Analysis of Corneal Epithelium-Specific Protein & its Clinical Application

我们首次利用重组SV40-腺病毒载体成功地将人角膜上皮细胞永生化。这些永生化细胞保留了一些与上皮细胞一致的特性，包括微绒毛和桥粒的形成以及乙醛脱氢酶的存在。逆转录-聚合酶链式反应证实在这些细胞中有1型转谷氨酰胺酶的表达。此外，免疫组织化学技术在细胞层化的区域发现角蛋白呈阳性。在P物质存在的情况下，Cad的表达增加，提示该细胞系可用于研究角膜上皮细胞的黏附和分化机制。当将这些永生化的角膜上皮细胞种植到含有人类角膜细胞的II型胶原基质上时，这些永生化的角膜上皮细胞形成了分层的层。该重建组织可作为角膜上皮细胞的暂时性替代物。此外，本实验室还开展了以乳酸脱氢酶为指示剂的滴眼液细胞毒性体外筛选试验，揭示了临床上已有的乌诺前列酮和噻吗洛尔对角膜上皮细胞的毒性作用。这种特殊的细胞系已经广泛分布于许多研究实验室，并被广泛用作人角膜上皮细胞的来源。

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.