JP2009294202A5 - - Google Patents
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- JP2009294202A5 JP2009294202A5 JP2009092355A JP2009092355A JP2009294202A5 JP 2009294202 A5 JP2009294202 A5 JP 2009294202A5 JP 2009092355 A JP2009092355 A JP 2009092355A JP 2009092355 A JP2009092355 A JP 2009092355A JP 2009294202 A5 JP2009294202 A5 JP 2009294202A5
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- 210000003491 skin Anatomy 0.000 claims description 61
- 210000001578 tight junction Anatomy 0.000 claims description 32
- 102000000591 Tight Junction Proteins Human genes 0.000 claims description 17
- 108010002321 Tight Junction Proteins Proteins 0.000 claims description 17
- 230000006378 damage Effects 0.000 claims description 17
- 208000027418 Wounds and injury Diseases 0.000 claims description 16
- 208000014674 injury Diseases 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 16
- 238000011282 treatment Methods 0.000 claims description 9
- 230000004069 differentiation Effects 0.000 claims description 5
- 238000011084 recovery Methods 0.000 claims description 3
- 210000000434 stratum corneum Anatomy 0.000 claims description 3
- YDNKGFDKKRUKPY-JHOUSYSJSA-N C16 ceramide Natural products CCCCCCCCCCCCCCCC(=O)N[C@@H](CO)[C@H](O)C=CCCCCCCCCCCCCC YDNKGFDKKRUKPY-JHOUSYSJSA-N 0.000 claims description 2
- LNLLNTMHVMIMOG-YUMQZZPRSA-N Gamma-glutamyl-Lysine Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CC[C@H](N)C(O)=O LNLLNTMHVMIMOG-YUMQZZPRSA-N 0.000 claims description 2
- CRJGESKKUOMBCT-VQTJNVASSA-N N-acetylsphinganine Chemical compound CCCCCCCCCCCCCCC[C@@H](O)[C@H](CO)NC(C)=O CRJGESKKUOMBCT-VQTJNVASSA-N 0.000 claims description 2
- 241000206755 Palmaria Species 0.000 claims description 2
- 238000010306 acid treatment Methods 0.000 claims description 2
- 229940106189 ceramide Drugs 0.000 claims description 2
- ZVEQCJWYRWKARO-UHFFFAOYSA-N ceramide Natural products CCCCCCCCCCCCCCC(O)C(=O)NC(CO)C(O)C=CCCC=C(C)CCCCCCCCC ZVEQCJWYRWKARO-UHFFFAOYSA-N 0.000 claims description 2
- 238000012850 discrimination method Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 claims description 2
- 210000001339 epidermal cell Anatomy 0.000 claims description 2
- VVGIYYKRAMHVLU-UHFFFAOYSA-N newbouldiamide Natural products CCCCCCCCCCCCCCCCCCCC(O)C(O)C(O)C(CO)NC(=O)CCCCCCCCCCCCCCCCC VVGIYYKRAMHVLU-UHFFFAOYSA-N 0.000 claims description 2
- 238000002372 labelling Methods 0.000 claims 1
- 125000005480 straight-chain fatty acid group Chemical group 0.000 claims 1
- 239000000126 substance Substances 0.000 description 3
- 208000018380 Chemical injury Diseases 0.000 description 2
- GHVNFZFCNZKVNT-UHFFFAOYSA-N decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 2
- 210000004207 dermis Anatomy 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- WWZKQHOCKIZLMA-UHFFFAOYSA-N octanoic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000005632 Capric acid (CAS 334-48-5) Substances 0.000 description 1
- 239000005635 Caprylic acid (CAS 124-07-2) Substances 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 102000002029 Claudin Human genes 0.000 description 1
- 108050009302 Claudin Proteins 0.000 description 1
- 231100000948 EpiDerm Skin Irritation Test Toxicity 0.000 description 1
- 102000003940 Occludin Human genes 0.000 description 1
- 108090000304 Occludin Proteins 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 210000002615 epidermis Anatomy 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 210000002510 keratinocyte Anatomy 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 229960002446 octanoic acid Drugs 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 229920006395 saturated elastomer Polymers 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 210000004927 skin cell Anatomy 0.000 description 1
- 230000037380 skin damage Effects 0.000 description 1
- 230000037204 skin physiology Effects 0.000 description 1
- FIWQZURFGYXCEO-UHFFFAOYSA-M sodium;decanoate Chemical compound [Na+].CCCCCCCCCC([O-])=O FIWQZURFGYXCEO-UHFFFAOYSA-M 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Description
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、皮膚又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）皮膚又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又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、（１）又は（２）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。
（４）前記皮膚又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）の何れか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）の何れか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。
（７）（１）〜（６）の何れかに記載の方法において、培地中に含有させた場合に、皮膚関連成分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、ε，γ−グルタミルリジン及び／又はパルマリア抽出物を含む、傷害タイトジャンクションの回復促進剤。
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, a tight junction injured skin model is prepared, cultured in a medium containing a labeled skin-related component, and the distribution of the label The inventors have found that such problems can be solved by using the situation as an indicator, 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 to create a tight junction injury skin model, cultured in a medium containing labeled skin-related components, and the distribution status of the label as an indicator A method for differentiating the degree of involvement of tight junctions in the distribution or transport of skin-related components.
(2) The degree of involvement of tight junctions in the distribution or transport of skin-related components according to (1), characterized in that a skin or cultured skin three-dimensional model not subjected to tight junction injury treatment is used as a control. Differentiation method.
(3) The distribution of the skin-related component according to (1) or (2) , wherein the discrimination method measures a labeled skin-related component released into the medium on the stratum corneum side. Alternatively , a method for distinguishing the degree of involvement of tight junctions in transportation.
(4) The skin or skin three-dimensional model is a cultured human skin three-dimensional model, and the tight junction to the distribution or transport of the skin-related component according to any one of (1) to (3) The method of differentiation of the degree of involvement.
(5) The tight junction to the distribution or transport of the skin-related component 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 The method of differentiation of the degree of involvement.
(6) labeled with skin-related component comprising is characterized by a fluorescence-labeled ceramide, (1) to (5) of any of the crab according tight junction to distribution or transport of skin-related components A method to differentiate the degree of involvement.
(7) In the method according to any one of (1) to (6), when contained in the medium, ε, which has an effect of accelerating the action of epidermal cells that secrete skin-related components in the horny layer direction A recovery promoter for injury tight junction, comprising γ-glutamyllysine and / or palmaria extract.
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、皮膚又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、ヒト又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。かかる皮膚又は培養
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よく、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の溶液を真皮側又は表皮基底層側から、５〜２４時間、さらに好ましくは１０〜１５時間作用させればよい。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又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、培地又は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を、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する。
The method of distinguishing the degree of involvement of tight junctions in the distribution or transport of skin-related components of the present invention is to treat tight junctions of skin or cultured skin three-dimensional models, create tight junction injury skin models, and label them. 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, a rat, Guinea pig, pig and rabbit skin fragments are preferred. In addition, as the cultured skin three-dimensional model, the skin cells such as normal (non-cancerous) keratinocytes and fibroblasts collected from human or non- human animal skin are cultured, and a three-dimensional structure is constructed. preferably those obtained by pseudo the structure of the skin, can also be used to purchase a commercially available product of such a form. 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 a three-dimensional model of skin or cultured skin damages a tight junction portion by taking an injury means from the granule layer side. For example, ultraviolet rays and chemical substances can be preferably exemplified as the injury means, and 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 . rather I be irradiated with the energy, if chemical treatments, 0 aliphatic saturated straight chain fatty acid or a monovalent metal salt of capric acid, chain length in such caprylic acid (8-12 carbon atoms) .1～10MM, more preferably a solution from the dermis side or epidermal basal layer side of 0.5 to 2 mm, 5 to 24 hours, more preferably not good if caused to act for 10-15 hours. 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. Thus the tight junctions injury skin model obtained, cultured in media containing labeled formed by skin-related components, and thereafter, tight junction injury skin model in unincorporated labeled wash skin associated components such as 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 We observe the distribution situation.
Claims (7)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2009092355A JP2009294202A (en) | 2008-05-09 | 2009-04-06 | Differentiation method for involvement of tight junction in material transport |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008123071 | 2008-05-09 | ||
| JP2009092355A JP2009294202A (en) | 2008-05-09 | 2009-04-06 | Differentiation method for involvement of tight junction in material transport |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013028561A Division JP2013100355A (en) | 2008-05-09 | 2013-02-18 | Promoter of recovery of injured tight junction |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2009294202A JP2009294202A (en) | 2009-12-17 |
| JP2009294202A5 true JP2009294202A5 (en) | 2012-05-31 |
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| Application Number | Title | Priority Date | Filing Date |
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| JP2009092355A Pending JP2009294202A (en) | 2008-05-09 | 2009-04-06 | Differentiation method for involvement of tight junction in material transport |
| JP2013028561A Pending JP2013100355A (en) | 2008-05-09 | 2013-02-18 | Promoter of recovery of injured tight junction |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2013028561A Pending JP2013100355A (en) | 2008-05-09 | 2013-02-18 | Promoter of recovery of injured tight junction |
Country Status (1)
| Country | Link |
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| JP (2) | JP2009294202A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009294200A (en) * | 2008-05-09 | 2009-12-17 | Pola Chem Ind Inc | Differentiation method for tight junction function restoring agent |
Family Cites Families (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2836772B2 (en) * | 1992-02-25 | 1998-12-14 | ポーラ化成工業株式会社 | Cosmetics |
| JP3807782B2 (en) * | 1995-06-22 | 2006-08-09 | ライオン株式会社 | Hyaluronidase inhibitor |
| JP4050799B2 (en) * | 1995-07-19 | 2008-02-20 | 雪印乳業株式会社 | Tight junction formation accelerator |
| JPH10236918A (en) * | 1997-02-21 | 1998-09-08 | Lion Corp | Skin patch |
| JP2005035926A (en) * | 2003-07-14 | 2005-02-10 | Japan Science & Technology Agency | Drug for promoting ceramide transport, base sequence for producing the drug, method for measuring activity for promoting ceramide release, and method for measuring activity for promoting transmembrane movement of ceramide |
| JP2005137307A (en) * | 2003-11-07 | 2005-06-02 | Japan Science & Technology Agency | Method and apparatus for evaluating damage of cells and tissues |
| JP4671721B2 (en) * | 2005-03-11 | 2011-04-20 | ポーラ化成工業株式会社 | Differentiation of skin effects |
| US20080194611A1 (en) * | 2005-06-03 | 2008-08-14 | Alverdy John C | Modulation of Cell Barrier Dysfunction |
| JP2007174931A (en) * | 2005-12-27 | 2007-07-12 | Pola Chem Ind Inc | Epidermal keratinocyte layer membrane and utilization of the epidermal keratinocyte layer membrane |
| DK2046382T3 (en) * | 2006-07-10 | 2016-12-12 | Esbatech Alcon Biomed Res Unit | scFv Antibodies which passes through the epithelial and / or endothelial layer |
| JP2008026092A (en) * | 2006-07-20 | 2008-02-07 | Pola Chem Ind Inc | Discrimination method of serviceability of material for improving skin barrier function |
| TW200831125A (en) * | 2006-12-08 | 2008-08-01 | Pola Chem Ind Inc | A discrimination method of skin barrier function, the screening method of skin barrier function reinforced material by using the discrimination method, the skin barrier function reinforced materials, and the cosmetic material containing the skin barrier |
-
2009
- 2009-04-06 JP JP2009092355A patent/JP2009294202A/en active Pending
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2013
- 2013-02-18 JP JP2013028561A patent/JP2013100355A/en active Pending
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