JP7057557B2 - Methods for assessing therapeutic and / or preventive efficacy against epithelial diseases, screening methods for therapeutic agents for epithelial diseases, and therapeutic agents for epithelial diseases - Google Patents

Methods for assessing therapeutic and / or preventive efficacy against epithelial diseases, screening methods for therapeutic agents for epithelial diseases, and therapeutic agents for epithelial diseases Download PDF

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JP7057557B2
JP7057557B2 JP2017532548A JP2017532548A JP7057557B2 JP 7057557 B2 JP7057557 B2 JP 7057557B2 JP 2017532548 A JP2017532548 A JP 2017532548A JP 2017532548 A JP2017532548 A JP 2017532548A JP 7057557 B2 JP7057557 B2 JP 7057557B2
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cyclophilin
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幸二 西田
竜平 林
陽一 本間
徹 大久保
峻 柴田
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Description

本発明は、上皮疾患に対する治療及び／又は予防有効性を評価する方法、上皮疾患治療剤のスクリーニング方法、及び上皮疾患治療剤に関する。 The present invention relates to a method for evaluating therapeutic and / or preventive efficacy for epithelial diseases, a method for screening an agent for treating epithelial diseases, and an agent for treating epithelial diseases.

上皮間葉転換（Ｅｐｉｔｈｅｌｉａｌ Ｍｅｓｅｎｃｈｙｍａｌ Ｔｒａｎｓｉｔｉｏｎ；ＥＭＴ）とは、上皮細胞がその細胞極性や周囲細胞との細胞接着機能を失い、遊走、浸潤能を得ることで間葉系様の細胞へと変化するプロセスである。上皮間葉転換は中胚葉形成や神経管形成等を含む様々な発生プロセス、創傷治癒において重要な役割を果たしている。また、上皮間葉転換が、組織の線維化、癌の浸潤、転移等の多くの疾患においても関与していることが近年明らかになってきている。 Epithelial-Messymal Transition (EMT) is a process in which epithelial cells lose their cell polarity and cell adhesion function with surrounding cells, and gain migration and invasion ability to transform into mesenchymal-like cells. Is. Epithelial-mesenchymal transition plays an important role in various developmental processes including mesoderm formation and neural tube formation, and wound healing. In recent years, it has become clear that epithelial-mesenchymal transition is also involved in many diseases such as tissue fibrosis, cancer infiltration, and metastasis.

例えば、殆どの癌において、転移の間、腫瘍細胞で上皮間葉転換が起こることが知られている（非特許文献１参照）。また、上皮間葉転換は、損傷組織が線維化反応を起こすための共通のメカニズムであると広く示唆されている。さらに、近年、トランスフォーミング増殖因子（ＴＧＦ）－βがｉｎ ｖｉｔｒｏ及びｉｎ ｖｉｖｏで肺胞上皮細胞（ＡＥＣ）、***上皮細胞、その他の上皮細胞におけるＥＭＴのインデューサーであることが示されている（非特許文献２～７参照）。このようにＥＭＴは、その基礎疾患の病因を問わず、線維化において共通の普遍的な役割を果たしている可能性がある。 For example, in most cancers, it is known that epithelial-mesenchymal transition occurs in tumor cells during metastasis (see Non-Patent Document 1). It has also been widely suggested that epithelial-mesenchymal transition is a common mechanism for injured tissue to undergo a fibrotic response. Furthermore, in recent years, transforming growth factor (TGF) -β has been shown to be an inducer of EMT in alveolar epithelial cells (AEC), breast epithelial cells, and other epithelial cells in vitro and in vivo (in vitro and in vivo). See Non-Patent Documents 2-7). Thus, EMT may play a common universal role in fibrosis, regardless of the etiology of its underlying disease.

癌の転移や線維化が起こる分子メカニズムについての研究は、ある程度進展しているにもかかわらず、癌転移、線維症には未だ十分に有効な治療剤が存在しない。特に、線維症を患う患者にとっての唯一の救済策は臓器移植であるため、種々の線維症に対して優れた効果を有する新規治療剤が強く求められている。 Although research on the molecular mechanism by which cancer metastasis and fibrosis occur has progressed to some extent, there is still no sufficiently effective therapeutic agent for cancer metastasis and fibrosis. In particular, since the only remedy for patients suffering from fibrosis is organ transplantation, there is a strong demand for new therapeutic agents having excellent effects on various fibrosis.

一方、細胞や細胞の培養上清を用いる治療方法として、間葉系幹細胞やその培養上清を含む医薬組成物を、標的組織の損傷部を修復するために用いる方法が知られている（特許文献１及び２参照）。また、間充織幹細胞の培養上清を含む医薬組成物を、疾病または損傷による続発性筋肉線維症等の治療に用いる方法も知られている（特許文献３参照）。しかし、これらの医薬組成物は癌転移やその他の線維症に十分有効な治療薬ではなく、さらには、細胞や細胞培養上清を含む医薬組成物の治療有効性を簡便に評価したり、医薬品開発への適性を迅速に判断してスクリーニングする方法は、知られていない。 On the other hand, as a therapeutic method using cells or a culture supernatant of cells, a method of using a mesenchymal stem cell or a pharmaceutical composition containing the culture supernatant to repair a damaged part of a target tissue is known (patented). See Documents 1 and 2). Further, there is also known a method of using a pharmaceutical composition containing a culture supernatant of mesenchymal stem cells for the treatment of secondary muscular fibrosis due to disease or injury (see Patent Document 3). However, these pharmaceutical compositions are not sufficiently effective therapeutic agents for cancer metastasis and other fibrosis, and further, the therapeutic efficacy of pharmaceutical compositions containing cells and cell culture supernatants can be easily evaluated, or pharmaceutical products can be used. No method is known to quickly determine and screen for development suitability.

細胞はその種類や処理条件等により種々の機能を備え、またその培養上清は細胞の種類やその処理条件等によって異なる種々多様な因子を含むため、医薬品成分、化粧品成分等として注目されている。このような種々多様な細胞や細胞培養上清について、上皮疾患に対する治療・予防効果を有するか否か、その効果はどの程度か、等について評価・判定するためには、例えば動物モデルを使用した実験を行う必要があり、評価に必要な時間、コストの面において負担が大きい。 Since cells have various functions depending on the type and treatment conditions, and the culture supernatant contains various factors that differ depending on the cell type and the treatment conditions, etc., they are attracting attention as pharmaceutical ingredients, cosmetic ingredients, and the like. .. For example, an animal model was used to evaluate and judge whether or not such various cells and cell culture supernatants have a therapeutic / preventive effect on epithelial diseases and how much the effect is. It is necessary to carry out an experiment, which is a heavy burden in terms of time and cost required for evaluation.

一方、サイクロフィリンは、免疫抑制剤サイクロスポリンＡに結合する蛋白質として見出され、免疫細胞の活性化に関与することが知られている（非特許文献８参照）。ヒトサイクロフィリンとしては、サイクロフィリンＡ（非特許文献９参照）、サイクロフィリンＢ（非特許文献１０参照）及びサイクロフィリンＣ（非特許文献１１参照）が知られている。しかし、サイクロフィリンとＥＭＴとの関連については何ら報告された例がない。 On the other hand, cyclophilin is found as a protein that binds to the immunosuppressant cyclosporin A and is known to be involved in the activation of immune cells (see Non-Patent Document 8). As human cyclophilins, cyclophilin A (see Non-Patent Document 9), cyclophilin B (see Non-Patent Document 10), and cyclophilin C (see Non-Patent Document 11) are known. However, there are no reports of the association between cyclophilin and EMT.

国際公開２０１１／１１８７９５号International Publication 2011/118795 特表２０１０－５４０６６２号公報Special Table 2010-540662 特表２００７－５２８７０３号公報Special Table 2007-528703

Ｔｈｉｅｒｙ，Ｊ．Ｐ．、Ｎａｔ．Ｒｅｖ．Ｃａｎｃｅｒ、２００２、ｖｏｌ．２：ｐ．４４２－４５４Thierry, J.M. P. , Nat. Rev. Cancer, 2002, vol. 2: p. 442-454 Ｍｉｅｔｔｉｎｅｎ，Ｐ．Ｊ．ｅｔ ａｌ．、Ｊｏｕｒｎａｌ ｏｆ ｃｅｌｌ Ｂｉｏｌｏｇｙ、１９９４、ｖｏｌ．１２７：ｐ．２０２１－２０３６Miettinen, P. et al. J. et al. , Journal of cell Biology, 1994, vol. 127: p. 2021-2036 Ｆａｎ，Ｊ．Ｍ．ｅｔ ａｌ．、Ｋｉｄｎｅｙ Ｉｎｔ、１９９９、ｖｏｌ．５６：ｐ．１４５５－１４６７Fan, J.M. M. et al. , Kidney Int, 1999, vol. 56: p. 1455-1467 Ｈａｌｅｓ，Ａ．Ｍ．ｅｔ ａｌ．、Ｃｕｒｒ Ｅｙｅ Ｒｅｓ、１９９４、ｖｏｌ．１３：ｐ．８８５－８９０Halles, A. M. et al. , Curr Eye Res, 1994, vol. 13: p. 885-890 Ｋａｓａｉ，Ｈ．ｅｔ ａｌ．、Ｒｅｓｐｉｒ Ｒｅｓ、２００５，ｖｏｌ．６：ｐ．５６Kai, H. et al. et al. , Respir Res, 2005, vol. 6: p. 56 Ｓａｉｋａ，Ｓ．ｅｔ ａｌ．、Ａｍ Ｊ Ｐａｔｈｏｌ， ２００４，ｖｏｌ．１６４：ｐ．６５１－６６３Saika, S.A. et al. , Am J Pathol, 2004, vol. 164: p. 651-663 Ｗｉｌｌｉｓ，ＢＣ．ｅｔ ａｌ．、Ａｍ Ｊ Ｐａｔｈｏｌ，２００５，ｖｏｌ．１６６：ｐ．１３２１－１３３２Willis, BC. et al. , Am J Pathol, 2005, vol. 166: p. 1321-1332 Ｈａｎｄｓｃｈｕｍａｃｈｅｒ，Ｒ．Ｅ．ｅｔ ａｌ．、Ｓｃｉｅｎｃｅ、１９８４、２２６：ｐ．５４４Handschumacher, R.M. E. et al. , Science, 1984, 226: p. 544 Ｈａｅｎｄｌｅｒ，Ｂ．、ＥＭＢＯ Ｊ、１９８７、６（４）：ｐ．９４７Handler, B. et al. , EMBO J, 1987, 6 (4): p. 947 Ｐｒｉｃｅ，Ｅ．Ｒ．ｅｔ ａｌ．、Ｐｒｏｃ．Ｎａｔｌ．Ａｃａｄ．Ｓｃｉ．Ｕ．Ｓ．Ａ．、１９９１、８８（５）：ｐ．１９０３Price, E.I. R. et al. , Proc. Natl. Acad. Sci. U. S. A. , 1991, 88 (5): p. 1903 Ｓｃｈｎｅｉｄｅｒ，Ｈ．ｅｔ ａｌ．、Ｂｉｏｃｈｅｍｉｓｔｒｙ、１９９４、３３（２７）：ｐ．８２１８Schneider, H. et al. et al. , Biochemistry, 1994, 33 (27): p. 8218

このような情況の中、種々多様な細胞又は細胞培養上清を含む製剤について、上皮疾患に対する治療・予防効果を有するか否か、その効果はどの程度か、等について、簡便かつ効率よく評価・判定できる方法が求められている。そこで、本発明は、上皮疾患に対する治療・予防効果を有する細胞又は細胞培養上清を簡便に選択でき、かつ細胞又は細胞培養上清を含む上皮疾患の治療剤において、薬理効果・所定の品質等を有するか否かの評価、判断が簡便かつ高精度で実施可能となる、上皮疾患に対する治療及び／又は予防有効性を評価する方法を提供することを課題とする。さらに、この方法を使用した、細胞又は細胞培養上清を含む上皮疾患治療剤のスクリーニング方法、及びこのスクリーニング方法により選択される上皮疾患治療剤を提供することも課題とする。 Under such circumstances, it is easy and efficient to evaluate whether or not a preparation containing various cells or cell culture supernatants has a therapeutic / preventive effect on epithelial diseases, and how much the effect is. There is a need for a method that can be determined. Therefore, the present invention can easily select cells or cell culture supernatants having therapeutic / preventive effects on epithelial diseases, and has pharmacological effects, predetermined qualities, etc. in therapeutic agents for epithelial diseases including cells or cell culture supernatants. It is an object of the present invention to provide a method for evaluating the therapeutic and / or preventive efficacy for epithelial diseases, which enables easy and highly accurate evaluation and judgment as to whether or not the cells have. Further, it is also an object to provide a screening method for an epithelial disease therapeutic agent containing cells or cell culture supernatant using this method, and an epithelial disease therapeutic agent selected by this screening method.

上記課題を解決するために種々検討した結果、本発明者らは、細胞におけるサイクロフィリンＢ発現量、又は細胞培養上清中のサイクロフィリンＢの含有量が、その細胞又は細胞培養上清が示す上皮疾患に対する治療・予防効果と相関することを見出し、本発明を完成するに至った。即ち、細胞又は細胞培養上清中のサイクロフィリンＢを指標とすることで、この細胞培養上清が上皮疾患に対する治療・予防効果を有するか否か、またその効果の程度について、簡便に評価できることを見出した。 As a result of various studies to solve the above problems, the present inventors indicate the expression level of cyclophilin B in the cell or the content of cyclophilin B in the cell culture supernatant by the cell or the cell culture supernatant. We have found that it correlates with the therapeutic / preventive effect on epithelial diseases, and have completed the present invention. That is, by using cyclophilin B in cells or cell culture supernatant as an index, it is possible to easily evaluate whether or not this cell culture supernatant has a therapeutic / preventive effect on epithelial diseases and the degree of the effect. I found.

本発明の要旨は、以下に記載する通りである。
〔１〕細胞及び／又はその培養上清を含む製剤において、サイクロフィリンＢを指標に、上記製剤の、上皮疾患に対する治療及び／又は予防有効性を評価する方法。
〔２〕上記上皮疾患が、上皮間葉転換が関与している疾患である、〔１〕記載の方法。
〔３〕上記上皮疾患が、眼部、肝臓、肺、腎臓、消化管、気道又は腹膜における疾患である、〔１〕又は〔２〕記載の方法。
〔４〕上記上皮疾患が、線維症である、〔１〕から〔３〕のいずれか記載の方法。
〔５〕上記上皮疾患が、角膜疾患、網膜疾患又は表皮疾患である、〔１〕から〔４〕のいずれか記載の方法。
〔６〕上記上皮疾患が、翼状片、瘢痕、ＥＢウイルス角膜炎、角膜上皮幹細胞疲弊症、硝子体網膜症、又は強皮症である、〔５〕記載の方法。
〔７〕上記上皮疾患が、癌である、〔１〕又は〔２〕記載の方法。
〔８〕上記細胞が、間葉系幹細胞である、〔１〕から〔７〕のいずれか記載の方法。
〔９〕上記間葉系幹細胞が、脂肪由来間葉系幹細胞又は臍帯由来間葉系幹細胞である、〔８〕記載の方法。
〔１０〕細胞におけるサイクロフィリンＢの発現量、又は細胞培養上清中のサイクロフィリンＢの含有量を測定する工程を含む、上記細胞又はその培養上清の、上皮疾患に対する治療及び／又は予防有効性を評価する方法。
〔１１〕得られたサイクロフィリンＢ量を、基準となるサイクロフィリンＢ量と比較する工程をさらに含む、〔１０〕記載の方法。
〔１２〕〔１〕から〔１１〕のいずれか記載の方法を用いる、上皮疾患治療剤のスクリーニング方法。
〔１３〕〔１２〕記載のスクリーニング方法により選択された上皮疾患治療剤であって、間葉系幹細胞又はその培養上清を含むことを特徴とする、上皮疾患治療剤。
〔１４〕上記上皮疾患が、上皮間葉転換が関与している疾患である、〔１３〕記載の上皮疾患治療剤。
〔１５〕上記上皮疾患が、眼部、肝臓、肺、腎臓、消化管、気道又は腹膜における疾患である、〔１３〕又は〔１４〕記載の上皮疾患治療剤。
〔１６〕上記上皮疾患が、線維症である、〔１３〕から〔１５〕のいずれか記載の上皮疾患治療剤。
〔１７〕上記上皮疾患が、角膜疾患、網膜疾患又は表皮疾患である、〔１３〕から〔１６〕のいずれか記載の上皮疾患治療剤。
〔１８〕上記上皮疾患が、翼状片、瘢痕、ＥＢウイルス角膜炎、角膜上皮幹細胞疲弊症、硝子体網膜症、又は強皮症である、〔１７〕記載の上皮疾患治療剤。
〔１９〕上記上皮疾患が、癌である、〔１３〕記載の上皮疾患治療剤。The gist of the present invention is as described below.
[1] A method for evaluating the therapeutic and / or preventive efficacy of the above-mentioned preparation for epithelial diseases using cyclophilin B as an index in a preparation containing cells and / or a culture supernatant thereof.
[2] The method according to [1], wherein the epithelial disease is a disease in which epithelial-mesenchymal transition is involved.
[3] The method according to [1] or [2], wherein the epithelial disease is a disease in the eye, liver, lung, kidney, gastrointestinal tract, respiratory tract or peritoneum.
[4] The method according to any one of [1] to [3], wherein the epithelial disease is fibrosis.
[5] The method according to any one of [1] to [4], wherein the epithelial disease is a corneal disease, a retinal disease or an epidermal disease.
[6] The method according to [5], wherein the epithelial disease is pterygium, scar, EB virus keratitis, corneal epithelial stem cell exhaustion, vitreous retinopathy, or scleroderma.
[7] The method according to [1] or [2], wherein the epithelial disease is cancer.
[8] The method according to any one of [1] to [7], wherein the cell is a mesenchymal stem cell.
[9] The method according to [8], wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells.
[10] Effectiveness for treating and / or preventing epithelial diseases of the above cells or the culture supernatant thereof, which comprises a step of measuring the expression level of cyclophilin B in the cells or the content of cyclophilin B in the cell culture supernatant. How to evaluate sex.
[11] The method according to [10], further comprising a step of comparing the obtained amount of cyclophilin B with the reference amount of cyclophilin B.
[12] A method for screening an epithelial disease therapeutic agent using the method according to any one of [1] to [11].
[13] An epithelial disease therapeutic agent selected by the screening method according to [12], which comprises mesenchymal stem cells or a culture supernatant thereof.
[14] The therapeutic agent for epithelial disease according to [13], wherein the epithelial disease is a disease in which epithelial-mesenchymal transition is involved.
[15] The therapeutic agent for epithelial disease according to [13] or [14], wherein the epithelial disease is a disease in the eye, liver, lung, kidney, gastrointestinal tract, respiratory tract or peritoneum.
[16] The agent for treating an epithelial disease according to any one of [13] to [15], wherein the epithelial disease is fibrosis.
[17] The agent for treating epithelial diseases according to any one of [13] to [16], wherein the epithelial disease is a corneal disease, a retinal disease or an epithelial disease.
[18] The therapeutic agent for epithelial diseases according to [17], wherein the epithelial disease is pterygium, scar, EB virus keratitis, corneal epithelial stem cell exhaustion, vitreous retinopathy, or scleroderma.
[19] The agent for treating epithelial diseases according to [13], wherein the epithelial disease is cancer.

本発明によると、上皮疾患に対する治療・予防効果を有する細胞又は細胞培養上清を簡便に選択でき、かつ細胞又は細胞培養上清を含む上皮疾患の治療剤が、薬理効果・所定の品質等を有するか否かの評価・判断が簡便かつ高精度で実施可能となる、上皮疾患に対する治療及び／又は予防有効性を評価する方法を提供することができる。また、この方法を用いたスクリーニング方法によると、上皮疾患に対する優れた治療・予防効果を有する細胞又は細胞培養上清を簡便かつ高精度で選択することができるため、新規の上皮疾患治療剤の探索研究において、有効に活用することができる。これにより、開発者の負担を顕著に低減することもできる。さらに、細胞及び／又は細胞培養上清を含む上皮疾患の治療剤が、薬理効果・所定の品質等を有するか否か、例えば薬事法上の承認を受ける際の規格を満たしているか否かの判断等においても、本発明の方法によると、簡便かつ高精度で実施可能となる。 According to the present invention, cells or cell culture supernatants having a therapeutic / preventive effect on epithelial diseases can be easily selected, and a therapeutic agent for epithelial diseases containing cells or cell culture supernatants has a pharmacological effect, predetermined quality, etc. It is possible to provide a method for evaluating the therapeutic and / or preventive effectiveness for epithelial diseases, which enables easy and highly accurate evaluation / judgment as to whether or not the cell has. In addition, according to the screening method using this method, cells or cell culture supernatants having excellent therapeutic / preventive effects on epithelial diseases can be selected easily and with high accuracy, so that a search for a new therapeutic agent for epithelial diseases can be performed. It can be effectively used in research. As a result, the burden on the developer can be significantly reduced. Furthermore, whether or not the therapeutic agent for epithelial diseases including cells and / or cell culture supernatant has a pharmacological effect, a predetermined quality, etc., for example, whether or not it meets the standards for approval under the Pharmaceutical Affairs Law. In terms of determination and the like, according to the method of the present invention, it can be carried out easily and with high accuracy.

各種細胞培養上清中のサイクロフィリンＢのウェスタンブロッティングの結果を示す図である。It is a figure which shows the result of Western blotting of cyclophilin B in the supernatant of various cell cultures. 各種細胞培養上清のＥＭＴ抑制効果を示す図である。It is a figure which shows the EMT suppression effect of the various cell culture supernatants. 間葉系幹細胞培養上清のＥＭＴ抑制効果を示す図である。It is a figure which shows the EMT suppression effect of the mesenchymal stem cell culture supernatant. 網膜色素上皮細胞に対するＥＭＴ誘導剤の作用を示す図である。It is a figure which shows the action of the EMT inducer on the retinal pigment epithelial cell. 間葉系幹細胞培養上清のＥＭＴ抑制効果を示す図である。It is a figure which shows the EMT suppression effect of the mesenchymal stem cell culture supernatant.

以下、本発明について詳細に説明する。 Hereinafter, the present invention will be described in detail.

＜細胞又はその培養上清を含む製剤の、上皮疾患に対する治療及び／又は予防有効性を評価する方法＞
本発明の評価方法は、細胞及び／又はその培養上清中のサイクロフィリンＢを指標に、上記細胞又はその培養上清の、上皮疾患に対する治療及び／又は予防有効性を評価できる方法である。以下に、本発明の評価方法を詳細に説明する。本発明の評価方法は、細胞におけるサイクロフィリンＢの発現量、又は細胞培養上清中のサイクロフィリンＢの含有量を測定する工程（以下、「測定工程」ともいう）を含む。また、得られたサイクロフィリンＢ量を、基準となるサイクロフィリンＢ量と比較する工程（以下、「比較工程」ともいう）を含むことが好ましい。さらに、細胞又は細胞培養上清の上皮疾患に対する治療・予防効果を確認するために上皮間葉転換（ＥＭＴ）抑制活性を測定する工程（以下、「確認工程」ともいう）を含むことがより好ましい。なお、上記サイクロフィリンＢ量とは、細胞におけるサイクロフィリンＢの発現量及び／又は細胞培養上清中のサイクロフィリンＢの含有量をいう。<Method for evaluating the therapeutic and / or preventive efficacy of a preparation containing cells or a culture supernatant thereof for epithelial diseases>
The evaluation method of the present invention is a method capable of evaluating the therapeutic and / or preventive effectiveness of the cells or the culture supernatant of the cells or the culture supernatant thereof for epithelial diseases by using cyclophilin B in the cells and / or the culture supernatant thereof as an index. Hereinafter, the evaluation method of the present invention will be described in detail. The evaluation method of the present invention includes a step of measuring the expression level of cyclophilin B in cells or the content of cyclophilin B in the cell culture supernatant (hereinafter, also referred to as “measurement step”). Further, it is preferable to include a step of comparing the obtained amount of cyclophilin B with the reference amount of cyclophilin B (hereinafter, also referred to as “comparison step”). Further, it is more preferable to include a step of measuring the epithelial-mesenchymal transition (EMT) inhibitory activity (hereinafter, also referred to as "confirmation step") in order to confirm the therapeutic / preventive effect of the cell or the cell culture supernatant on the epithelial disease. .. The amount of cyclophilin B refers to the expression level of cyclophilin B in cells and / or the content of cyclophilin B in the cell culture supernatant.

上記測定工程においては、細胞におけるサイクロフィリンＢの発現量、又は細胞培養上清中のサイクロフィリンＢの含有量を測定する。 In the above measurement step, the expression level of cyclophilin B in cells or the content of cyclophilin B in the cell culture supernatant is measured.

本発明の方法により評価される細胞としては、動物細胞であってもよいし、植物細胞であってもよいが、細胞を含む医薬組成物を使用する対象と同種由来の細胞であることが好ましい。特に、ヒトの上皮疾患に対する治療、予防のために使用する場合には、ヒトの細胞であることが好ましい。このような細胞としては、本発明者により培養上清に上皮疾患に対する効果（上皮間葉転換の抑制効果）が確認された間葉系幹細胞がより好ましく、例えば、骨髄、脂肪、筋肉、神経、皮膚、羊膜、胎盤、絨毛膜、脱落膜又は臍帯由来の間葉系幹細胞が挙げられるが、これらのうち、サイクロフィリンＢの分泌量が多い細胞である臍帯由来間葉系幹細胞、脂肪由来間葉系幹細胞、骨髄由来間葉系幹細胞がさらに好ましく、臍帯由来間葉系幹細胞、脂肪由来間葉系幹細胞が特に好ましい。 The cell evaluated by the method of the present invention may be an animal cell or a plant cell, but is preferably a cell derived from the same species as the subject using the pharmaceutical composition containing the cell. .. In particular, when used for the treatment or prevention of human epithelial diseases, human cells are preferable. As such cells, mesenchymal stem cells whose culture supernatant has been confirmed to have an effect on epithelial diseases (inhibitory effect on epithelial mesenchymal conversion) are more preferable, for example, bone marrow, fat, muscle, nerve, and the like. Mesenchymal stem cells derived from skin, sheep membrane, placenta, chorionic villi, decidua or umbilical cord can be mentioned. Among these, mesenchymal stem cells derived from umbilical cord and adipose-derived mesenchymal stem cells, which are cells that secrete a large amount of cyclophilin B. Lineage stem cells and bone marrow-derived mesenchymal stem cells are more preferable, and umbilical cord-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells are particularly preferable.

本発明の方法により評価され得る細胞培養上清としては、細胞を培養液中で培養して得られる培養上清であれば特に限定されない。細胞が増殖、又は維持され得る条件の下、細胞を培養して得られた培養液（培養後の培養液）から細胞を除去したものを意味するが、このような培養上清から、例えば、残存培地成分（培養前の培養液の成分のうち、培養後の培養液中に残存している成分）、培養液の水分などの、本発明の効果に寄与しない成分の少なくとも一部をさらに除去したものも、便宜上、本明細書における細胞培養上清に含まれるものとする。なお、簡便性の観点からは、培養後の培養液から細胞を除去したものをそのまま培養上清として用いることが好ましい。上記培養上清を得るための細胞としては、全段落と同様の細胞を挙げることができる。 The cell culture supernatant that can be evaluated by the method of the present invention is not particularly limited as long as it is a culture supernatant obtained by culturing cells in a culture medium. It means that the cells are removed from the culture medium (culture solution after culturing) obtained by culturing the cells under the condition that the cells can be proliferated or maintained, and from such a culture supernatant, for example, Further remove at least a part of the components that do not contribute to the effect of the present invention, such as residual medium components (components remaining in the culture solution after culture among the components of the culture solution before culture) and water content of the culture solution. For the sake of convenience, these are also included in the cell culture supernatant in the present specification. From the viewpoint of convenience, it is preferable to use the culture supernatant obtained by removing cells from the culture solution after culturing. Examples of the cells for obtaining the culture supernatant include cells similar to those in the whole paragraph.

本発明の評価方法により測定するサイクロフィリンＢは、当業者に公知の任意のサイクロフィリンＢである。種は特に限定されないが、評価対象の細胞又は細胞培養上清と同種由来のサイクロフィリンＢを測定対象とすることが好ましい。例えば、本発明の評価対象をヒトの上皮疾患の治療に用いる場合には、ヒト由来のサイクロフィリンＢを特異的に検出する手段を用いて測定することが好ましい。基準として用いるサイクロフィリンＢとしては、例えば、天然供給源からの抽出、サイクロフィリンＢをコードする組換え核酸の発現、又は化学合成によって得られ得る。サイクロフィリンＢをコードする組換え核酸の発現においては、真核生物、原核生物のいずれを使用してもよい。純度は、カラムクロマトグラフィー、ポリアクリルアミドゲル電気泳動のような任意の適切な方法又はＨＰＬＣ分析等によって測定され得る。 The cyclophilin B measured by the evaluation method of the present invention is any cyclophilin B known to those skilled in the art. The species is not particularly limited, but it is preferable to use cyclophilin B derived from the same species as the cell to be evaluated or the cell culture supernatant as the measurement target. For example, when the evaluation target of the present invention is used for the treatment of human epithelial diseases, it is preferable to measure by using a means for specifically detecting cyclophilin B derived from humans. The cyclophilin B used as a reference can be obtained, for example, by extraction from a natural source, expression of a recombinant nucleic acid encoding cyclophilin B, or chemical synthesis. Either eukaryotes or prokaryotes may be used in the expression of the recombinant nucleic acid encoding cyclophilin B. Purity can be measured by any suitable method such as column chromatography, polyacrylamide gel electrophoresis, HPLC analysis and the like.

ヒト由来のサイクロフィリンＢとしては、配列表の配列番号１のアミノ酸配列を含むものが挙げられる。マウス由来のサイクロフィリンＢとしては、配列表の配列番号２のアミノ酸配列を含むものが挙げられる。 Examples of the human-derived cyclophilin B include those containing the amino acid sequence of SEQ ID NO: 1 in the sequence listing. Examples of the mouse-derived cyclophilin B include those containing the amino acid sequence of SEQ ID NO: 2 in the sequence listing.

サイクロフィリンＢ量のうち、細胞におけるサイクロフィリンＢの発現量としては、細胞内又は細胞表面におけるタンパクの発現量でもよいし、ｍＲＮＡ等の遺伝子の発現量でもよい。細胞におけるサイクロフィリンＢの発現量の測定方法としては、当業者に従来公知の方法を用いることができる。例えば、細胞破砕液についてウェスタンブロッティングを行う方法、ＥＬＩＳＡによる方法等が挙げられる。また、細胞表面におけるタンパク発現の測定方法としては、ＦＡＣＳ解析等が挙げられる。上記ウェスタンブロッティングを行う方法、ＥＬＩＳＡによる方法、ＦＡＣＳ解析のいずれにおいても、サイクロフィリンＢに特異的に結合する抗サイクロフィリンＢ抗体を使用し、サイクロフィリンＢの発現量を精度よく測定することができる。なお、ウェスタンブロッティングを行う方法においては、得られるサイクロフィリンＢ特異的なブロットの強度を、濃度のわかる標準標品（ポジティブコントロール）の強度と比較することで、サンプル中のサイクロフィリンＢ発現量を算出することができる。 Among the amounts of cyclophilin B, the expression level of cyclophilin B in cells may be the expression level of a protein in the cell or on the cell surface, or the expression level of a gene such as mRNA. As a method for measuring the expression level of cyclophilin B in cells, a method conventionally known to those skilled in the art can be used. For example, a method of performing Western blotting on a cell disruption solution, a method by ELISA, and the like can be mentioned. Further, as a method for measuring protein expression on the cell surface, FACS analysis or the like can be mentioned. In any of the above Western blotting method, ELISA method, and FACS analysis, an anti-cyclophilin B antibody that specifically binds to cyclophilin B can be used to accurately measure the expression level of cyclophilin B. .. In the method of performing Western blotting, the intensity of the obtained cyclophilin B-specific blot is compared with the intensity of the standard standard (positive control) whose concentration is known, so that the expression level of cyclophilin B in the sample can be determined. Can be calculated.

サイクロフィリンＢ量のうち、細胞培養上清中のサイクロフィリンＢの含有量の測定方法としては、当業者に従来公知の方法を用いることができる。例えば、細胞培養上清についてウェスタンブロッティングを行う方法、ＥＬＩＳＡによる方法等が挙げられる。上記ウェスタンブロッティングを行う方法、ＥＬＩＳＡによる方法のいずれにおいても、サイクロフィリンＢに特異的に結合する抗サイクロフィリンＢ抗体を使用し、サイクロフィリンＢの発現量を精度よく測定することができる。なお、ウェスタンブロッティングを行う方法においては、得られるサイクロフィリンＢ特異的なブロットの強度を、濃度のわかる標準標品（ポジティブコントロール）の強度と比較することで、サンプル中のサイクロフィリンＢ含有量を算出することができる。 As a method for measuring the content of cyclophilin B in the cell culture supernatant among the amount of cyclophilin B, a method conventionally known to those skilled in the art can be used. For example, a method of performing Western blotting on the cell culture supernatant, a method by ELISA, and the like can be mentioned. In both the Western blotting method and the ELISA method, an anti-cyclophilin B antibody that specifically binds to cyclophilin B can be used to accurately measure the expression level of cyclophilin B. In the method of performing Western blotting, the cyclophilin B content in the sample is determined by comparing the strength of the obtained cyclophilin B-specific blot with the strength of the standard standard (positive control) whose concentration is known. Can be calculated.

本発明の評価方法においては、本比較工程により、評価対象の細胞におけるサイクロフィリンＢの発現量又は細胞培養上清中のサイクロフィリンＢ含有量と、基準とするサイクロフィリンＢの発現量又は含有量とを比較する。評価の目的、対象に合わせて、基準とするサイクロフィリンＢ量を適宜選択することができる。 In the evaluation method of the present invention, the expression level of cyclophilin B in the cells to be evaluated or the content of cyclophilin B in the cell culture supernatant and the expression level or content of reference cyclophilin B in the cells to be evaluated by this comparison step. And compare. The amount of cyclophilin B as a reference can be appropriately selected according to the purpose of evaluation and the subject.

さらに、本発明の評価方法による評価結果を確認するために、評価対象の細胞又は細胞培養上清について、上皮間葉転換（Ｅｐｉｔｈｅｌｉａｌ Ｍｅｓｅｎｃｈｙｍａｌ Ｔｒａｎｓｉｔｉｏｎ；ＥＭＴ）抑制効果を測定する「確認工程」を有することが好ましい。 Further, in order to confirm the evaluation result by the evaluation method of the present invention, the cell to be evaluated or the cell culture supernatant has a "confirmation step" for measuring the epithelial-mesenchymal transition (EMT) inhibitory effect. Is preferable.

ここで上皮間葉転換とは、上皮細胞がその細胞極性や周囲細胞との細胞接着機能を失い、遊走、浸潤能を得ることで間葉系様の細胞へと変化するプロセスである。この上皮間葉転換は、癌の転移や組織の線維化においても関与していることが近年明らかになってきている。従って、この上皮細胞における上皮間葉転換を抑制する効果がある細胞培養上清は、癌、線維症等の上皮疾患の治療・予防薬として有効である可能性が高い。そこで、本発明の評価方法においては、評価結果の確認のために、ＥＭＴ抑制効果を測定することが好ましい。 Here, epithelial-mesenchymal transition is a process in which epithelial cells lose their cell polarity and cell adhesion function with surrounding cells, and change into mesenchymal-like cells by gaining migration and invasion ability. In recent years, it has become clear that this epithelial-mesenchymal transition is also involved in cancer metastasis and tissue fibrosis. Therefore, the cell culture supernatant having the effect of suppressing epithelial-mesenchymal transition in these epithelial cells is highly likely to be effective as a therapeutic / preventive agent for epithelial diseases such as cancer and fibrosis. Therefore, in the evaluation method of the present invention, it is preferable to measure the EMT suppression effect in order to confirm the evaluation result.

上記ＥＭＴ抑制効果を測定する方法としては、例えば、ｉｎ ｖｉｔｒｏにおけるＥＭＴ抑制実験が挙げられる。即ち、任意の上皮細胞（組織から単離したプライマリーの細胞であっても、確立された細胞株であってもよい）に対して、ＥＭＴを誘導する処理を行う。このような処理としては、例えば、ＴＧＦ－β及びＴＮＦ－αによる処理が代表的な処理方法として挙げられる。これらの誘導剤によってＥＭＴが誘導されたことは、上皮細胞に特異的な遺伝子発現が低下し、間葉細胞に特異的な遺伝子発現が増強することにより確認することができる。上皮細胞に特異的に発現する遺伝子としては、例えば、Ｃｌａｕｄｉｎ－１、Ｃｌａｕｄｉｎ－３、Ｃｌａｕｄｉｎ－４、Ｅ－ｃａｄｈｅｒｉｎ、Ｃｙｔｏｋｅｒａｔｉｎ等が挙げられる。また、間葉細胞に特異的に発現する遺伝子としては、例えば、Ｖｉｍｅｎｔｉｎ、Ｓｌｕｇ、α－Ｓｍｏｏｔｈ Ｍｕｓｃｌｅ Ａｃｔｉｎ、Ｎ－ｃａｄｈｅｒｉｎ、Ｆｉｂｒｏｎｅｃｔｉｎ等が挙げられる。本発明の医薬組成物は、ＥＭＴを抑制する効果を有するので、ＥＭＴを誘導した細胞に作用させた場合に、又はＥＭＴを起す前の細胞に作用させた後にＥＭＴを誘導する処理をした場合に、上皮細胞特異的な遺伝子発現の低減を抑え、間葉細胞特異的な遺伝子発現の増強を抑制することができる。 Examples of the method for measuring the EMT suppression effect include an in vitro EMT suppression experiment. That is, any epithelial cell (which may be a primary cell isolated from a tissue or an established cell line) is subjected to a treatment for inducing EMT. As such a treatment, for example, a treatment with TGF-β and TNF-α can be mentioned as a typical treatment method. The induction of EMT by these inducers can be confirmed by the decrease in gene expression specific to epithelial cells and the enhancement of gene expression specific to mesenchymal cells. Examples of genes specifically expressed in epithelial cells include Claudin-1, Claudin-3, Claudin-4, E-cadherin, Cytokeratin and the like. Examples of genes specifically expressed in mesenchymal cells include Vimentin, Slug, α-Smooth Muscle Actin, N-cadherin, Fibronectin and the like. Since the pharmaceutical composition of the present invention has an effect of suppressing EMT, when it is applied to cells in which EMT is induced, or when it is treated to induce EMT after it is applied to cells before causing EMT. , It is possible to suppress the decrease in gene expression specific to epithelial cells and suppress the enhancement of gene expression specific to mesenchymal cells.

＜上皮疾患治療剤のスクリーニング方法＞
本発明の上皮疾患治療剤のスクリーニング方法は、本発明の上記評価方法を用いる。本発明の評価方法により、評価対象の細胞又は細胞培養上清の上皮疾患に対する治療・予防有効性を評価した後、上皮疾患治療剤として次の開発段階に進めるか否かを判断する方法である。本発明のスクリーニング方法によると、上皮疾患に対する治療・予防効果を有する細胞又は細胞培養上清を簡便に選択できるため、新規の上皮疾患治療剤の探索研究において、有効に活用することができる。<Screening method for therapeutic agents for epithelial diseases>
As the screening method for the therapeutic agent for epithelial diseases of the present invention, the above-mentioned evaluation method of the present invention is used. According to the evaluation method of the present invention, after evaluating the therapeutic / preventive efficacy of the cell to be evaluated or the cell culture supernatant for epithelial diseases, it is determined whether or not to proceed to the next development stage as a therapeutic agent for epithelial diseases. .. According to the screening method of the present invention, cells or cell culture supernatants having a therapeutic / preventive effect on epithelial diseases can be easily selected, and therefore, they can be effectively utilized in the exploratory research of new therapeutic agents for epithelial diseases.

＜上皮疾患治療剤＞
本発明は、本発明のスクリーニング方法により選択された上皮疾患治療剤も含む。
本発明の上皮疾患治療剤が含む細胞又は細胞培養上清における「細胞」としては、動物細胞であってもよいし、植物細胞であってもよいが、細胞を含む医薬組成物を使用する対象と同種由来の細胞であることが好ましい。特に、ヒトの上皮疾患に対する治療、予防のために使用する場合には、ヒトの細胞であることが好ましい。このような細胞としては、本発明者により培養上清に上皮疾患に対する効果（上皮間葉転換の抑制効果）が確認された間葉系幹細胞が好ましい。上記間葉系幹細胞としては、例えば、骨髄、脂肪、筋肉、神経、皮膚、羊膜、胎盤、絨毛膜、脱落膜又は臍帯由来の間葉系幹細胞が挙げられるが、これらのうち、サイクロフィリンＢの分泌量が多い細胞である臍帯由来間葉系幹細胞、脂肪由来間葉系幹細胞、骨髄由来間葉系幹細胞が好ましく、臍帯由来間葉系幹細胞、脂肪由来間葉系幹細胞がより好ましい。<Epithelial disease therapeutic agent>
The present invention also includes a therapeutic agent for epithelial diseases selected by the screening method of the present invention.
The "cell" in the cell or cell culture supernatant contained in the therapeutic agent for epithelial disease of the present invention may be an animal cell or a plant cell, but a subject for which a pharmaceutical composition containing the cell is used. It is preferable that the cells are derived from the same species as. In particular, when used for the treatment or prevention of human epithelial diseases, human cells are preferable. As such cells, mesenchymal stem cells whose culture supernatant has been confirmed to have an effect on epithelial diseases (inhibitory effect on epithelial-mesenchymal transition) by the present inventor are preferable. Examples of the mesenchymal stem cells include mesenchymal stem cells derived from bone marrow, fat, muscle, nerve, skin, sheep membrane, placenta, chorionic villi, decidua or umbilical cord, and among these, mesenchymal stem cells of cyclophilin B. Umbilical band-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, and bone marrow-derived mesenchymal stem cells, which are cells with a large amount of secretion, are preferable, and umbilical cord-derived mesenchymal stem cells and adipose-derived mesenchymal stem cells are more preferable.

本発明者らは、本発明のスクリーニング方法を用いて、間葉系幹細胞又はその培養上清がサイクロフィリンＢを含み、上皮間葉転換を抑制する効果を有することから、上皮疾患に対して優れた治療・予防効果を奏することを見出した。 Using the screening method of the present invention, the present inventors are excellent in treating epithelial diseases because the mesenchymal stem cells or the culture supernatant thereof contain cyclophilin B and have an effect of suppressing epithelial-mesenchymal transition. It was found to have a therapeutic / preventive effect.

本発明における上皮疾患治療剤として、以下のようにして得られる間葉系幹細胞又はその培養上清を含めることができる。上記間葉系幹細胞は、例えば以下の方法によって培養する。即ち、組織由来の間葉系幹細胞、株化された間葉系幹細胞等の間葉系幹細胞を馴化培地中で培養し、続いて目的に応じた特定の培地中で間葉系幹細胞を培養する。 As the therapeutic agent for epithelial diseases in the present invention, mesenchymal stem cells obtained as follows or a culture supernatant thereof can be included. The mesenchymal stem cells are cultured by, for example, the following method. That is, tissue-derived mesenchymal stem cells, mesenchymal stem cells that have been established, and the like are cultured in a conditioned medium, and then the mesenchymal stem cells are cultured in a specific medium according to the purpose. ..

上記馴化培地としては、当業者に従来公知の培地を間葉系幹細胞の種類毎にそれぞれ選択して用いることができ、特に限定されない。馴化培地としては、例えば、Ｐｒｏｍｏ Ｃｅｌｌ社、Ｌｉｆｅ Ｌｉｎｅ社、Ｌｏｎｚａ社等の間葉系幹細胞培養用培地等が挙げられる。馴化培地は、生物由来原料（例えば、動物血清）を含有してもよいが、得られる細胞やその培養上清を動物（ヒトを含む）の疾患の治療のために用いることを考慮すると、生物由来原料を含まない培地（例えば、無血清培地）であることが好ましい。 As the acclimation medium, a medium conventionally known to those skilled in the art can be selected and used for each type of mesenchymal stem cells, and is not particularly limited. Examples of the conditioned medium include media for culturing mesenchymal stem cells of Promo Cell, Life Line, Lonza and the like. The conditioned medium may contain biological material (eg, animal serum), but given that the resulting cells and culture supernatants thereof are used for the treatment of diseases of animals (including humans), organisms. A medium containing no derived raw material (for example, a serum-free medium) is preferable.

上記特定の培地は、目的や、間葉系幹細胞の種類毎にそれぞれ選択して用いることができる。例えば、通常間葉系幹細胞の培養に用いられる培地、通常間葉系幹細胞以外の培養に用いられる培地、馴化培地から特定の成分を除いた培地、又は馴化培地に特定の成分を加えた培地を用いることができる。例えば、Ｌｏｎｚａ社のＴｈｅｒａＰＥＡＫ（登録商標） ＭＳＣＧＭ－ＣＤ（登録商標） Ｍｅｓｅｎｃｈｙｍａｌ Ｓｔｅｍ Ｃｅｌｌ Ｍｅｄｉｕｍ， Ｃｈｅｍｉｃａｌｌｙ Ｄｅｆｉｎｅｄ等を用いることができるが、これに限定されない。ＦＣＳの含有量は、通常０．１％～２０％であり、０．２％～１０％であることが好ましい。なお、馴化培地と、この特定の培地とが同じ組成であってもよい。 The specific medium can be selected and used according to the purpose and the type of mesenchymal stem cells. For example, a medium used for culturing normal mesenchymal stem cells, a medium used for culturing other than normal mesenchymal stem cells, a medium obtained by removing a specific component from an acclimatized medium, or a medium obtained by adding a specific component to an acclimatized medium. Can be used. For example, Lonza's TheraPEAK (registered trademark) MSCGM-CD (registered trademark) Mesenchymal Stem Cell Medium, Chemically Defined, and the like can be used, but the present invention is not limited thereto. The content of FCS is usually 0.1% to 20%, preferably 0.2% to 10%. The conditioned medium and this specific medium may have the same composition.

上記無血清培地としては、添加剤としての動物血清を含まない培地であればよく、特に限定されない。公知の基本培地に、動物血清を除くその他添加剤を含有した組成を有するものを用いることができる。基本培地の組成は、培養するべき細胞の種類に応じて適宜選択することができる。例えば、イーグル培地のような最小必須培地（ＭＥＭ）、ダルベッコ改変イーグル培地（ＤＭＥＭ）、最小必須培地α（ＭＥＭ－α）、間葉系細胞基礎培地（ＭＳＣＢＭ）、Ｈａｍ’ｓ Ｆ－１２及びＦ－１０培地、ＤＭＥＭ／Ｆ１２培地、Ｗｉｌｌｉａｍｓ培地Ｅ、ＲＰＭＩ－１６４０培地、ＭＣＤＢ培地、１９９培地、Ｆｉｓｈｅｒ培地、Ｉｓｃｏｖｅ改変ダルベッコ培地（ＩＭＤＭ）、ＭｃＣｏｙ改変培地等が挙げられる。 The serum-free medium may be any medium that does not contain animal serum as an additive, and is not particularly limited. A known basal medium having a composition containing other additives other than animal serum can be used. The composition of the basal medium can be appropriately selected depending on the type of cells to be cultured. For example, minimum essential medium (MEM) such as Eagle's medium, Dalveco modified Eagle's medium (DMEM), minimum essential medium α (MEM-α), mesenchymal cell basal medium (MSCBM), Ham's F-12 and F. -10 medium, DMEM / F12 medium, Williams medium E, RPMI-1640 medium, MCDB medium, 199 medium, Fisher medium, Iscove-modified Dulbecco medium (IMDM), McCoy-modified medium and the like can be mentioned.

基本培地に加えるその他の添加剤としては、アミノ酸類、無機塩類、ビタミン類及び炭素源や抗生物質等の他の添加剤を挙げることができる。これらの添加剤の使用濃度は特に限定されず、通常の哺乳動物細胞用培地に用いられる濃度で用いることができる。 Other additives added to the basal medium include amino acids, inorganic salts, vitamins and other additives such as carbon sources and antibiotics. The concentration of these additives used is not particularly limited, and can be used at the concentration used for a normal medium for mammalian cells.

アミノ酸類としては、例えば、グリシン、Ｌ－アラニン、Ｌ－アルギニン、Ｌ－アスパラギン、Ｌ－アスパラギン酸、Ｌ－システイン、Ｌ－シスチン、Ｌ－グルタミン酸、Ｌ－グルタミン、Ｌ－ヒスチジン、Ｌ－イソロイシン、Ｌ－ロイシン、Ｌ－リジン、Ｌ－メチオニン、Ｌ－フェニルアラニン、Ｌ－プロリン、Ｌ－セリン、Ｌ－スレオニン、Ｌ－トリプトファン、Ｌ－チロシン、Ｌ－バリン等が挙げられる。 Examples of amino acids include glycine, L-alanine, L-arginine, L-aspartin, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-histidine, and L-isoleucine. Examples thereof include L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine.

無機塩類としては、例えば、塩化カルシウム、硫酸銅、硝酸鉄（ＩＩＩ）、硫酸鉄、塩化マグネシウム、硫酸マグネシウム、塩化カリウム、炭酸水素ナトリウム、塩化ナトリウム、リン酸水素二ナトリウム、リン酸二水素ナトリウム等が挙げられる。 Examples of the inorganic salts include calcium chloride, copper sulfate, iron (III) nitrate, iron sulfate, magnesium chloride, magnesium sulfate, potassium chloride, sodium hydrogen carbonate, sodium chloride, disodium hydrogen phosphate, sodium dihydrogen phosphate and the like. Can be mentioned.

ビタミン類としては、例えば、コリン、ビタミンＡ、ビタミンＢ１、ビタミンＢ２、ビタミンＢ３、ビタミンＢ４、ビタミンＢ５、ビタミンＢ６、ビタミンＢ７、ビタミンＢ１２、ビタミンＢ１３、ビタミンＢ１５、ビタミンＢ１７、ビタミンＢｈ、ビタミンＢｔ、ビタミンＢｘ、ビタミンＣ、ビタミンＤ、ビタミンＥ、ビタミンＦ、ビタミンＫ、ビタミンＭ、ビタミンＰ等が挙げられる。 Examples of vitamins include choline, vitamin A, vitamin B1, vitamin B2, vitamin B3, vitamin B4, vitamin B5, vitamin B6, vitamin B7, vitamin B12, vitamin B13, vitamin B15, vitamin B17, vitamin Bh, and vitamin Bt. , Vitamin Bx, Vitamin C, Vitamin D, Vitamin E, Vitamin F, Vitamin K, Vitamin M, Vitamin P and the like.

他の添加剤としては、繊維芽細胞増殖因子（ＦＧＦ）、内皮細胞増殖因子（ＥＧＦ）、血小板由来増殖因子（ＰＤＧＦ）、上皮成長因子（ＥＧＦ）、インスリン様成長因子（ＩＧＦ）、トランスフォーミング成長因子（ＴＧＦ）、神経成長因子（ＮＧＦ）、脳由来神経栄養因子（ＢＤＮＦ）、血管内皮細胞増殖因子（ＶＥＧＦ）、顆粒球コロニー刺激因子（Ｇ－ＣＳＦ）、顆粒球マクロファージコロニー刺激因子（ＧＭ－ＣＳＦ）、エリスロポエチン（ＥＰＯ）、トロンボポエチン（ＴＰＯ）、肝細胞増殖因子（ＨＧＦ）等の増殖因子；ペニシリン、ストレプトマイシン、ゲンタマイシン、カナマイシン等の抗生物質；グルコース、ガラクトース、フルクトース、スクロース等の炭素源；マグネシウム、鉄、亜鉛、カルシウム、カリウム、ナトリウム、銅、セレン、コバルト、スズ、モリブデン、ニッケル、ケイ素等の微量金属；β－グリセロリン酸、デキサメタゾン、ロシグリタゾン、イソブチルメチルキサンチン、５－アザシチジン等の幹細胞分化誘導剤；２－メルカプトエタノール、カタラーゼ、スーパーオキシドジスムターゼ、Ｎ－アセチルシステイン等の抗酸化剤；アデノシン５’－一リン酸、コルチコステロン、エタノールアミン、インスリン、還元型グルタチオン、リポ酸、メラトニン、ヒポキサンチン、フェノールレッド、プロゲステロン、プトレシン、ピルビン酸、チミジン、トリヨードチロニン、トランスフェリン、ラクトフェリン等が挙げられる。 Other additives include fibroblast growth factor (FGF), endothelial cell growth factor (EGF), platelet-derived growth factor (PDGF), epithelial growth factor (EGF), insulin-like growth factor (IGF), transforming growth. Factor (TGF), Nerve Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), Vascular Endothelial Cell Growth Factor (VEGF), Granulocyte Colony Stimulator (G-CSF), Granulocyte Macrophage Colony Stimulator (GM-) Growth factors such as CSF), erythropoetin (EPO), thrombopoetin (TPO), hepatocellular growth factor (HGF); antibiotics such as penicillin, streptomycin, gentamycin, canamycin; carbon sources such as glucose, galactose, fructose, sucrose; magnesium , Iron, zinc, calcium, potassium, sodium, copper, selenium, cobalt, tin, molybdenum, nickel, silicon and other trace metals; β-glycerophosphate, dexamethazone, rosiglytazone, isobutylmethylxanthin, 5-azacitidine and other stem cell differentiation Inducers; Antioxidants such as 2-mercaptoethanol, catalase, superoxide dismutase, N-acetylcysteine; adenosine 5'-monophosphate, corticosterone, ethanolamine, insulin, reduced glutathione, lipoic acid, melatonin, Examples thereof include hypoxanthin, phenol red, progesterone, putresin, pyruvate, thymidin, triiodotyronin, transferase, lactoferrin and the like.

本発明における間葉系幹細胞に好適な無血清培地として、市販の無血清培地を使用することもできる。この無血清培地は、抗酸化剤、動物血清アルブミン、成長因子、界面活性剤、Ｅｄｇリガンド、セロトニンリガンド等をさらに含有してもよい。 As a serum-free medium suitable for mesenchymal stem cells in the present invention, a commercially available serum-free medium can also be used. This serum-free medium may further contain an antioxidant, animal serum albumin, a growth factor, a surfactant, an Edg ligand, a serotonin ligand and the like.

間葉系幹細胞の培養条件は、それぞれの間葉系幹細胞に適した方法であれば特に限定されず、従来と同様の方法が用いられる。通常、３０℃～３７℃の温度、２％～７％ＣＯ２環境下、５％～２１％Ｏ２環境下で行われる。また、間葉系幹細胞の継代の時期及び方法もそれぞれの細胞に適していれば特に限定されず、細胞の様子を見ながら、従来と同様に行うことができる。The culture conditions for the mesenchymal stem cells are not particularly limited as long as they are suitable for each mesenchymal stem cell, and the same method as before is used. It is usually carried out at a temperature of 30 ° C. to 37 ° C. under a 2% to 7% CO 2 environment and a 5% to 21% O 2 environment. Further, the time and method of passage of the mesenchymal stem cells is not particularly limited as long as it is suitable for each cell, and it can be carried out in the same manner as before while observing the state of the cells.

上記特定の培地中で培養した間葉系幹細胞について、細胞の状態を勘案して適切な回数の継代を行った後、通常その１日後～５日後、好ましくは２日後～５日後、より好ましくは２日後～４日後、さらに好ましくは３日後～４日後に、遠心分離して細胞培養上清を回収する。細胞培養上清は、１回のみの回収でもよいし、複数日に渡って複数回回収してもよい。 Mesenchymal stem cells cultured in the above-mentioned specific medium are passaged an appropriate number of times in consideration of the state of the cells, and then usually 1 to 5 days later, preferably 2 to 5 days later, more preferably. After 2 to 4 days, more preferably 3 to 4 days, centrifuge and collect the cell culture supernatant. The cell culture supernatant may be collected only once or may be collected multiple times over a plurality of days.

本発明の上皮疾患治療剤は、本発明の効果を損なわない範囲でその他の成分を含んでいてもよい。その他の成分としては、その他の有効成分や、一般的な医薬品、医薬部外品等が含むことができる有効成分以外の薬学的に許容される担体等の成分が挙げられる。 The therapeutic agent for epithelial diseases of the present invention may contain other components as long as the effects of the present invention are not impaired. Examples of other ingredients include other active ingredients and ingredients such as pharmaceutically acceptable carriers other than active ingredients that can be contained in general pharmaceutical products, quasi-drugs, and the like.

［上皮疾患治療剤の調製方法］
本発明の上皮疾患治療剤は、本発明のスクリーニング方法により選択された細胞又は細胞培養上清と、その他の成分を常法により混合して製造することができる。[Preparation method for therapeutic agents for epithelial diseases]
The therapeutic agent for epithelial disease of the present invention can be produced by mixing the cells or cell culture supernatant selected by the screening method of the present invention with other components by a conventional method.

［上皮疾患治療剤の用途］
本発明の上皮疾患治療剤は、線維症及び線維症関連疾患、並びに癌及び癌関連疾患等の上皮疾患に対して好適に用いられる。なかでも、ＥＭＴが関与する疾患、さらには、眼部、肝臓、肺、腎臓、消化管、気道、腹膜等においてＥＭＴが関与する疾患に対して好ましく用いられる。[Use of therapeutic agents for epithelial diseases]
The therapeutic agent for epithelial diseases of the present invention is suitably used for fibrosis and fibrosis-related diseases, and epithelial diseases such as cancer and cancer-related diseases. Among them, it is preferably used for diseases related to EMT, and further for diseases related to EMT in the eyes, liver, lungs, kidneys, gastrointestinal tract, respiratory tract, peritoneum and the like.

本発明の上皮疾患治療剤が用いられる具体的な疾患としては、例えば、癌、前癌性症状、炎症性疾患、代謝疾患、心血管疾患、胃腸疾患、腹膜疾患、肺疾患、気道疾患、肝疾患、腎疾患、消化管疾患、眼部疾患（角膜疾患、網膜疾患）、表皮疾患等が挙げられる。 Specific diseases in which the therapeutic agent for epithelial diseases of the present invention is used include, for example, cancer, precancerous symptoms, inflammatory diseases, metabolic diseases, cardiovascular diseases, gastrointestinal diseases, peritoneal diseases, lung diseases, airway diseases, and liver. Diseases, renal diseases, gastrointestinal diseases, ocular diseases (corneal diseases, retinal diseases), epidermal diseases and the like can be mentioned.

上記疾患の詳細な具体例としては、食道癌、胃食道逆流症、バレット食道、胃癌、十二指腸癌、小腸癌、虫垂癌、大腸癌、結腸癌、直腸癌、肛門癌、膵臓癌、肝臓癌、胆嚢癌、脾臓癌、腎癌、膀胱癌、前立腺癌、精巣癌、子宮癌、卵巣癌、乳癌、肺癌、甲状腺癌、軟骨分解、関節リウマチ、乾癬性関節炎、脊椎関節炎、変形性関節症、痛風、乾癬、多発性硬化症、筋萎縮性側索硬化症、アルツハイマー病、パーキンソン病、うっ血性心不全、脳卒中、大動脈弁狭窄症、腎不全、狼瘡、膵炎、アレルギー、線維症、貧血、アテローム性動脈硬化症、再狭窄、化学療法／放射線関連合併症、Ｉ型糖尿病、II型糖尿病、自己免疫性肝炎、Ｃ型肝炎、原発性胆汁性肝硬変、原発性硬化性胆管炎、劇症肝炎、セリアック病、非特異性大腸炎、アレルギー性結膜炎、糖尿病性網膜症、シェーグレン症候群、ブドウ膜炎アレルギー性鼻炎、喘息、石綿症、珪肺、慢性閉塞性肺疾患、慢性肉芽腫性炎症、嚢胞性線維症、サルコイドーシス、糸球体腎炎、脈管炎、皮膚炎、ＨＩＶ関連悪液質、大脳マラリア、強直性脊椎炎、らい病、肺線維症、線維筋痛、翼状片、瘢痕、ＥＢウイルス角膜炎、角膜上皮幹細胞疲弊症、硝子体網膜症、強皮症等が挙げられる。 Specific examples of the above-mentioned diseases include esophageal cancer, gastroesophageal reflux disease, Barrett's esophagus, gastric cancer, duodenal cancer, small intestinal cancer, pituitary cancer, colon cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, liver cancer, and the like. Bile sac cancer, spleen cancer, kidney cancer, bladder cancer, prostate cancer, testis cancer, uterine cancer, ovarian cancer, breast cancer, lung cancer, thyroid cancer, chondrosis, rheumatoid arthritis, psoriatic arthritis, spondyloarthritis, osteoarthritis, gout , Psoriasis, multiple sclerosis, muscular atrophic lateral sclerosis, Alzheimer's disease, Parkinson's disease, congestive heart failure, stroke, aortic valve stenosis, renal failure, ulcer, pancreatitis, allergies, fibrosis, anemia, atherosclerotic arteries Sclerosis, re-stenosis, chemotherapy / radiation-related complications, type I diabetes, type II diabetes, autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, fulminant hepatitis, celiac disease , Non-specific colitis, allergic conjunctivitis, diabetic retinopathy, Schegren's syndrome, vegetative rhinitis, asthma, asbestos, sickle lung, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, Sarcoidosis, glomerular nephritis, vasculitis, dermatitis, HIV-related malaise, cerebral malaria, tonic spondylitis, leprosy, pulmonary fibrosis, fibromyalgia, winglets, scars, EB virus keratitis, corneal epithelium Examples include stem cell exhaustion, vitreous retinopathy, and scleroderma.

本発明の上皮疾患治療剤は、ＥＭＴに関与する疾患の治療又は予防において好ましく用いられるという観点から、特に、角膜疾患、網膜疾患、表皮疾患に好適に用いられる。具体的には、翼状片、瘢痕、ＥＢウイルス角膜炎、硝子体網膜症、強皮症等が挙げられる。さらに、本発明の医薬組成物は、癌及び前癌性症状からなる群より選択される疾患においても好ましく用いられる。具体的には、食道癌、胃食道逆流症、バレット食道、胃癌、十二指腸癌、小腸癌、虫垂癌、大腸癌、結腸癌、直腸癌、肛門癌、膵臓癌、肝臓癌、胆嚢癌、脾臓癌、腎癌、膀胱癌、前立腺癌、精巣癌、子宮癌、卵巣癌、乳癌、肺癌、甲状腺癌等が挙げられる。 The therapeutic agent for epithelial diseases of the present invention is particularly preferably used for corneal diseases, retinal diseases, and epidermal diseases from the viewpoint of being preferably used in the treatment or prevention of diseases related to EMT. Specific examples include pterygium, scars, EB virus keratitis, vitreous retinopathy, scleroderma and the like. Furthermore, the pharmaceutical composition of the present invention is also preferably used in diseases selected from the group consisting of cancer and precancerous symptoms. Specifically, esophageal cancer, gastroesophageal reflux disease, Barrett esophagus, gastric cancer, duodenal cancer, small intestinal cancer, pituitary cancer, colon cancer, colon cancer, rectal cancer, anal cancer, pancreatic cancer, liver cancer, bile sac cancer, spleen cancer. , Renal cancer, bladder cancer, prostate cancer, testis cancer, uterine cancer, ovarian cancer, breast cancer, lung cancer, thyroid cancer and the like.

本発明の上皮疾患治療剤は、他の抗癌剤、抗線維症剤、抗炎症剤等の他の薬剤と併用して用いることもできる。他の薬剤と同時に投与してもよいし、他の薬剤を投与する前後の適切な時期に投与してもよい。 The therapeutic agent for epithelial diseases of the present invention can also be used in combination with other agents such as other anticancer agents, antifibrotic agents and anti-inflammatory agents. It may be administered at the same time as other drugs, or may be administered at an appropriate time before and after administration of other drugs.

本発明の上皮疾患治療剤は、常法によって適宜の製剤とすることができる。本発明の上皮疾患治療剤が細胞を含む場合には、一般的な細胞製剤が含む成分を配合することができる。また、本発明の上皮疾患治療剤が細胞培養上清を含む場合には、製剤の剤型としては散剤、顆粒剤などの固形製剤であってもよいが、優れた予防・治療効果を得る観点からは、溶液剤、乳剤、懸濁剤などの液剤とすることが好ましい。特に点眼剤とする場合には、溶液剤であることがより好ましい。上記液剤の製造方法としては、例えば間葉系幹細胞の培養上清をそのまま使用する方法、その他溶剤と混合する方法や、さらに懸濁化剤や乳化剤を混合する方法を好適に例示することができる。以上のように、本発明における上皮疾患治療剤の製剤においては、製剤上の必要に応じて、適宜の薬学的に許容される担体、例えば、賦形剤、結合剤、溶剤、溶解補助剤、懸濁化剤、乳化剤、等張化剤、緩衝剤、安定化剤、無痛化剤、防腐剤、抗酸化剤、着色剤、滑沢剤、崩壊剤、湿潤剤、吸着剤、甘味剤、希釈剤などの任意成分を配合することができる。 The therapeutic agent for epithelial diseases of the present invention can be appropriately prepared by a conventional method. When the therapeutic agent for epithelial disease of the present invention contains cells, the components contained in a general cell preparation can be blended. When the therapeutic agent for epithelial disease of the present invention contains a cell culture supernatant, the dosage form of the preparation may be a solid preparation such as a powder or a granule, but from the viewpoint of obtaining an excellent preventive / therapeutic effect. Therefore, it is preferable to use a liquid agent such as a solution agent, an emulsion or a suspension agent. In particular, when it is used as an eye drop, it is more preferably a solution. As a method for producing the above liquid agent, for example, a method of using the culture supernatant of mesenchymal stem cells as it is, a method of mixing with another solvent, and a method of further mixing a suspending agent or an emulsifier can be preferably exemplified. .. As described above, in the preparation of the therapeutic agent for epithelial disease according to the present invention, an appropriate pharmaceutically acceptable carrier, for example, an excipient, a binder, a solvent, a solubilizing agent, etc. Suspension agents, emulsifiers, tonicity agents, buffers, stabilizers, soothing agents, preservatives, antioxidants, colorants, lubricants, disintegrants, wetting agents, adsorbents, sweeteners, dilutions Any ingredient such as an agent can be blended.

本発明の上皮疾患治療剤の投与方法としては特に制限されないが、血管内投与（好ましくは静脈内投与）、腹腔内投与、腸管内投与、皮下投与、点眼による投与等が好ましい。 The method for administering the therapeutic agent for epithelial disease of the present invention is not particularly limited, but intravascular administration (preferably intravenous administration), intraperitoneal administration, intestinal administration, subcutaneous administration, instillation and the like are preferable.

本発明の医薬組成物の製剤の投与量は、疾患の種類やその症状の度合い、剤型、投与対象の体重等によって変わり得る。本発明の上皮疾患治療剤が細胞を含む場合には、１日当たり、細胞を１Ｘ１０３個～１Ｘ１０９個の範囲で投与することができる。なお、本発明の予防・治療剤の投与は、１日のうち１～複数回に分けて行ってもよい。また、本発明の医薬組成物の製剤の投与は、単回投与でもよいし、継続的に行ってもよい。継続的に行う場合は、例えば、３日に１回以上の頻度で、２回以上継続して投与することができ、中でも、２日に１回以上の頻度で、３回以上継続して投与することが好ましく、１日に１回以上の頻度で４回以上継続して投与することがより好ましい。The dose of the pharmaceutical composition of the present invention may vary depending on the type of disease, the degree of symptoms thereof, the dosage form, the body weight of the subject to be administered, and the like. When the therapeutic agent for epithelial disease of the present invention contains cells, the cells can be administered in the range of 1X10 3 to 1X10 9 per day. The prophylactic / therapeutic agent of the present invention may be administered once or multiple times a day. In addition, the pharmaceutical composition of the present invention may be administered as a single dose or continuously. In the case of continuous administration, for example, it can be administered twice or more continuously at a frequency of once or more every three days, and above all, it can be continuously administered three times or more at a frequency of once or more every two days. It is preferable to administer it at least once a day, and it is more preferable to administer it continuously at least 4 times a day.

本発明の上皮疾患治療剤が点眼剤である場合、点眼剤に汎用されている技術を用い、必要に応じて製薬学的に許容され得る添加剤を用いて調製することができる。 When the therapeutic agent for epithelial disease of the present invention is an eye drop, it can be prepared by using a technique generally used for eye drops and, if necessary, using a pharmaceutically acceptable additive.

例えば、塩化ナトリウム、濃グリセリンなどの等張化剤；塩酸、水酸化ナトリウムなどのｐＨ調整剤；リン酸ナトリウム、酢酸ナトリウムなどの緩衝化剤；ポリオキシエチレンソルビタンモノオレート、ステアリン酸ポリオキシル４０、ポリオキシエチレン硬化ヒマシ油などの界面活性剤；クエン酸ナトリウム、エデト酸ナトリウムなどの安定化剤；塩化ベンザルコニウム、パラベンなどの防腐剤などから必要に応じて選択して用い、調製することができる。本点眼液のｐＨは眼科製剤に許容される範囲内にあればよいが、通常４～８の範囲内が好ましい。 For example, isotonic agents such as sodium chloride and concentrated glycerin; pH regulators such as hydrochloric acid and sodium hydroxide; buffering agents such as sodium phosphate and sodium acetate; polyoxyethylene sorbitan monooleate, polyoxyl 40 stearate, poly Surfactants such as oxyethylene hydrogenated castor oil; stabilizers such as sodium citrate and sodium edetate; preservatives such as benzalconium chloride and paraben can be selected and used as necessary to prepare. .. The pH of the present ophthalmic solution may be within the range acceptable for the ophthalmic preparation, but is usually preferably within the range of 4 to 8.

本発明の上皮疾患治療剤の投与対象となる動物としては、特に制限されないが、ヒト、サル、マウス、ラット、ハムスター、モルモット、ウシ、ブタ、ウマ、ウサギ、ヒツジ、ヤギ、ネコ、イヌ等が好ましく、中でもヒトがより好ましい。また、本発明の上皮疾患治療剤が含む細胞培養上清の細胞は、投与対象となる動物の種類と一致した由来であることが、疾患に対するより安定して優れた予防及び／又は治療効果を得る観点から好ましい。 The animal to which the therapeutic agent for the epithelial disease of the present invention is administered is not particularly limited, but humans, monkeys, mice, rats, hamsters, guinea pigs, cows, pigs, horses, rabbits, sheep, goats, cats, dogs and the like can be used. It is preferable, and human is more preferable. In addition, the cells in the cell culture supernatant contained in the therapeutic agent for epithelial disease of the present invention are derived from the same type as the animal to be administered, which provides a more stable and excellent preventive and / or therapeutic effect on the disease. It is preferable from the viewpoint of obtaining.

以下に本発明を実施例に基づいて更に詳細に説明するが、本発明はこれら実施例に限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples.

＜細胞培養上清のサイクロフィリンＢ含有量の測定及び角膜上皮細胞（ＨＣＥＣ）におけるＥＭＴ抑制効果の評価＞
間葉系幹細胞は脂肪由来幹細胞（ＡＤ－ＭＳＣ， Ｌｏｎｚａ）、骨髄由来幹細胞（ＢＭ－ＭＳＣ， Ｌｏｎｚａ）、臍帯由来幹細胞（ＵＣ－ＭＳＣ，Ｌｉｆｅｌｉｎｅ Ｃｅｌｌ Ｔｅｃｈｎｏｌｏｇｙ）を使用し、培養液はヒト間葉系幹細胞専用完全合成培地キット（ＭＳＣ－ＧＭＣＤ，Ｌｏｎｚａ）を用いた。線維芽細胞は、ヒト皮膚線維芽細胞（ＮＨＤＦ，クラボウ）を使用し、培養液は１０％ ＦＢＳ含有 ＤＭＥＭ（Ｌｉｆｅｔｅｃｈｎｏｌｏｇｉｅｓ）を用いた。それぞれの細胞を培養液で培養した後、ＭＳＣ－ＧＭＣＤ培地に交換し、さらに２－５日間培養した後、上清を３００ｘｇで遠心処理後、上清を評価用の培養上清として取得した。各細胞の培養上清を総タンパク量が２５μｇ（陽性細胞のＨｅｐＧ２ ｃｅｌｌ ｌｙｓａｔｅは１２．５μｇ）となるように調製し、５％β－ｍｅｒｃａｐｔｏｅｔｈａｎｏｌ を含有した４ ｘ ＮｕＰＡＧＥ ＬＤＳ Ｓａｍｐｌｅ Ｂｕｆｆｅｒ（Ｂｉｏ－Ｒａｄ）を総量の１／４量加え、７０℃で１０分間インキュベートし、サンプルとした。ＳＤＳ－ＰＡＧＥは４－１２％のＮｕＰＡＧＥ Ｎｏｖｅｘ Ｂｉｓ－Ｔｒｉｓゲル（ｉｎｖｉｔｒｏｇｅｎ）を用いて電気泳動した。ｉＢｌｏｔシステム（ｉｎｖｉｔｒｏｇｅｎ）を用いてＰＶＤＦメンブレンに転写した後、５％ ｓｋｉｍ ｍｉｌｋ／ＰＢＳ中で室温にて１時間ブロッキングした。０．０５％ Ｔｗｅｅｎ２０含有ＴＢＳ（ＴＢＳ－Ｔ）で５分間３回洗浄したメンブレンを一次抗体に室温で１～４時間または４℃で一晩反応させ、ＴＢＳ－Ｔで５分間３回洗浄した後、二次抗体に室温で４５分間反応させた。一次抗体には、抗Ｃｙｃｌｏｐｈｉｌｉｎ Ｂ抗体（＃ａｂ１６０４５， ａｂｃａｍ）（１，２００倍希釈）を使用した。二次抗体には、ＨＲＰ標識抗ウサギＩｇＧ抗体（１０，０００倍希釈）を使用した．抗体はいずれもＴＢＳで希釈して使用した。発光にはＥＣＬ ｐｒｉｍｅ（ＧＥ ｈｅａｌｔｈｃａｒｅ Ｂｉｏ－Ｓｃｉｅｎｃｅｓ）を使用し、ＣｈｅｍｉＤｏｃ ＸＲＳ（Ｂｉｏ－Ｒａｄ） にて検出した。結果を図１に示す。<Measurement of cyclophilin B content in cell culture supernatant and evaluation of EMT inhibitory effect on corneal epithelial cells (HCEC)>
As mesenchymal stem cells, adipose-derived stem cells (AD-MSC, Lonza), bone marrow-derived stem cells (BM-MSC, Lonza), and umbilical cord-derived stem cells (UC-MSC, Lifeline Cell Technology) are used, and the culture medium is human mesenchymal stem cells. A complete synthetic medium kit for stem cells (MSC-GMCD, Lonza) was used. As fibroblasts, human skin fibroblasts (NHDF, Kurabou) were used, and DMEM (Lifetechnologies) containing 10% FBS was used as the culture medium. After culturing each cell in a culture medium, the cells were replaced with MSC-GMCD medium, and the cells were further cultured for 2-5 days. The supernatant was centrifuged at 300 xg, and the supernatant was obtained as a culture supernatant for evaluation. The culture supernatant of each cell was prepared so that the total protein amount was 25 μg (HepG2 cell lysate of positive cells was 12.5 μg), and 4 x NuPAGE LDS Sample Buffer (Bio-Rad) containing 5% β-mercaptoethanol. Was added in 1/4 of the total amount and incubated at 70 ° C. for 10 minutes to prepare a sample. SDS-PAGE was electrophoresed on a 4-12% NuPAGE Novex Bis-Tris gel (invitrogen). After transfer to a PVDF membrane using the iBlot system (invitrogen), blocking was performed in 5% skim milk / PBS at room temperature for 1 hour. The membrane washed 3 times for 5 minutes with TBS (TBS-T) containing 0.05% Tween 20 was reacted with the primary antibody at room temperature for 1 to 4 hours or overnight at 4 ° C., and then washed 3 times with TBS-T for 5 minutes. , The secondary antibody was reacted at room temperature for 45 minutes. As the primary antibody, an anti-Cyclophilin B antibody (# ab16045, abcam) (diluted 1,200 times) was used. As the secondary antibody, HRP-labeled anti-rabbit IgG antibody (diluted 10,000 times) was used. All antibodies were diluted with TBS before use. ECL prime (GE healthcare Bio-Sciences) was used for luminescence, and it was detected by ChemiDoc XRS (Bio-Rad). The results are shown in FIG.

また、それぞれの培養上清のＥＭＴ抑制効果を次のように調べた。４８ ｗｅｌｌ ｐｌａｔｅ（ＢＤ Ｆａｌｃｏｎ）に角膜上皮細胞（ＨＣＥＣ）を２．０×１０４ ｃｅｌｌｓ／ｗｅｌｌ （２．６７×１０４ ｃｅｌｌｓ／ｃｍ２）の細胞密度で播種し、３７℃、５ ％ ＣＯ２条件下にて培養し、２４時間後、１０ ｎｇ／ｍＬ濃度のＴＧＦ－β１およびＴＮＦ－αを添加した培養液を加え、さらに４日間培養を行った後、ＡＤ、ＢＭ、またはＵＣ－ＭＳＣ、およびＮＨＤＦの培養上清（すべてＭＳＣ－ＧＭＣＤ培地）を培地全体の半量となるように添加し、さらに２日間培養を行った後、 ＱＩＡｚｏｌ Ｌｙｓｉｓ Ｒｅａｇｅｎｔ（ＱＩＡＧＥＮ）を用いてＲＮＡを抽出・精製し、ＳｕｐｅｒＳｃｒｉｐｔ（登録商標） ＩＩＩ Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ ＳｕｐｅｒＭｉｘ ｆｏｒ ｑＲＴ－ＰＣＲ （Ｉｎｖｉｔｒｏｇｅｎ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてＴａｑｍａｎアッセイ（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ）により遺伝子発現解析を行った。結果を図２に示す。
また、両方の結果を表１に合わせて示した。In addition, the EMT inhibitory effect of each culture supernatant was investigated as follows. Corneal epithelial cells (HCEC) were seeded in 48 well plates (BD Falcon) at a cell density of 2.0 × 10 4 cells / well (2.67 × 10 4 cells / cm 2 ) and 37 ° C., 5% CO 2 After culturing under the conditions, after 24 hours, a culture medium containing 10 ng / mL concentration of TGF-β1 and TNF-α was added, and after further culturing for 4 days, AD, BM, or UC-MSC, And NHDF culture supernatant (all MSC-GMCD medium) was added to half the volume of the whole medium, and after further culturing for 2 days, RNA was extracted and purified using QIAzol Lysis Reagent (QIAGEN). CDNA was synthesized using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Gene expression analysis was performed by Taqman assay (Life technologies) using the synthesized cDNA. The results are shown in FIG.
Both results are also shown in Table 1.

Figure 0007057557000001

ＡＤ－ＭＳＣ、ＡＤ－ＭＳＣ２：脂肪由来間葉系幹細胞
ＢＭ－ＭＳＣ：骨髄由来間葉系幹細胞
ＵＣ－ＭＳＣ：臍帯由来間葉系幹細胞
ＮＨＤＦ：皮膚線維芽細胞
Figure 0007057557000001
AD-MSC, AD-MSC2: Fat-derived mesenchymal stem cells BM-MSC: Bone marrow-derived mesenchymal stem cells UC-MSC: Umbilical band-derived mesenchymal stem cells NHDF: Skin fibroblasts

ＨＣＥＣをＴＧＦ－β及びＴＮＦ－αで処理することにより、ＥＭＴが起こり、上皮マーカーであるＣｌａｕｄｉｎ－１の発現低下が、また、間葉マーカーであるＶｉｍｅｎｔｉｎ及びＳｌｕｇの発現増強が見られたが（サンプルＮｏ．６）、各種ＭＳＣ培養上清を添加すると、ＥＭＴ抑制効果が見られた。このＥＭＴ抑制効果は、培養上清中のサイクロフィリンＢ含有量と相関していた。従って、培養上清中のサイクロフィリンＢを指標として、ＥＭＴ抑制効果の有無、その程度について規定すると共に、評価、判定することが可能であることがわかった。 Treatment of HCEC with TGF-β and TNF-α resulted in EMT, with decreased expression of the epithelial marker Claudin-1 and increased expression of the mesenchymal markers Vimentin and Slug ( When sample No. 6) and various MSC culture supernatants were added, an EMT inhibitory effect was observed. This EMT inhibitory effect was correlated with the cyclophilin B content in the culture supernatant. Therefore, it was found that using cyclophilin B in the culture supernatant as an index, the presence or absence of the EMT inhibitory effect and its degree can be defined, and evaluated and judged.

また、上記ＭＳＣ培養上清のうち、サイクロフィリンＢの含有量が多いＡＤ－ＭＳＣ、ＵＣ－ＭＳＣの培養上清は、ＥＭＴ抑制効果が高いことから、上皮疾患治療剤として、好適に用いられる。特に、眼部、肝臓、肺、腎臓、消化管、気道又は腹膜における疾患、中でも、角膜疾患、具体的には、翼状片、瘢痕、ＥＢウイルス角膜炎等に有効である。 Further, among the above-mentioned MSC culture supernatants, the culture supernatants of AD-MSC and UC-MSC, which have a high content of cyclophilin B, have a high EMT inhibitory effect and are therefore suitably used as a therapeutic agent for epithelial diseases. In particular, it is effective for diseases in the eye, liver, lung, kidney, gastrointestinal tract, airway or peritoneum, especially corneal disease, specifically, pterygium, scar, EB virus keratitis and the like.

次に、上記試験にて、角膜上皮細胞（ＨＣＥＣ）に対して高いＥＭＴ抑制効果を示したＡＤ－ＭＳＣの培養上清と同様のものについて、表皮角化細胞（ＮＨＥＫ）、網膜色素上皮細胞株（ＡＲＰＥ－１９）に対する効果を評価した。 Next, in the above test, the same as the culture supernatant of AD-MSC which showed a high EMT inhibitory effect on corneal epithelial cells (HCEC), epidermal keratinocytes (NHEK), retinal pigment epithelial cell line. The effect on (ARPE-19) was evaluated.

＜表皮角化細胞（ＮＨＥＫ）におけるＥＭＴ抑制効果の評価＞
サイクロフィリンＢ含有培養上清の表皮角化細胞（ＮＨＥＫ）のＥＭＴ抑制効果を次のように調べた。すなわち、２４ ｗｅｌｌ ｐｌａｔｅ（ＢＤ Ｆａｌｃｏｎ）に表皮角化細胞（ＮＨＥＫ）を２．０×１０４ ｃｅｌｌｓ／ｗｅｌｌ （１．０６×１０４ ｃｅｌｌｓ／ｃｍ２）の細胞密度で播種し、３７℃、５％ ＣＯ２条件下にて培養し、２４時間後、１０ ｎｇ／ｍＬ濃度のＴＧＦ－β１およびＴＮＦ－αを添加した培養液を加え、さらに３日間培養を行った後、サイクロフィリンＢを含有するＡＤ－ＭＳＣの培養上清（すべてＭＳＣ－ＧＭＣＤ培地）を培地全体の半量となるように添加し、さらに３日間培養を行った後、ＲＮｅａｓｙ ｋｉｔ（ＱＩＡＧＥＮ）を用いてＲＮＡを抽出・精製し、ＳｕｐｅｒＳｃｒｉｐｔ（登録商標） ＩＩＩ Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ ＳｕｐｅｒＭｉｘ ｆｏｒ ｑＲＴ－ＰＣＲ （Ｉｎｖｉｔｒｏｇｅｎ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてＴａｑｍａｎアッセイ（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ）により遺伝子発現解析を行った。その結果を図３に示す。<Evaluation of EMT inhibitory effect on epidermal keratinocytes (NHEK)>
The EMT inhibitory effect of epidermal keratinocytes (NHEK) in the cyclophilin B-containing culture supernatant was investigated as follows. That is, epidermal keratinized cells (NHEK) were seeded in 24 well plates (BD Falcon) at a cell density of 2.0 × 10 4 cells / well (1.06 × 10 4 cells / cm 2 ) at 37 ° C. and 5 % CO 2 conditions, 24 hours later, 10 ng / mL concentration of TGF-β1 and TNF-α-added culture medium is added, and after further culturing for 3 days, cyclophilin B is contained. AD-MSC culture supernatant (all MSC-GMCD medium) was added to half the total amount of the medium, and after further culturing for 3 days, RNA was extracted and purified using RNeasy kit (QIAGEN). CDNA was synthesized using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Gene expression analysis was performed by Taqman assay (Life technologies) using the synthesized cDNA. The results are shown in FIG.

図３に示すとおり、ＮＨＥＫをＴＧＦ－β１及びＴＮＦ－αで処理することにより、ＥＭＴが起こり、間葉マーカーであるＮ－ｃａｄｈｅｒｉｎ及びＳｌｕｇの発現増強が見られたが（Ｃｏｎｔｒｏｌ）、ＡＤ－ＭＳＣ培養上清（ＭＳＣ－ＣＭ）を添加すると、角膜上皮細胞（ＨＣＥＣ）に対してと同様に、高いＥＭＴ抑制効果が見られた。このことから、サイクロフィリンＢの含有量が多いＭＳＣの培養上清は、上皮疾患治療剤として好適に用いられ、特に表皮疾患、具体的には、強皮症等に有効であることがわかった。 As shown in FIG. 3, treatment of NHEK with TGF-β1 and TNF-α resulted in EMT, and enhanced expression of the mesenchymal markers N-cadherin and Slug was observed (Control), but AD-MSC. When the culture supernatant (MSC-CM) was added, a high EMT inhibitory effect was observed as in the case of corneal epithelial cells (HCEC). From this, it was found that the culture supernatant of MSC having a high content of cyclophilin B is suitably used as a therapeutic agent for epithelial diseases, and is particularly effective for epidermal diseases, specifically scleroderma and the like. ..

＜網膜色素上皮細胞株（ＡＲＰＥ－１９）におけるＥＭＴ抑制効果の評価＞
網膜色素上皮細胞株（ＡＲＰＥ－１９）のＴＧＦ－β１添加によるＥＭＴ様遺伝子変動を、以下のように確認した。すなわち、２４ ｗｅｌｌ ｐｌａｔｅ（ＢＤ Ｆａｌｃｏｎ）に網膜色素上皮細胞株（ＡＲＰＥ－１９）を２．５×１０４ ｃｅｌｌｓ／ｃｍ２の細胞密度で播種し、３７℃、５％ ＣＯ２条件下にて、５または１０ｎｇ／ｍＬ濃度のＴＧＦ－β１を含む培養液（１０％ＦＢＳ含有ＤＭＥＭ／Ｆ１２）で４日間培養を行った後、ＱＩＡｚｏｌ Ｌｙｓｉｓ Ｒｅａｇｅｎｔ（ＱＩＡＧＥＮ）を用いてＲＮＡを抽出・精製し、ＳｕｐｅｒＳｃｒｉｐｔ（登録商標） ＩＩＩ Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ ＳｕｐｅｒＭｉｘ ｆｏｒ ｑＲＴ－ＰＣＲ （Ｉｎｖｉｔｒｏｇｅｎ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてＴａｑｍａｎアッセイ（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ）により遺伝子発現解析を行った。その結果を図４に示す。<Evaluation of EMT inhibitory effect in retinal pigment epithelial cell line (ARPE-19)>
EMT-like gene changes due to the addition of TGF-β1 in the retinal pigment epithelial cell line (ARPE-19) were confirmed as follows. That is, a retinal pigment epithelial cell line (ARPE-19) was seeded on a 24-well plate (BD Falcon) at a cell density of 2.5 × 10 4 cells / cm 2 at 37 ° C. and 5% CO 2 conditions. After culturing for 4 days in a culture medium containing TGF-β1 at a concentration of 5 or 10 ng / mL (DMEM / F12 containing 10% FBS), RNA was extracted and purified using QIAzol Lysis Reagent (QIAGEN), and SuperScript (10% FBS-containing DMEM / F12) was used to extract and purify RNA. CDNA was synthesized using III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Gene expression analysis was performed by Taqman assay (Life technologies) using the synthesized cDNA. The results are shown in FIG.

次に、網膜色素上皮細胞株（ＡＲＰＥ－１９）に対するサイクロフィリンＢ含有ＭＳＣ培養上清のＥＭＴ抑制効果を次のように調べた。すなわち、２４ ｗｅｌｌ ｐｌａｔｅ（ＢＤ Ｆａｌｃｏｎ）に網膜色素上皮細胞株（ＡＲＰＥ－１９）を２．５×１０４ ｃｅｌｌｓ／ｃｍ２の細胞密度で播種し、３７℃、ＦＢＳ含有ＤＭＥＭ／Ｆ１２）を用いて４日間培養を行った後、サイクロフィリンＢを含有するＡＤ－ＭＳＣの培養上清（すべてＭＳＣ－ＧＭＣＤ培地）を培地全体の半量となるように添加し、さらに３日間培養を行い、ＱＩＡｚｏｌ Ｌｙｓｉｓ Ｒｅａｇｅｎｔ（ＱＩＡＧＥＮ）を用いてＲＮＡを抽出・精製し、ＳｕｐｅｒＳｃｒｉｐｔ（登録商標） ＩＩＩ Ｆｉｒｓｔ－Ｓｔｒａｎｄ Ｓｙｎｔｈｅｓｉｓ ＳｕｐｅｒＭｉｘ ｆｏｒ ｑＲＴ－ＰＣＲ （Ｉｎｖｉｔｒｏｇｅｎ）を用いてｃＤＮＡを合成した。合成したｃＤＮＡを用いてＴａｑｍａｎアッセイ（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ）により遺伝子発現解析を行った。その結果を図５に示す。Next, the EMT inhibitory effect of the cyclophilin B-containing MSC culture supernatant on the retinal pigment epithelial cell line (ARPE-19) was investigated as follows. That is, a retinal pigment epithelial cell line (ARPE-19) was seeded on a 24-well plate (BD Falcon) at a cell density of 2.5 × 10 4 cells / cm 2 and used at 37 ° C., FBS-containing DMEM / F12). After culturing for 4 days, the culture supernatant of AD-MSC containing cyclophilin B (all MSC-GMCD medium) was added so as to be half the total amount of the medium, and the cells were further cultured for 3 days, and QIAzol Lysis Regent. RNA was extracted and purified using (QIAGEN), and cDNA was synthesized using SuperScript® III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). Gene expression analysis was performed by Taqman assay (Life technologies) using the synthesized cDNA. The results are shown in FIG.

図４に示すとおり、ＡＲＰＥ－１９をＴＧＦ－β１及びＴＮＦ－αで処理することにより、ＥＭＴが起こり、間葉マーカーであるＦｉｂｒｏｎｅｃｔｉｎ及びＮ－ｃａｄｈｅｒｉｎの発現増強が見られたが、ＡＤ－ＭＳＣ培養上清（ＭＳＣ－ＣＭ）を添加すると、角膜上皮細胞（ＨＣＥＣ）に対してと同様に、高いＥＭＴ抑制効果が見られ、これらの遺伝子発現が抑制された。他の間葉マーカーであるＳｎａｉｌ及びＳｌｕｇの発現も同様に抑制された（図５）。このことから、サイクロフィリンＢの含有量が多いＭＳＣの培養上清は、上皮疾患治療剤として好適に用いられ、特に眼部における疾患、中でも網膜疾患、具体的には硝子体網膜症等に有効であることがわかった。 As shown in FIG. 4, treatment of ARPE-19 with TGF-β1 and TNF-α caused EMT, and enhanced expression of the mesenchymal markers fibronectin and N-cadherin was observed, but AD-MSC culture was observed. When the supernatant (MSC-CM) was added, a high EMT inhibitory effect was observed as in the case of corneal epithelial cells (HCEC), and the expression of these genes was suppressed. Expression of other mesenchymal markers Snail and Slug was similarly suppressed (Fig. 5). For this reason, the culture supernatant of MSC having a high content of cyclophilin B is suitably used as a therapeutic agent for epithelial diseases, and is particularly effective for diseases in the eye, especially retinal diseases, specifically vitreous retinopathy and the like. It turned out to be.

本発明によると、上皮疾患に対する治療・予防効果を有する細胞又は細胞培養上清を簡便に選択でき、かつ細胞又は細胞培養上清を含む上皮疾患の治療剤が、薬理効果・所定の品質等を有するか否かの評価・判断が簡便かつ高精度で実施可能となる、上皮疾患に対する治療及び／又は予防有効性を評価する方法を提供することができる。また、この方法を用いたスクリーニング方法によると、上皮疾患に対する優れた治療・予防効果を有する細胞又は細胞培養上清を簡便かつ高精度で選択することができるため、新規の上皮疾患治療剤の探索研究において、有効に活用することができる。 According to the present invention, cells or cell culture supernatants having a therapeutic / preventive effect on epithelial diseases can be easily selected, and a therapeutic agent for epithelial diseases containing cells or cell culture supernatants has a pharmacological effect, predetermined quality, etc. It is possible to provide a method for evaluating the therapeutic and / or preventive effectiveness for epithelial diseases, which enables easy and highly accurate evaluation / judgment as to whether or not the cell has. In addition, according to the screening method using this method, cells or cell culture supernatants having excellent therapeutic / preventive effects on epithelial diseases can be selected easily and with high accuracy, so that a search for a new therapeutic agent for epithelial diseases can be performed. It can be effectively used in research.

Claims (14)

細胞及び／又はその培養上清を含む製剤において、サイクロフィリンＢを指標に、上記製剤の、上皮疾患に対する治療及び／又は予防有効性を評価する方法。 A method for evaluating the therapeutic and / or prophylactic efficacy of the above-mentioned preparation for epithelial diseases using cyclophilin B as an index in a preparation containing cells and / or a culture supernatant thereof. 上記上皮疾患が、上皮間葉転換が関与している疾患である、請求項１記載の方法。 The method according to claim 1, wherein the epithelial disease is a disease in which epithelial-mesenchymal transition is involved. 上記上皮疾患が、眼部、肝臓、肺、腎臓、消化管、気道又は腹膜における疾患である、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the epithelial disease is a disease in the eye, liver, lungs, kidneys, gastrointestinal tract, respiratory tract or peritoneum. 上記上皮疾患が、線維症である、請求項１から３のいずれか１項記載の方法。 The method according to any one of claims 1 to 3, wherein the epithelial disease is fibrosis. 上記上皮疾患が、角膜疾患、網膜疾患又は表皮疾患である、請求項１から４のいずれか１項記載の方法。 The method according to any one of claims 1 to 4, wherein the epithelial disease is a corneal disease, a retinal disease or an epidermal disease. 上記上皮疾患が、翼状片、瘢痕、ＥＢウイルス角膜炎、角膜上皮幹細胞疲弊症、硝子体網膜症、又は強皮症である、請求項５記載の方法。 The method according to claim 5, wherein the epithelial disease is pterygium, scar, EB virus keratitis, corneal epithelial stem cell exhaustion, vitreous retinopathy, or scleroderma. 上記上皮疾患が、癌である、請求項１又は２記載の方法。 The method according to claim 1 or 2, wherein the epithelial disease is cancer. 上記細胞が、間葉系幹細胞である、請求項１から７のいずれか１項記載の方法。 The method according to any one of claims 1 to 7, wherein the cell is a mesenchymal stem cell. 上記間葉系幹細胞が、脂肪由来間葉系幹細胞又は臍帯由来間葉系幹細胞である、請求項８記載の方法。 The method according to claim 8, wherein the mesenchymal stem cells are adipose-derived mesenchymal stem cells or umbilical cord-derived mesenchymal stem cells. 細胞におけるサイクロフィリンＢの発現量、又は細胞培養上清中のサイクロフィリンＢの含有量を測定する工程
を含む、上記細胞又は細胞培養上清の、上皮疾患に対する治療及び／又は予防有効性を評価する方法。 To evaluate the therapeutic and / or preventive efficacy of the above cells or cell culture supernatant for epithelial diseases, which comprises the step of measuring the expression level of cyclophilin B in cells or the content of cyclophilin B in the cell culture supernatant. how to. 得られたサイクロフィリンＢ量を、基準となるサイクロフィリンＢ量と比較する工程
をさらに含む、請求項１０記載の方法。 The method according to claim 10, further comprising a step of comparing the obtained amount of cyclophilin B with the reference amount of cyclophilin B. 請求項１から１１のいずれか１項記載の方法を用いる、上皮疾患治療剤のスクリーニング方法。 A method for screening an epithelial disease therapeutic agent using the method according to any one of claims 1 to 11. 請求項１２記載のスクリーニング方法により選択された上皮疾患治療剤であって、間葉系幹細胞又はその培養上清を含むことを特徴とし、上記間葉系幹細胞が、臍帯由来間葉系幹細胞であり、上記上皮疾患が、網膜疾患である上皮疾患治療剤。 A therapeutic agent for epithelial disease selected by the screening method according to claim 12, characterized in that it contains mesenchymal stem cells or a culture supernatant thereof, and the mesenchymal stem cells are mesenchymal stem cells derived from the umbilical cord. , The above-mentioned epithelial disease is a therapeutic agent for epithelial disease, which is a retinal disease. 上記上皮疾患が、硝子体網膜症である、請求項１３記載の上皮疾患治療剤。 The therapeutic agent for epithelial disease according to claim 13 , wherein the epithelial disease is vitreous retinopathy.
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