed under a fluorescent microscope.
観察に際しては、タイトジャンクションを傷害せずに、同様の処理で作成した、タイトジャンクション非傷害皮膚モデルを対照において観察することにより、タイトジャンクションの皮膚生理関連成分の輸送と分布に対する寄与が明確になり好ましい。 When observing a tight junction non-injured skin model created in the same manner without damaging the tight junction in the control, the contribution of the tight junction to the transport and distribution of skin physiology-related components becomes clear. preferable.
以下に、実施例を挙げて、本発明について更に詳細に説明を加える。 Hereinafter, the present invention will be described in more detail with reference to examples.
＜工程１＞「EPI-200」を培養プレートに移し、表皮基底層側に２００μｌの5μM 「BODIPY FL-C5-Ceramide-BSA」を含む維持培地(EPI-100 クラボウ製)を加え、３７℃、５%二酸化炭素で４時間培養した後、4℃冷蔵庫で１時間静置した。
＜工程２＞５００μｌの新鮮な維持培地に置換し、３７℃、５% CO2で２０時間培養した。
＜工程３＞３００μｌの５%脱脂牛血清アルブミンを含む維持培地に置換し、４℃で３０分間静置した。
＜工程４＞工程３を２回繰り返した。
＜工程５＞新鮮な維持培地及び1mMカプリン酸ナトリウムを含む維持培地にそれぞれ置換し、３７℃、５%二酸化炭素で培養した。
＜工程６＞培養１２時間後、1mM C10を含まない新鮮な維持培地に置換した(C10R)。経時的に培養上清の蛍光強度（励起波長４８５ｎｍ、蛍光波長５３５ｎｍ）を測定した。
<
<Step 2> The medium was replaced with 500 μl of a fresh maintenance medium, and cultured at 37 ° C. and 5% CO 2 for 20 hours.
<Step 3> The medium was replaced with a maintenance medium containing 300 μl of 5% defatted bovine serum albumin and allowed to stand at 4 ° C. for 30 minutes.
<Step 4> Step 3 was repeated twice.
<
<Step 6> After 12 hours of culture, the culture medium was replaced with a fresh maintenance medium not containing 1 mM C10 (C10R). The fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) of the culture supernatant was measured over time.
図１は培養上清の蛍光強度を測定することにより放出されたセラミドの相対量を示したグラフである。カプリン酸ナトリウムで処理していない場合（図１中Control）は時間経過とともに培養上清中に放出されるセラミドが増加していくが、１ｍMカプリン酸ナトリウム処理により（図１中C10）、セラミドの増加が妨げられた。培養５時間後にカプリン酸ナトリウムを含まない新鮮な維持培地に交換することにより（図１中C10R）、セラミドの放出量が再び増加した。 FIG. 1 is a graph showing the relative amount of ceramide released by measuring the fluorescence intensity of the culture supernatant. When not treated with sodium caprate (Control in FIG. 1), the amount of ceramide released into the culture supernatant increases with time. By treatment with 1 mM sodium caprate (C10 in FIG. 1), The increase was hindered. By changing to a fresh maintenance medium not containing sodium caprate after 5 hours of culture (C10R in FIG. 1), the amount of ceramide released increased again.
＜工程１＞「EPI-200」を培養プレートに移し、表皮基底層側に２００μｌの5μM 「BODIPY FL-C5-Ceramide-BSA」を含む維持培地（EPI-100、クラボウ製）を加え、３７℃、５%二酸化炭素で４時間培養した後、４℃冷蔵庫で１時間静置した。
＜工程２＞５００μｌの新鮮な維持培地に置換し、３７℃、５% CO2で２０時間培養した。
＜工程３＞３００μｌの５%脱脂牛血清アルブミンを含む維持培地に置換し、４℃で３０分間静置した。
＜工程４＞工程３を２回繰り返した。
＜工程５＞新鮮な維持培地及び１mMカプリン酸ナトリウムを含む維持培地にそれぞれ置換し、３７℃、５%二酸化炭素で培養した。
＜工程６＞培養１２時間後、１mMカプリン酸ナトリウムを含まない新鮮な維持培地に置換した。
＜工程７＞一定時間培養後「EPI-200」を支持体から切り離し、「OCTコンパウンド」（サクラファインテック社製）に包埋して液体窒素で凍結した。
＜工程８＞凍結切片を作製し、蛍光顕微鏡下、励起波長４７０ｎｍ/蛍光波長５２５ｎｍで観察した。
<
<Step 2> The medium was replaced with 500 μl of a fresh maintenance medium, and cultured at 37 ° C. and 5% CO 2 for 20 hours.
<Step 3> The medium was replaced with a maintenance medium containing 300 μl of 5% defatted bovine serum albumin and allowed to stand at 4 ° C. for 30 minutes.
<Step 4> Step 3 was repeated twice.
<
<Step 6> After 12 hours of culture, the culture medium was replaced with a fresh maintenance medium not containing 1 mM sodium caprate.
<Step 7> After culturing for a certain period of time, “EPI-200” was detached from the support, embedded in “OCT compound” (manufactured by Sakura Finetech), and frozen with liquid nitrogen.
<Step 8> Frozen sections were prepared and observed under a fluorescence microscope at an excitation wavelength of 470 nm / fluorescence wavelength of 525 nm.
図２はカプリン酸ナトリウム未処理、図３は１ｍMカプリン酸ナトリウム処理１２時間後、図４は１ｍMカプリン酸ナトリウム処理１２時間後、カプリン酸ナトリウムを含まない新鮮な維持培地に置換したときの「EPI-200」の凍結切片の蛍光画像である。角層（図中点線で囲まれた部分）直下の表皮顆粒層（矢印）の蛍光（セラミド）がカプリン酸ナトリウム未処理では細胞間の上部分に多く認められるのに対して（図２）、カプリン酸ナトリウム処理により消失していることが判る（図３）。この後カプリン酸ナトリウムを含まない新鮮な維持培地に置換することにより、再び表皮基底層の細胞間の上部分（矢印）に蛍光（セラミド）が認められる（図４）。 FIG. 2 shows an untreated sodium caprate, FIG. 3 shows 12 hours after treatment with 1 mM sodium caprate, and FIG. 4 shows “EPI when replaced with a fresh maintenance medium not containing sodium caprate after 12 hours of 1 mM sodium caprate treatment. -200 "is a fluorescence image of a frozen section. Whereas the fluorescence (ceramide) of the epidermal granule layer (arrow) just below the stratum corneum (part surrounded by a dotted line in the figure) is not observed with sodium caprate, it is often observed in the upper part between cells (FIG. 2). It turns out that it has disappeared by the treatment with sodium caprate (FIG. 3). Thereafter, by replacing with a fresh maintenance medium not containing sodium caprate, fluorescence (ceramide) is observed again in the upper part (arrow) between cells of the epidermal basal layer (FIG. 4).
実施例２と同様に＜工程６＞まで行うが、＜工程６＞の後、別個の「EPI-200」を、５００μｇ／ｍｌε，γ−グルタミルリジン、０．１％パルマリア抽出物それぞれ単独で含有する維持培地に置換し６時間培養を続けた。その後培地を回収し、細胞外に分泌された「BODIPY FL-C5-Ceramide-BSA」の蛍光強度（励起波長４８５ｎｍ、蛍光波長５３５ｎｍ）を、ARVO SX Multilabel Counter（PerkinElmer社製）を用いて測定した。さらに、その後＜工程８＞まで進めた。 <Step 6> is performed in the same manner as in Example 2, but after <Step 6>, separate "EPI-200" is contained alone at 500 µg / mlε, γ-glutamyllysine and 0.1% palmaria extract, respectively. The culture medium was replaced with the maintenance medium to be cultured for 6 hours. Thereafter, the medium was collected, and the fluorescence intensity (excitation wavelength: 485 nm, fluorescence wavelength: 535 nm) of “BODIPY FL-C5-Ceramide-BSA” secreted outside the cells was measured using ARVO SX Multilabel Counter (manufactured by PerkinElmer). . Furthermore, it advanced to <process 8> after that.
結果を図５に示すが、ε，γ−グルタミルリジン、パルマリア抽出物は、いずれも無添加コントロールよりも有意に蛍光標識セラミド「BODIPY FL-C5-Ceramide-BSA」の細胞外への分泌量を促進する。また図６は１ｍＭカプリン酸ナトリウム処理１２時間後、維持培地に置換して１２時間後、図７は１ｍMカプリン酸ナトリウム処理１２時間後、５００μｇ／ｍｌε，γ−グルタミルリジンを含有する維持培地に置換して１２時間後、図８は１ｍMカプリン酸ナトリウム処理１２時間後、０．１％パルマリア抽出物を含有する維持培地に置換して１２時間後の「EPI-200」の凍結切片の蛍光画像である。角層直下の表皮顆粒層の蛍光（セラミド）が図６では細胞間の上部分には多く認められないのに対して、図７、８ではそれぞれ表皮顆粒層の細胞間の上部分（矢印）に多く認められる。つまり、ε，γ−グルタミルリジン及びパルマリア抽出物はそれぞれ単独で、セラミドを角層方向に分泌する表皮細胞の働きを早める効果を有することが判る。即ち本鑑別法は、セラミドの組織内輸送へのタイトジャンクションの関与の程度の評価に適していることが判る。 The results are shown in FIG. 5, and ε, γ-glutamyllysine and palmaria extract all significantly increased the amount of secretion of fluorescently labeled ceramide “BODIPY FL-C5-Ceramide-BSA” to the outside of the control without addition. Facilitate. 6 shows 12 hours after treatment with 1 mM sodium caprate and 12 hours after replacement with maintenance medium. FIG. 7 shows 12 hours after treatment with 1 mM sodium caprate and replacement with maintenance medium containing 500 μg / ml ε, γ-glutamyllysine. 12 hours later, FIG. 8 is a fluorescence image of a frozen section of “EPI-200” after 12 hours after treatment with 1 mM sodium caprate and after replacement with a maintenance medium containing 0.1% palmaria extract. is there. In FIG. 6, the fluorescence (ceramide) in the epidermal granule layer just below the stratum corneum is not observed in the upper part between cells, whereas in FIGS. 7 and 8, the upper part (arrow) between the cells in the epidermal granule layer, respectively. Many are recognized. That is, it turns out that (epsilon), (gamma) -glutamyl lysine and a palmaria extract each have the effect which accelerates | stimulates the function of the epidermal cell which secretes ceramide toward a stratum corneum. That is, it can be seen that this discrimination method is suitable for evaluating the degree of involvement of tight junctions in the intracerebral transport of ceramide.
本発明は、化粧料などの皮膚外用剤の有効成分の評価に応用できる。 The present invention can be applied to the evaluation of active ingredients of external preparations for skin such as cosmetics.
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