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.

我们通过使用重组SV40-腺病毒载体首次成功地使人角膜上皮细胞成为永生。这些永生的细胞保留了与上皮细胞一致的几种特性，包括形成微绒毛和脱粒体以及醛脱氢酶的存在。 RT-PCR证明了这些细胞中1型转谷氨酰胺酶的表达。同样，通过在细胞分层的区域通过免疫组织化学技术发现蜂集阳性。在存在P的情况下，钙粘着蛋白的表达增加，这表明该细胞系可用于研究角膜上皮的粘附和分化的马赫主义。当播种到含有人角膜角细胞的II型胶原蛋白基质上时，这些永生的角膜上皮细胞形成了分层的Layr。这种重建的组织可能适用于角膜上皮细胞的短暂替代物。此外，在我们的实验室中，使用乳酸脱氢酶作为指示剂在我们的实验室开发了筛选眼质溶液的细胞毒性的体外测试，并已在临床情况下披露了无前列腺酮和timolol对角膜上皮细胞的细胞毒性。该特定细胞系已被广泛分布在研究实验室的数量上，并广泛用作人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。”

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。

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。”

Araki K,Ohashi Y,Kinoshita S,Kuwayama Y,Tano Y: "Epithelial wound healing in the denervated cornea." Curr Eye Res. 13-2. 203-211 (1994)

Araki K、Ohashi Y、Kinoshita S、Kuwayama Y、Tano Y：“去神经支配角膜中的上皮伤口愈合。”