MOLECULAR ASPECTS OF CORNEAL EPITHELIAL MIGRATION

角膜上皮迁移的分子方面

• 批准号: 2162301
• 负责人: Mary Ann Stepp
• 金额: $ 20.29万
• 依托单位: GEORGE WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-07-01 至 1998-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2162301
• 关键词: antiserum; blocking antibody; cell adhesion molecules; cell cell interaction; cell migration; corneal epithelium; cytoskeletal proteins; fluorimetry; gene expression; immunoprecipitation; integrins; intercellular connection; laboratory rabbit; laboratory rat; molecular biology; neutrophil; organ culture; phosphopyruvate hydratase; phosphorylation; protein biosynthesis; protein structure; proteolysis; tissue /cell culture; western blottings; wound healing

The corneal epithelium has a variety of cell:cell and cell:substrate contacts which maintain much of its integrity even as it migrates in response to injury. Among the cell:substrate junctions, the hemidesmosomes (HDs) are responsible for most of the adhesion of the epithelium to its substrate. HDs contain the alpha6beta4 integrin as one of their components and other integrin heterodimers are present in cell:cell boundaries of the corneal epithelium. Aim 1 of this proposal is to determine whether the expression and structure of beta1, beta4, alpha3, alpha5 and alpha6 integrin mRNAs are altered during migration by a) amplifying rat integrin cDNAs from a rat corneal epithelial cell library, b) determining if the expression of rat integrin mRNAs is altered during migration, c) determining the relative abundance of the cytoplasmic integrin mRNA alternative splicing variants and whether there are changes during migration, and d) correlating the data on integrin mRNA expression with data on integrin protein synthesis. Aim 2 is to determine if the delay in corneal epithelial migration induced by addition of extracts from rat polymorphonuclear leukocytes (PMNs) to debridement wounded corneal organ cultures involves alterations in the amounts or the localization of integrins by a) using immunoblotting, immunoprecipitation, and mRNA quantitation to discover if the PMN extract added to debridement wounds affects protein synthesis in the epithelium by determining the level of integrins, vinculin, alpha-actin, alpha- enolase, and ICAM-1 and b) determining if the addition of purified inflammatory cytokines to corneal organ cultures with epithelial debridement wounds results in a delay in epithelial migration by a mechanism similar to PMN extract. Aim 3 is to determine whether the disassembly of hemidesmosomes and migration of the corneal epithelium involves phosphorylation and/or proteolytic cleavage of the beta4 subunit by a) determining whether the HD alpha6 and beta4 subunits are phosphorylated on tyrosine in the unwounded cornea and if their phosphorylation state is altered during migration, b) developing polyclonal antisera with specificity against either the entire extracellular domain or the entire cytoplasmic portion of the beta4 molecule, and c) using the antisera to determine if the beta4 molecule undergoes cleavage during epithelial cell migration. Aim 4 is to determine if integrins at regions of cell:cell interaction are functionally important in maintaining cell:cell contacts in the corneal epithelium by a) establishing cell culture conditions for bovine corneal epithelial cells, b) determining the state of assembly of desmosomes, adherins junctions, and alphav- and the beta1-containing cell:cell junctions in cells cultured in low calcium and after shifting cells to high calcium medium, c) determining the effect of adhesion blocking integrin peptides and antibodies on the ability of cultured corneal epithelial cells to maintain cell:cell contact in low and in high calcium media, and d) determining whether addition of PMN extract to cultured cells disrupts cell:cell contacts and the cell:cell localization of alphav and the beta1 integrins in low and high calcium media. These experiments will help accomplish our goal of obtaining a better understanding, at the molecular level, of the role of integrins in the normal cornea and in epithelial cell migration during wound healing.

角膜上皮有多种细胞:细胞和细胞:基质 即使在迁移时仍保持其大部分完整性的接触 对伤害的反应。 在细胞：基质连接处， 半桥粒 (HD) 负责大部分粘附 上皮与其基底。 HD 含有 α6β4 整合素作为一个 它们的成分和其他整合素异二聚体存在于 细胞：角膜上皮的细胞边界。 本提案的目标 1 是确定β1、β4、 α3、α5 和 α6 整合素 mRNA 在迁移过程中发生改变 a) 从大鼠角膜上皮细胞中扩增大鼠整合素 cDNA 文库，b) 确定大鼠整合素 mRNA 的表达是否 在迁移过程中发生改变，c) 确定 细胞质整合素 mRNA 选择性剪接变异体以及是否存在 是迁移过程中的变化，d) 将整合素数据关联起来 mRNA 表达以及整合素蛋白合成数据。 目标 2 是 确定角膜上皮迁移延迟是否由以下因素引起 将大鼠多形核白细胞 (PMN) 的提取物添加到 清创损伤的角膜器官培养物涉及角膜组织的改变 a) 使用免疫印迹法确定整合素的量或定位， 免疫沉淀和 mRNA 定量以确定 PMN 提取物是否 添加到清创伤口中会影响上皮中的蛋白质合成 通过测定整合素、纽蛋白、α-肌动蛋白、α- 烯醇化酶和 ICAM-1，b) 确定是否添加纯化的 炎症细胞因子对角膜器官培养物上皮细胞的影响 清创伤口导致上皮细胞迁移延迟 机制类似于 PMN 提取物。 目标 3 是确定是否 半桥粒的分解和角膜上皮的迁移 涉及 β4 亚基的磷酸化和/或蛋白水解裂解 a) 确定 HD α6 和 beta4 亚基是否 未受伤的角膜中的酪氨酸被磷酸化，如果它们 磷酸化状态在迁移过程中改变，b) 发育 多克隆抗血清，对整个 beta4 的胞外结构域或整个胞质部分 分子，以及 c) 使用抗血清确定 beta4 分子是否 在上皮细胞迁移过程中经历裂解。 目标 4 是 确定细胞区域的整合素：细胞相互作用 对于维持角膜中细胞与细胞的接触具有重要功能 a) 建立牛角膜细胞培养条件 上皮细胞，b) 确定桥粒的组装状态， 粘附蛋白连接，以及含有 alphav 和 beta1 的细胞：细胞 低钙培养的细胞中的连接以及将细胞转移到 高钙培养基，c) 测定粘附阻断的效果 整合素肽和抗体对培养角膜能力的影响 上皮细胞在低钙和高钙条件下维持细胞：细胞接触 培养基，以及 d) 确定是否将 PMN 提取物添加到培养物中 细胞破坏细胞与细胞的接触以及细胞与细胞的定位 低钙和高钙培养基中的 alphav 和 beta1 整合素。 这些 实验将有助于实现我们获得更好的目标 在分子水平上了解整合素在 正常角膜和伤口愈合期间上皮细胞迁移。