MOLECULAR ASPECTS OF CORNEAL EPITHELIAL MIGRATION

角膜上皮迁移的分子方面

• 批准号: 3265840
• 负责人: Mary Ann Stepp
• 金额: $ 19.14万
• 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/3265840
• 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 这些接触即使在迁移时也能保持其完整性 对伤害的反应。 在细胞之间：基底连接， 半桥粒（HD）负责大部分的粘附， 上皮细胞与其基质的结合。 HD含有α 6 β 4整联蛋白作为一个 它们的组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 是为了确定β 1，β 4， α 3、α 5和α 6整联蛋白mRNA在迁移过程中被改变， a）从大鼠角膜上皮细胞扩增大鼠整联蛋白cDNA 文库，B）确定大鼠整联蛋白mRNA的表达是否 在迁移过程中发生变化，c）确定 细胞质整合素mRNA选择性剪接变体以及是否存在 是迁移过程中的变化，以及d）将整合素上的数据 mRNA表达与整合素蛋白合成数据。 目标二是 确定是否延迟角膜上皮迁移诱导的 将大鼠多形核白细胞（PMNs）提取物添加到 清创损伤的角膜器官培养涉及到 整合素的量或定位通过a）使用免疫印迹， 免疫沉淀和mRNA定量，以发现是否PMN提取物 添加到清创伤口影响上皮中的蛋白质合成 通过测定整联蛋白、黏着斑蛋白、α-肌动蛋白、α-肌动蛋白和α-肌动蛋白的水平， 和B）确定是否加入纯化的 炎性细胞因子对角膜器官培养物的影响 清创伤口导致上皮迁移延迟， 机制类似于PMN提取物。 目标3是确定 半桥粒解体和角膜上皮迁移 涉及β 4亚基的磷酸化和/或蛋白水解裂解 通过a）确定HD α 6和β 4亚基是否 在未受伤的角膜中酪氨酸磷酸化， 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 β 4的胞外域或整个胞质部分 分子，和c）使用抗血清确定β 4分子是否 在上皮细胞迁移期间经历分裂。 目标4： 确定细胞：细胞相互作用区域的整合素是否 在维持角膜中的细胞：细胞接触方面功能重要 a）建立牛角膜上皮细胞培养条件 上皮细胞，B）确定桥粒的组装状态， 粘附素连接，以及含有α v和β 1的细胞：细胞 在低钙培养的细胞中， 高钙介质，c）确定粘连阻断的效果 整合素肽和抗体对培养角膜上皮细胞增殖能力的影响 上皮细胞维持细胞：细胞接触在低和高钙 d）确定是否将PMN提取物添加到培养的 细胞破坏细胞：细胞接触和细胞：细胞定位， α V和β 1整联蛋白。 这些 实验将有助于实现我们的目标， 在分子水平上理解整合素在肿瘤细胞中的作用， 正常角膜和伤口愈合期间上皮细胞迁移。