Analysis of Corneal Epithelium-Specific Protein & its Clinical Application

角膜上皮特异性蛋白质的分析

• 批准号: 06454497
• 负责人: OHASHI Yuichi
• 金额: $ 3.97万
• 依托单位: Ehime University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06454497/
• 关键词: Corneal epithelium; Immortalization; Corneal Transplantation; Three dimension culture; Functional differentiation; SV40; SV 40-アデノウイルスベクター; 不死化細胞; サイトケラチン; アルデヒドデヒドロゲナーゼ; 細胞培養

We have succeeded in immortalizing human corneal epithelial cells for the first time by using a recombinant SV40-adenovirus vector. These immortalized cells retained several properties consistent with epithelial cells, including formation of microvilli and desmosome as well as existence of aldehyde dehydrogenase. RT-PCR has demonstrated the expression of type 1 transglutaminase in these cells. Also, cornifin was found positive by immunohistochemical technique at the area where the cells became stratified. The expression of cadherin was increased in the presence of substance P,suggesting this cell line can be used for studying the machanism of adhesion and differentiation of corneal epithelium. When seeded onto a human keratocyte-containing type II collagen matrix, these immortalized corneal epithelial cells formed a stratified layr. This reconstructed tissue may be applicable as a transient substitute of corneal epithelial cells. In addition, in vitro test for screening the cytotoxicity of eyedrop solution has been developed in our laboratory using lactic dehydrogenase as a indicator, and has disclosed the cytotoxicity of unoprostone and timolol to corneal epithelial cells as has been experienced in clinical situation. This particular cell line has been widely distributed to number of research laboratories and extensively used as a source of human corneal epithelial cells.

我们已经成功地永生化人角膜上皮细胞的第一次使用重组SV 40腺病毒载体。这些永生化细胞保留了一些与上皮细胞一致的性质，包括微绒毛和桥粒的形成以及醛脱氢酶的存在。RT-PCR证实了1型转氨酶在这些细胞中表达。免疫组化结果显示，角蛋白在细胞复层处呈阳性。P物质存在时，钙粘蛋白表达增加，提示该细胞系可用于研究角膜上皮的粘附和分化机制。当接种到含有人角膜基质细胞的II型胶原基质上时，这些永生化的角膜上皮细胞形成复层。这种重建的组织可能适用于作为角膜上皮细胞的暂时替代物。此外，本实验室还开发了以乳酸脱氢酶为指标筛选滴眼液细胞毒性的体外试验，并揭示了乌诺前列酮和噻吗洛尔对角膜上皮细胞的细胞毒性，与临床情况相同。这种特殊的细胞系已被广泛分发到许多研究实验室，并广泛用作人类角膜上皮细胞的来源。

项目成果

Sasaki-Araki K,Ohashi Y,Sasabe T,Hayashi K,Watanabe H,Tano Y,Handa H.: "An SV40-immortalized human corneal epithelial cell line and its characterization." Invest Ophthalmol Vis Sci. 36(3). 614-621 (1995)

Sasaki-Araki K、Ohashi Y、Sasabe T、Hayashi K、Watanabe H、Tano Y、Handa H.：“SV40 永生化人角膜上皮细胞系及其表征。”

Yao Y-F,Inoue Y,Hara Y,Kiritoshi A,Tano Y,Ohashi Y: "Suppression of graft rejection in rat keratoepithelio-plasty by anterior camber inoculation of donor lymphocytes." Jpn J Ophthalmol. 38-4. 345-352 (1994)

Yao Y-F，Inoue Y，Hara Y，Kiritoshi A，Tano Y，Ohashi Y：“通过接种供体淋巴细胞的前弧度来抑制大鼠角膜上皮成形术中的移植物排斥。”

Yao Y-F,Inoue Y,Hara Y,Kiritoshi A,Tano Y,Ohashi Y.: "Suppression of graft rejection ina rat keratopeithelioplasty by anterior chamber inoculation of donor lymphocytes." Jpn J Ophthalmol. 38-4. 345-352 (1994)

Yao Y-F，Inoue Y，Hara Y，Kiritoshi A，Tano Y，Ohashi Y.：“通过前房接种供体淋巴细胞抑制大鼠角膜移植排斥反应。”

Kaoru Araki: "SV40-Immortalized human corneal epithelial cells expressing aldehydedehydrogenase activity" Investigative Ophthalmology and Visual Sciences. In Press. (1994)

Kaoru Araki：“SV40-表达乙醛脱氢酶活性的永生化人角膜上皮细胞”研究眼科和视觉科学。

大橋裕一編: "点眼薬-常識と非常識" メジカルビュー社, 126 (1994)

Yuichi Ohashi 编辑：“滴眼液 - 常识和误解” Medical View Publishing，126 (1994)