JP2009240271A - Method for producing three-dimensional skin model of parakeratosis and epidermal growth abnormality - Google Patents
Method for producing three-dimensional skin model of parakeratosis and epidermal growth abnormality Download PDFInfo
- Publication number
- JP2009240271A JP2009240271A JP2008093131A JP2008093131A JP2009240271A JP 2009240271 A JP2009240271 A JP 2009240271A JP 2008093131 A JP2008093131 A JP 2008093131A JP 2008093131 A JP2008093131 A JP 2008093131A JP 2009240271 A JP2009240271 A JP 2009240271A
- Authority
- JP
- Japan
- Prior art keywords
- keratinization
- skin model
- dimensional skin
- uvb
- skin
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
Description
本発明は、不全角化及び／又は表皮増殖異常三次元皮膚モデルの作製方法及びその方法により作製された不全角化及び／又は表皮増殖異常三次元皮膚モデルを提供する。 The present invention provides a method for producing a three-dimensional skin model for abnormal keratinization and / or abnormal epidermal proliferation, and a three-dimensional skin model for abnormal keratinization and / or abnormal epidermal proliferation produced by the method.
皮膚が正常の角化を行うと顆粒層から角層へと移行する段階で核が消滅する「脱核」が生ずる。表皮細胞は基底層で増殖し、上層に移行して成熟し角層となる。しかしながら、例えば乾癬、炎症性異常角化症、アトピー性皮膚炎といった皮膚病に罹り、正常な角化が行われない肌荒れした皮膚においては、角質細胞に核が未消化のまま残り、角質細胞が核を有した未熟な状態で角層中に存在することがあり、これを「不全角化」又は「錯角化」（ｐａｒａｋｅｒａｔｏｓｉｓ）という(J. Am. Acad. Dermatol. Vol.50, No.1, pp.77-84 (2004)：非特許文献１）。不全角化の現象は古くから知られているものの、不全角化が生じるメカニズムや生化学的な指標についてはよく知られていなかった。不全角化は肌荒れの顕著な状態であり、その治療・予防に有効な薬剤の評価方法、新規の不全角化改善薬剤の研究・開発が求められ、そのために有効な不全角化モデルの出現が望まれている。 When the skin undergoes normal keratinization, “nucleation” occurs in which the nucleus disappears at the stage of transition from the granule layer to the stratum corneum. Epidermal cells proliferate in the basal layer, migrate to the upper layer, mature and become the stratum corneum. However, in rough skin that does not have normal keratinization due to skin diseases such as psoriasis, inflammatory keratosis, and atopic dermatitis, the keratinocytes remain undigested, It may be present in the stratum corneum in an immature state having a nucleus, and this is called “dyskeratosis” or “parakeratosis” (J. Am. Acad. Dermatol. Vol. 50, No. 1). , pp.77-84 (2004): Non-Patent Document 1). Although the phenomenon of keratinization has been known for a long time, the mechanism and biochemical indicators that cause keratinization have not been well known. Abnormal keratinization is a prominent condition of rough skin, and there is a need for methods for evaluating drugs that are effective in the treatment and prevention of it, as well as research and development of new drugs for improving keratinization. It is desired.
紫外線照射、乾燥、界面活性剤など皮膚に有害な外部刺激による皮膚傷害、アトピー性皮膚炎、乾癬、接触性皮膚炎などといった種々の皮膚疾患に見られる肌荒れ症状においては、皮膚バリアー機能が低下し、表皮増殖異常が起こることが報告されている。皮膚の皮膚機能の低下による表皮増殖異常を防止するために有効な新規の皮膚バリアー機能回復促進剤に開発が求められ、そのために有効な表皮増殖異常モデルの出現が望まれている。 Skin barrier function is reduced in rough skin symptoms such as skin damage caused by external stimuli harmful to the skin such as ultraviolet irradiation, drying, surfactant, atopic dermatitis, psoriasis, contact dermatitis, etc. It has been reported that abnormal epidermal proliferation occurs. Development of a novel skin barrier function recovery accelerator effective to prevent epidermal proliferation abnormality due to a decrease in skin function of the skin is required, and therefore, an effective epidermal proliferation abnormality model is desired.
不全角化モデルの作成方法としては例えばマウスなどの動物モデルの皮膚にオレイン酸溶液を繰り返し塗布することで不全角化を人為的に起こさせる手法がある（非特許文献１）。また、表皮増殖異常モデルの作成方法としては例えばマウスなどの動物モデルに粘着テープを貼り／剥がすといった工程を繰り返す行うことによりの表皮の肥厚化を人為的に起こさせる手法（いわゆるテープストリッピング法［特開２０００−１５９６６６：非特許文献２］）がある。しかしながら、このような方法は動物愛護の観点から敬遠され、またマウスなどの皮膚細胞とヒトの細胞との間には表皮細胞の角質化に関与していると思われるカスパーゼファミリーやその他の生理活性物質の種類・作用の点において相違するものと考えられ、動物モデルで得られた結果が必ずしもヒトに適用できるものでもない。従って、生きた動物を使用せず、好ましくはヒト細胞から調製した不全角化三次元皮膚モデルや表皮増殖異常モデルの出現が特に所望される。 As a method for creating a keratinization model, for example, there is a method of artificially causing keratinization by repeatedly applying an oleic acid solution to the skin of an animal model such as a mouse (Non-Patent Document 1). In addition, as a method for creating an epidermal proliferation abnormality model, for example, a method of artificially causing thickening of the epidermis by repeating a process of applying / peeling an adhesive tape to / from an animal model such as a mouse (so-called tape stripping method [special Open 2000-159666: Non-Patent Document 2]). However, this method is avoided from the viewpoint of animal welfare, and the caspase family and other physiological activities that are thought to be involved in keratinization of epidermal cells between skin cells such as mice and human cells. It is considered that the types and effects of substances differ, and the results obtained with animal models are not necessarily applicable to humans. Therefore, the emergence of a keratinized three-dimensional skin model or epidermal proliferation abnormality model prepared from human cells, preferably without using live animals, is particularly desirable.
本発明の課題は、不全角化及び／又は表皮増殖異常三次元皮膚モデル、好ましくはヒト細胞由来の不全角化及び／又は表皮増殖異常三次元皮膚モデルの提供にある。かかる不全角化及び／又は表皮増殖異常三次元皮膚モデルは不全角化及び／又は表皮増殖異常に効果的な薬剤評価方法、不全角化及び／又は表皮増殖異常改善薬剤のスクリーニング方法などに有効である。 An object of the present invention is to provide a three-dimensional skin model with abnormal keratinization and / or abnormal epidermal proliferation, preferably a three-dimensional skin model with abnormal keratinization and / or abnormal epidermal proliferation derived from human cells. Such a three-dimensional skin model with aberrant keratinization and / or epidermal proliferation abnormality is effective for a drug evaluation method effective for aberrant keratinization and / or epidermal proliferation abnormality, and a screening method for a drug for improving aberrant keratinization and / or epidermal proliferation abnormality. is there.
本発明は、上記課題に着目し、鋭意検討した結果、ケラチノサイト及び繊維芽細胞を含んで成る三次元皮膚モデルに対しＵＶＢを所定の条件下で照射することで、有効な不全角化及び／又は表皮増殖異常三次元皮膚モデルを提供できることを見出し、本発明を完成するに至った。詳しくは、本願は以下の発明を包含する。
（１）ケラチノサイト及び繊維芽細胞を含んで成る三次元皮膚モデルに対しＵＶＢを下記の条件において照射することで不全角化及び／又は表皮増殖異常三次元皮膚モデルを作製する方法：
５≦Ｘ×Ｙ／Ｚ≦１５
（式中、
ＸはＵＶＢの強度（ｍＪ）、
ＹはＵＶＢ照射回数、そして
ＺはＵＶＢ照射時間（日数）を表し、
但し、１０≦Ｘであるか、又は
Ｘ≦１０のとき、２≦Ｙである。）。
（２）前記ケラチノサイトがヒト細胞である、（１）の方法。
（３）前記三次元皮膚モデルが、支持体に繊維芽細胞が分散され、その上にケラチノサイトが播種されることで構成される、（１）又は（２）の方法。
（４）前記支持体がコラーゲンゲルである、（３）の方法。
（５）（１）〜（４）のいずれかの方法で作製された不全角化及び／又は表皮増殖異常三次元皮膚モデル。
The present invention pays attention to the above-mentioned problem, and as a result of intensive studies, effective irradiation of keratinization and / or by irradiating UVB to a three-dimensional skin model comprising keratinocytes and fibroblasts under predetermined conditions. The present inventors have found that a three-dimensional skin model with abnormal epidermal proliferation can be provided, and have completed the present invention. Specifically, this application includes the following inventions.
(1) A method for producing a three-dimensional skin model with abnormal keratinization and / or abnormal epidermal proliferation by irradiating UVB with the following conditions on a three-dimensional skin model comprising keratinocytes and fibroblasts:
5 ≦ X × Y / Z ≦ 15
(Where
X is the intensity of UVB (mJ),
Y represents the number of UVB irradiations, and Z represents the UVB irradiation time (days),
However, when 10 ≦ X, or when X ≦ 10, 2 ≦ Y. ).
(2) The method according to (1), wherein the keratinocyte is a human cell.
(3) The method according to (1) or (2), wherein the three-dimensional skin model is configured by dispersing fibroblasts on a support and seeding keratinocytes thereon.
(4) The method of (3), wherein the support is a collagen gel.
(5) A three-dimensional skin model with keratinization and / or abnormal epidermal proliferation produced by any of the methods (1) to (4).
本発明に従い提供される不全角化及び／又は表皮増殖異常三次元皮膚モデルにより、不全角化及び／又は表皮増殖異常に有効な薬剤の評価、新規不全角化及び／又は表皮増殖異常改善薬剤のスクリーニングなどが、動物実験に頼ることなく、簡便に行うことが可能となる。 According to the three-dimensional skin model of keratinization and / or epidermal proliferation abnormality provided according to the present invention, evaluation of a drug effective for keratinization and / or epidermal proliferation abnormality, novel keratinization and / or epidermal proliferation abnormality improving drug Screening and the like can be easily performed without relying on animal experiments.
三次元皮膚モデル
本発明の方法に使用できる不全角化及び／又は表皮増殖異常三次元皮膚モデルは、ケラチノサイト及び繊維芽細胞を含んで成る三次元皮膚モデルから作製される。かかる三次元皮膚モデルは当業者にとって周知の方法（例えば、Amano S. et al., Exp. Cell Res., Vol.271, pp.249-262, 2001やTsunenga M. et al., Matrix. Biol., Vol.17, pp.603-613, 1998参照のこと）により調製することができ、例えばインサートメッシュ上において繊維芽細胞を支持体、例えばコラーゲンゲルに混ぜ込んだものを播いた後、その上にケラチノサイトを播き、培養することで調製したものであってよい。三次元皮膚モデルは例えば繊維芽細胞を１×１０4〜１０8個／ｃｍ2、好ましくは０．１〜１０×１０5個／ｃｍ2の量で含み、またケラチノサイトを１×１０2〜１０6個／ｃｍ2、好ましくは１．０〜１０×１０4個／ｃｍ2、より好ましくは約４〜８×１０4個／ｃｍ2の量で含むものであってよい。
このような三次元皮膚モデルは市販もされており、特に限定されるわけではないが、例えばＥＰＩ−ＭＯＤＥＬ（Ｊ−ＴＥＣ）、ＴＥＳＴＳＫＩＮ（商標）（ＴＯＹＯＢＯ）などが使用できる。
Three-dimensional skin model A three-dimensional skin model with keratinization and / or abnormal epidermal proliferation that can be used in the method of the present invention is created from a three-dimensional skin model comprising keratinocytes and fibroblasts. Such three-dimensional skin models are obtained by methods well known to those skilled in the art (for example, Amano S. et al., Exp. Cell Res., Vol.271, pp.249-262, 2001 and Tsunega M. et al., Matrix. Biol Vol. 17, pp. 603-613, 1998), for example, after seeding fibroblasts on a support such as a collagen gel on an insert mesh, It may be prepared by seeding and culturing keratinocytes on top. The three-dimensional skin model contains, for example, fibroblasts in an amount of 1 × 10 4 to 10 8 cells / cm 2 , preferably 0.1 to 10 × 10 5 cells / cm 2 , and keratinocytes 1 × 10 2 to 10 6 pieces / cm 2 , preferably 1.0 to 10 × 10 4 pieces / cm 2 , more preferably about 4 to 8 × 10 4 pieces / cm 2 .
Such a three-dimensional skin model is also commercially available and is not particularly limited. For example, EPI-MODEL (J-TEC), TESTSKIN (trademark) (TOYOBO), and the like can be used.
三次元皮膚モデルの培養は、例えば培養液として通常のケラチノサイト培養に用いられる培養液、例えばＫＧ培地、ＥｐｉｌｉｆｅＫＧ２（クラボウ）、Ｈｕｍｅｄｉａ−ＫＧ２（クラボウ）、アッセイ培地（ＴＯＹＯＢＯ）などを用い、約３７℃で０〜１４日間かけて行うことができる。培地としては、その他にＤＭＥＭ培地（ＧＩＢＣＯ）又は２−０−ａ−Ｄ−グルコピラノシル−Ｌ−アスコルビン酸含有ＫＧＭとＤＭＥＭを１：１１混合した培地などが使用できる。 For the culture of the three-dimensional skin model, for example, a culture solution used for normal keratinocyte culture such as KG medium, Epilife KG2 (Kurabo), Humedia-KG2 (Kurabo), assay medium (TOYOBO), etc. is used. Can be carried out over 0-14 days. As the medium, a DMEM medium (GIBCO) or a medium in which 2-0-aD-glucopyranosyl-L-ascorbic acid-containing KGM and DMEM are mixed at 1:11 can be used.
繊維芽細胞及びケラチノサイトは同種系でも異種系であってもよく、あらゆる哺乳動物に由来してよい。しかしながら、限定するわけではないが、ケラチノサイトがヒト由来であることが好ましい。ケラチノサイトがヒト由来である方が、それにより形成される不全角化及び／又は表皮増殖異常三次元皮膚モデルの性状をヒト皮膚のそれに一層近づけることができるからである。また、使用する繊維芽細胞とケラチノサイトの割合は特に限定されるわけではないが、例えばケラチノサイト４〜８×１０4〜6細胞に対して繊維芽細胞１×１０5〜7細胞、好ましくはケラチノサイト４〜８×１０5細胞に対し繊維芽細胞１×１０6細胞とすることができる。 Fibroblasts and keratinocytes may be homologous or heterologous and may be derived from any mammal. However, although not necessarily limited, it is preferred that the keratinocytes are derived from humans. This is because, when the keratinocyte is derived from human, the properties of the three-dimensional skin model formed by the keratinization and / or abnormal epidermal proliferation can be made closer to that of human skin. The ratio of fibroblasts and keratinocytes to be used is not particularly limited. For example, 1 × 10 5 to 7 cells, preferably keratinocytes 4 to 4 to 8 × 10 4 to 6 keratinocytes. Fibroblast 1 × 10 6 cells can be used for ˜8 × 10 5 cells.
ＵＶＢ照射
不全角化及び／又は表皮増殖異常形成のためのＵＶＢ照射は、上記三次元皮膚モデルに対し以下の条件を満たすようにして行う。
５≦Ｘ×Ｙ／Ｚ≦１５
（式中、
ＸはＵＶＢの強度（ｍＪ）、
ＹはＵＶＢ照射回数、そして
ＺはＵＶＢ照射時間（日数）を表し、
但し、１０≦Ｘであるか、又は
Ｘ≦１０のとき、２≦Ｙである。）。
UVB irradiation for UVB irradiation deficiency keratinization and / or epidermal proliferation abnormality formation is performed so as to satisfy the following conditions for the above three-dimensional skin model.
5 ≦ X × Y / Z ≦ 15
(Where
X is the intensity of UVB (mJ),
Y represents the number of UVB irradiations, and Z represents the UVB irradiation time (days),
However, when 10 ≦ X, or when X ≦ 10, 2 ≦ Y. ).
なお、ＵＶＢ照射は例えばＴｒａｎｓｉｌｌｕｍｉｎａｔｏｒ（ＴＰＲＥＸ ＦＬ２０５−Ｅ−３０／ＤＭＲ； Ｔｏｓｈｉｂａ Ｍｅｄｉｃａｌ Ｓｕｐｐｌｙ）を用いて行うことができる。また、ＵＶＢ照射はケラチノサイト培養の０〜１４日後に無菌的に行うことが好ましい。上記条件において、「Ｘ×Ｙ／Ｚ」（以下、「不全角化作製及び／又は表皮増殖異常誘導指数」を称する場合がある）は５未満であると不全角化の形成及び／又は表皮増殖異常の誘導が認められず、また不全角化作製及び／又は表皮増殖異常誘導指数が１５超である場合、棘融解等が生じ、皮膚ダメージが大きすぎてしまい、不全角化の形成及び／又は表皮増殖異常の誘導が困難となる。特に理論に拘束されるものではないが、不全角化及び／又は表皮増殖異常は適切な強度のＵＶＢを連続又は数日置きに照射することで形成されるものと考えられる。 In addition, UVB irradiation can be performed using Transilluminator (TPREX FL205-E-30 / DMR; Toshiba Medical Supply), for example. Moreover, it is preferable to perform UVB irradiation aseptically 0 to 14 days after keratinocyte culture. In the above conditions, if “X × Y / Z” (hereinafter, sometimes referred to as “abnormal keratinization and / or epidermal proliferation abnormality induction index”) is less than 5, formation of aberrant keratinization and / or epidermal proliferation If no induction of abnormality is observed, and preparation of aberrant keratinization and / or epidermal proliferation abnormality induction index is greater than 15, spinal fusion or the like occurs, skin damage is too great, and formation of aberrant keratinization and / or Induction of epidermal proliferation abnormality becomes difficult. Although not particularly bound by theory, it is considered that the keratinization and / or epidermal proliferation abnormality is formed by irradiating UVB having an appropriate intensity continuously or every several days.
本発明に係る不全角化及び／又は表皮増殖異常三次元皮膚モデルは、肌荒れ、例えば紫外線照射を原因とする皮膚老化、皮膚バリアー機能低下に伴う肌保湿力の低下による乾燥肌、乾癬などの炎症性異常角化症やアトピー性皮膚炎を原因とする不全角化及び／又は表皮増殖異常など、さまざまな皮膚性状の治療、改善及び／又は予防を目的とした薬剤の評価、スクリーニングに有効である。かかる不全角化及び／又は表皮増殖異常三次元皮膚モデルはまた、対象の薬剤が不全角化及び／又は表皮増殖異常を惹起するものであるか否かの判定のためにも利用できる。 The three-dimensional skin model for keratinization and / or abnormal epidermal proliferation according to the present invention is used for rough skin, for example, skin aging due to ultraviolet irradiation, dry skin due to reduced skin moisturizing power due to reduced skin barrier function, inflammation such as psoriasis, etc. Effective for the evaluation and screening of drugs for the treatment, improvement and / or prevention of various skin properties such as keratinization due to keratosis and atopic dermatitis and / or abnormal epidermal proliferation . Such a keratinization and / or epidermal proliferation abnormality three-dimensional skin model can also be used to determine whether the subject drug causes keratinization and / or epidermal proliferation abnormality.
以下、具体例を挙げて、本発明を更に具体的に説明する。なお、本発明はこれにより限定されるものではない。また。％と表示されている場合、特に断りのない限り、質量％を表す。 Hereinafter, the present invention will be described more specifically with specific examples. In addition, this invention is not limited by this. Also. In the case of “%”, “% by mass” is indicated unless otherwise specified.
三次元皮膚モデル
三次元皮膚モデルを用い、不全角化及び／又は表皮増殖異常を形成する培養条件、ＵＶＢ照射条件について検討を行った。三次元皮膚モデルは、（１）インサートメッシュ（大きさ：約２ｃｍ）上にケラチノサイトが直接播種され（商品名：ＥＰＩＭＯＤＥＬ；Ｊ−ＴＥＣ社製;）、培養されたもの又は（２）インサートメッシュ（大きさ：約２ｃｍ）上にコラーゲン及び繊維芽細胞を播いた後にケラチノサイトが播種され（商品名：ＴＥＳＴＳＫＩＮ（ＴＯＹＯＢＯ））、培養されたものを使用した。培養は、培養液（商品名：アッセイ培地（ＴＯＹＯＢＯ））を用いて３７℃で０〜１４日間行った。培養インサートモデルを図１に示す。
Three-dimensional skin model A three-dimensional skin model was used to examine culture conditions and UVB irradiation conditions for forming keratinization and / or abnormal epidermal proliferation. The three-dimensional skin model is either (1) a keratinocyte seeded directly on an insert mesh (size: about 2 cm) (trade name: EPIMODEL; manufactured by J-TEC), or (2) an insert mesh ( After culturing collagen and fibroblasts on the size (about 2 cm), keratinocytes were seeded (trade name: TESTSKIN (TOYOBO)) and cultured. The culture was carried out at 37 ° C. for 0 to 14 days using a culture solution (trade name: assay medium (TOYOBO)). A culture insert model is shown in FIG.
ＵＶＢ照射
上記三次元皮膚モデルの各々に対しＵＶＢ照射を行った。ＵＶＢ照射はＴｒａｎｓｉｌｌｕｍｉｎａｔｏｒ（ＴＰＲＥＸ ＦＬ２０５−Ｅ−３０／ＤＭＲ； Ｔｏｓｈｉｂａ Ｍｅｄｉｃａｌ Ｓｕｐｐｌｙ）を用い、ケラチノサイト培養０〜１４日後に無菌的に行った。
UVB irradiation UVB irradiation was performed on each of the above three-dimensional skin models. UVB irradiation was carried out aseptically using a Transilluminator (TPREX FL205-E-30 / DMR; Toshiba Medical Supply) 0 to 14 days after culturing keratinocytes.
組織学的検討
三次元皮膚モデルは、培養後４％パラホルムアルデヒド（ＰＦＡ）にて４時間固定した後、パラフィンで包埋した。３μｍに薄切した切片を、脱パラフィンした後にヘマトキシリン・エオジン染色（ＨＥ染色）して不全角化と表皮増殖異常の形成を確認した。
Histological examination The three-dimensional skin model was fixed with 4% paraformaldehyde (PFA) for 4 hours after culturing and then embedded in paraffin. Sections sliced into 3 μm were deparaffinized and then stained with hematoxylin and eosin (HE staining) to confirm the formation of aberrant keratinization and epidermal proliferation abnormality.
生化学的検討
乾癬表皮の上層において発現の亢進が認められるタンパク質として扁平上皮細胞癌関連抗原（ＳＣＣＡ）が知られる（Takeda A.ら、 J. Invest. Dermatol. (2002) 118(1), 147-154）。本発明者は以前、ＳＣＣＡが関与する表皮の生理学的メカニズムの解明を目的とする研究を行い、ＳＣＣＡの発現が乾癬などの不全角化の生じた皮膚において特異的に亢進し、ＳＣＣＡが不全角化を含む皮膚の肌荒れ性状の一因であることを見出し、皮膚角層細胞の扁平上皮細胞癌関連抗原（ＳＣＣＡ）の発現を指標とする皮膚性状の評価方法に係る発明の特許出願を行っている（特願２００５−０８０５３３）。このように、ＳＣＣＡ、特にＳＣＣＡ−１は不全角化を原因とする肌荒れなどの皮膚性状の指標となるため、本実験においても角化不全の形成の指標としてＳＣＣＡ−１の検出を試みた。
Biochemical study Squamous cell carcinoma-associated antigen (SCCA) is known as a protein whose expression is increased in the upper layer of psoriatic epidermis (Takeda A. et al., J. Invest. Dermatol. (2002) 118 (1), 147 -154). The present inventor has previously conducted research aimed at elucidating the physiological mechanism of the epidermis in which SCCA is involved, and the expression of SCCA is specifically increased in keratinized skin such as psoriasis. A patent application for an invention relating to a method for evaluating skin properties using the expression of squamous cell carcinoma-associated antigen (SCCA) in skin horny layer cells as an index (Japanese Patent Application No. 2005-080533). Thus, since SCCA, especially SCCA-1, becomes an index of skin properties such as rough skin caused by keratinization, detection of SCCA-1 was also attempted in this experiment as an index of formation of keratinization failure.
上記「組織学的検討」と同様に固定し、薄切した切片を、抗ＳＣＣＡ１モノクローナル抗体（Ｓａｎｔａ Ｃｒｕｚ社）を一次抗体に用いて染色し、ＳＣＣＡ１の発現の挙動を確認した。
詳しくは、上記切片をその切片の非特異的結合部位を１０％正常ウサギ血清(Histofine, Tokyo, Japan)でブロックした。次にその切片を抗−ＳＣＣＡ−１モノクローナル抗体（1：500に希釈）とインキュベーションし、ＰＢＳで洗浄後、核をヘマトキシリンでカウンター染色し、ＤＡＫＯ Ｅｎｖｉｓｉｏｎ Ｓｙｓｔｅｍ(DAKO Corp., CA, USA)で発色して観察を行った。
The section fixed and sliced in the same manner as in the above “histological examination” was stained with an anti-SCCA1 monoclonal antibody (Santa Cruz) as a primary antibody, and the behavior of SCCA1 expression was confirmed.
Specifically, the above-mentioned sections were blocked with 10% normal rabbit serum (Histofine, Tokyo, Japan) at non-specific binding sites on the sections. The sections are then incubated with anti-SCCA-1 monoclonal antibody (diluted 1: 500), washed with PBS, nuclei counterstained with hematoxylin, and developed with DAKO Envision System (DAKO Corp., CA, USA). And observed.
実験結果
１．ＵＶＢ照射した三次元皮膚モデルについて組織学的検討したところ、ケラチノサイトと繊維芽細胞の両者を含むモデルのみにおいて、表皮細胞における核の残存、即ち不全角化の形成と、表皮の肥厚化、即ち表皮増殖異常が認められ（図２参照）、ケラチノサイトだけから構成される三次元皮膚モデルでは、不全角化の形成や表皮増殖異常は認められず、しかもＵＶＢ １０ｍＪ単回照射で強度の表皮傷害が生じてしまった。（図３参照）。尚、コントロールはＵＶＢ照射を行わなかったものである。このことから、ヒト皮膚のＵＶへの反応を反映した良好な不全角化及び表皮増殖異常モデルを作製するにはケラチノサイトと繊維芽細胞の両者が必要であることが明らかとなった。また、ケラチノサイトと繊維芽細胞の両者を含むモデルでは表皮ＳＣＣＡ−１発現局在が不全角化形成皮膚の表皮上層で確認され、発現が広い範囲で亢進していることが明らかになった（図４）。
Experimental results Histological examination of the UVB-irradiated three-dimensional skin model revealed that only in the model containing both keratinocytes and fibroblasts, the survival of the nucleus in the epidermal cells, that is, the formation of keratinization, and the thickening of the epidermis, that is, the epidermis. In the three-dimensional skin model composed only of keratinocytes, no abnormal keratinization or epidermal proliferation abnormality was observed, and severe UVD 10mJ single irradiation caused severe epidermal injury. I have. (See FIG. 3). Note that the control was not subjected to UVB irradiation. From this, it became clear that both keratinocytes and fibroblasts are required to produce a model of abnormal keratinization and epidermal proliferation abnormality reflecting the response of human skin to UV. Moreover, in the model containing both keratinocytes and fibroblasts, the expression localization of the epidermal SCCA-1 was confirmed in the upper epidermis of the keratinized skin, and it was revealed that the expression was enhanced in a wide range (Fig. 4).
２．ＵＶＢ照射条件を検討したところ、下記表１に示すような結果が得られた。
上記の表に示されている結果を参照すると、不全角化作製及び／又は表皮増殖異常誘導指数が下記の条件：
５≦不全角化作製及び／又は表皮増殖異常誘導指数≦１５
条件１：１０≦Ｘであるか、又は
条件２：Ｘ≦１０のとき、２≦Ｙである
を満たす場合、不全角化の形成や表皮増殖異常が誘導されることが示された。
Referring to the results shown in the table above, preparation of keratinization and / or epidermal proliferation abnormality induction index is as follows:
5 ≦ Inadequate keratinization and / or epidermal proliferation abnormality induction index ≦ 15
Condition 1: 10 ≦ X, or Condition 2: When X ≦ 10, 2 ≦ Y is satisfied. It was shown that formation of keratinization and abnormal skin growth were induced.
薬剤評価
１−ピペリジンプロピオン酸（１-ＰＰ）は皮膚の不全角化に有効であることが知られ（特開２００６−３１９５２７）、また本発明者はそれが表皮増殖異常を抑制するという知見も得ている。詳しくは、３％の１−ＰＰを含有する薬剤を一方の頬、及びコントロールを他方の頬に塗布（２ヶ月連用）して、光干渉トモグラフィー（OCT：optical coherence tomography, ISIS optronicｓ社、ドイツ）を用いて表皮厚をそれぞれ測定したとことろ、１−ＰＰの塗布に表皮増殖異常の抑制が認められたとの知見を得ている（データーは示さない）。
Drug Evaluation 1-piperidinepropionic acid (1-PP) is known to be effective for keratinization of the skin (Japanese Patent Laid-Open No. 2006-319527), and the present inventor has also found that it suppresses epidermal proliferation abnormality It has gained. Specifically, a drug containing 3% 1-PP was applied to one cheek, and the control was applied to the other cheek (for two months), and optical coherence tomography (OCT: ISIS optronics, Germany) In addition, the thickness of each skin was measured, and the application of 1-PP showed that suppression of abnormal skin growth was observed (data not shown).
インサートメッシュ（大きさ：約２ｃｍ）上にコラーゲン及び繊維芽細胞の播れた三次元皮膚モデル（商品名：ＴＥＳＴＳＫＩＮ（ＴＯＹＯＢＯ））を、１−ＰＰを１％含有する培地（アッセイ培地（ＴＯＹＯＢＯ））中で３７℃にて３日間培養し、しかる後１５mＪ/cm2にて１回ＵＶＢ照射を施し（３日目）、さらに翌日同様に１５mＪ/cm2にて１回ＵＶＢ照射を施し（４日目）、そして同様の１−ＰＰ添加培地中で培養してからＰＦＡ固定し、上記のとおりにして組織学的検討を行った。コントロ−ルとして、１−ＰＰの添加されていない同様の培地で皮膚三次元モデルを同様にして培養し、組織学的検討を行った。 A three-dimensional skin model (trade name: TESTSKIN (TOYOBO)) seeded with collagen and fibroblasts on an insert mesh (size: about 2 cm), a medium containing 1% 1-PP (assay medium (TOYOBO)) ) At 37 ° C. for 3 days, then once irradiated with UVB once at 15 mJ / cm 2 (3rd day), and then once again irradiated with UVB once at 15 mJ / cm 2 (4 On the first day), the cells were cultured in the same 1-PP-added medium, fixed with PFA, and histologically examined as described above. As a control, a three-dimensional skin model was cultured in the same medium to which 1-PP was not added, and histological examination was performed.
その結果を図５に示す。１−ＰＰの無添加培地は顕著な表皮異常増殖と、細胞における核の残存により示される不全角化が観察された。一方、１−ＰＰ添加培地中で培養された三次元皮膚モデルにおいては、表皮異常増殖の抑制や、表皮細胞中の核の残存で示される不全角化の抑制、即ちＵＶＢによる皮膚障害の抑制が認められた。従って、本発明に係る三次元皮膚モデルは不全角化及び／又は表皮増殖異常改善薬剤のスクリーニングに有効であることがわかる。 The result is shown in FIG. In the medium without addition of 1-PP, marked epidermal abnormal growth and aberrant keratinization due to nuclei remaining in the cells were observed. On the other hand, in a three-dimensional skin model cultured in a medium supplemented with 1-PP, suppression of epidermal abnormal growth, suppression of aberrant keratinization indicated by remaining nuclei in epidermal cells, that is, suppression of skin damage by UVB. Admitted. Therefore, it can be seen that the three-dimensional skin model according to the present invention is effective in screening for a drug for improving keratinization and / or epidermal proliferation abnormality.
１ トランスウェル（インサート）
２ ケラチノサイト
３ 微小孔膜
４ 培地
５ コラーゲン＋繊維芽細胞
1 Transwell (insert)
2 Keratinocytes 3 Microporous membrane 4 Medium 5 Collagen + fibroblast
Claims (5)
５≦Ｘ×Ｙ／Ｚ≦１５
（式中、
ＸはＵＶＢの強度（ｍＪ）、
ＹはＵＶＢ照射回数、そして
ＺはＵＶＢ照射時間（日数）を表し、
但し、１０≦Ｘであるか、又は
Ｘ≦１０のとき、２≦Ｙである。）。 A method for producing a three-dimensional skin model with abnormal keratinization and / or abnormal epidermal proliferation by irradiating UVB with the following conditions on a three-dimensional skin model comprising keratinocytes and fibroblasts:
5 ≦ X × Y / Z ≦ 15
(Where
X is the intensity of UVB (mJ),
Y represents the number of UVB irradiations, and Z represents the UVB irradiation time (days),
However, when 10 ≦ X, or when X ≦ 10, 2 ≦ Y. ).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008093131A JP5219586B2 (en) | 2008-03-31 | 2008-03-31 | Method for producing a three-dimensional skin model with keratinization and epidermal proliferation abnormality |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2008093131A JP5219586B2 (en) | 2008-03-31 | 2008-03-31 | Method for producing a three-dimensional skin model with keratinization and epidermal proliferation abnormality |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2009240271A true JP2009240271A (en) | 2009-10-22 |
| JP5219586B2 JP5219586B2 (en) | 2013-06-26 |
Family
ID=41302963
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2008093131A Expired - Fee Related JP5219586B2 (en) | 2008-03-31 | 2008-03-31 | Method for producing a three-dimensional skin model with keratinization and epidermal proliferation abnormality |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP5219586B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009242345A (en) * | 2008-03-31 | 2009-10-22 | Shiseido Co Ltd | Scca-1 production inhibitor having 1-piperidinepropionic acid and/or its salt as active ingredient |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002218971A (en) * | 2001-01-29 | 2002-08-06 | Kanebo Ltd | Artificial skin model, method for preparing the same and its use |
| JP2006254875A (en) * | 2005-03-18 | 2006-09-28 | Shiseido Co Ltd | Method for screening substance suppressing parakeratosis, substance screened by the same method and method for suppressing parakeratosis |
-
2008
- 2008-03-31 JP JP2008093131A patent/JP5219586B2/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2002218971A (en) * | 2001-01-29 | 2002-08-06 | Kanebo Ltd | Artificial skin model, method for preparing the same and its use |
| JP2006254875A (en) * | 2005-03-18 | 2006-09-28 | Shiseido Co Ltd | Method for screening substance suppressing parakeratosis, substance screened by the same method and method for suppressing parakeratosis |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2009242345A (en) * | 2008-03-31 | 2009-10-22 | Shiseido Co Ltd | Scca-1 production inhibitor having 1-piperidinepropionic acid and/or its salt as active ingredient |
Also Published As
| Publication number | Publication date |
|---|---|
| JP5219586B2 (en) | 2013-06-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ge et al. | The aging skin microenvironment dictates stem cell behavior | |
| Jain | Dermatology: illustrated study guide and comprehensive board review | |
| Desmet et al. | In vitro psoriasis models with focus on reconstructed skin models as promising tools in psoriasis research | |
| Bertolotti et al. | Skin wound healing in different aged Xenopus laevis | |
| Huang et al. | Lgr6 marks epidermal stem cells with a nerve-dependent role in wound re-epithelialization | |
| Jean et al. | Effects of retinoic acid on keratinocyte proliferation and differentiation in a psoriatic skin model | |
| Munkhbayar et al. | Role of arachidonic acid in promoting hair growth | |
| Tran et al. | In vitro and in vivo studies with tetra‐hydro‐jasmonic acid (LR2412) reveal its potential to correct signs of skin ageing | |
| Kim et al. | Glucosamine improved atopic dermatitis‐like skin lesions in NC/Nga mice by inhibition of Th2 cell development | |
| Boisnic et al. | Repair of UVA-induced elastic fiber and collagen damage by 0.05% retinaldehyde cream in an ex vivo human skin model | |
| Kimura et al. | Tissue-scale tensional homeostasis in skin regulates structure and physiological function | |
| Smith et al. | The skin as a route of allergen exposure: part I. Immune components and mechanisms | |
| AU2020320171A1 (en) | YAP inhibition for wound healing | |
| Li et al. | Characterization of slow-cycling cells in the mouse cochlear lateral wall | |
| Nilforoushzadeh et al. | Soft tissue augmentation by autologous cultured fibroblasts transplantation for treatment of wrinkles and scars: a case series of 20 patients | |
| Zhao et al. | DNA damage-inducible transcript 4 is an innate surveillant of hair follicular stress in vitamin D receptor knockout mice and a regulator of wound re-epithelialization | |
| Rognoni et al. | Role of distinct fibroblast lineages and immune cells in dermal repair following UV radiation-induced tissue damage | |
| Ajani et al. | Cellular responses to disruption of the permeability barrier in a three-dimensional organotypic epidermal model | |
| Pageon et al. | UVA exposure combined with glycation of the dermis are two catalysts for skin aging and promotes a favorable environment to the appearance of elastosis | |
| Yoshida et al. | Changes in the Expression of Smooth Muscle Cell–Related Genes in Human Dermal Sheath Cup Cells Associated with the Treatment Outcome of Autologous Cell–Based Therapy for Male and Female Pattern Hair Loss | |
| JP5219586B2 (en) | Method for producing a three-dimensional skin model with keratinization and epidermal proliferation abnormality | |
| Harrison et al. | Use of an in vitro model of tissue‐engineered human skin to study keratinocyte attachment and migration in the process of reepithelialization | |
| Negrini et al. | Clinical and histological findings of cutaneous wound healing in the red‐eared slider turtle (Trachemys scripta elegans) housed in unheated outdoor enclosures | |
| JP6182728B2 (en) | Methods for evaluating efficacy and toxicity targeting stem cells | |
| JP2011120577A (en) | Method for preparing epithelium-reconstructed object, and method of screening by using epithelium-reconstructed object |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20110310 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20130205 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20130305 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20160315 Year of fee payment: 3 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 5219586 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| LAPS | Cancellation because of no payment of annual fees |