CELL/MATRIX INTERACTIONS IN CORNEAL STROMAL DEVELOPMENT

该建议的主要目的是阐明细胞基质 在角膜基质的发育过程中重要的相互作用。我们 假设是细胞-基质相互作用介导了形态学和 生理变化发生在这个时候。 我们将描述 整合素和非整合素受体在四个阶段的表达 发展：（1）在眼周间充质内迁移之前， 角膜成纤维细胞前体;（2）角膜成纤维细胞前体迁移开始后， 成纤维细胞前体;（3）当分化开始时，在接触 （4）当完全分化的角膜成纤维细胞被 沉积次生基质的有序基质。整合素是 异二聚体细胞表面受体，其影响形态学和 与细胞外基质结合后的生理变化。 我们 假设静止表型和有序基质沉积 角膜成纤维细胞的特征将与精确的 整合素异二聚体的组织。这将通过以下方式进行分析： 免疫印迹、免疫沉淀、各种免疫定位 技术和流式细胞术。我们记录了空间和时间 迁移开始期间基质沉积的差异， 角膜成纤维细胞/前体的分化。我们假设 这些变化将与整合素表达的变化相关， 将使用相同的技术进行分析。非整联蛋白受体可能 在角膜发育过程中也很重要。接下来，我们将描述 非整合素基质受体的表达：NG 2，其结合VI型 胶原蛋白和透明质酸受体。VI型胶原和透明质酸盐 在角膜发育过程中表现出分布的变化。因此我们 假设这两种配体的受体在 这次然后将确定什么样的体外培养条件 维持基质受体的表达。使用新鲜分离的细胞 可以让我们做短期的功能性论文，但我们假设， 通过调节培养条件，我们将能够维持 长期研究。最后，功能研究将确定 受体实际上与特定的配体结合。 我们假设 不同发育阶段的细胞会表现出不同的 附着、扩展和迁移，与基质有关 受体表达抗体抑制试验将评估 这些细胞中的各种整合素亚基/异二聚体和非整合素 流程.角膜移植过程中细胞-基质相互作用的表征 发展将提供有关基本过程的信息， 调节角膜透明度以及参与伤口修复的那些。

The major objective of this proposal is to elucidate cell-matrix interactions important during development of the cornea stroma. Our hypothesis is that cell-matrix interactions mediate the morphological and physiological changes which occur at this time. We will characterize the expression of integrin and non-integrin receptors during four stages of development: (1) within the periocular mesenchyme prior to migration of corneal fibroblast precursors; (2) upon initiation of migration of corneal fibroblast precursors; (3) when differentiation begins, upon contact with the primary stroma; (4) when fully differentiated corneal fibroblasts are depositing the orderly matrix of the secondary stroma. Integrins are heterodimeric cell surface receptors which effect morphological and physiological changes upon binding to extracellular matrix. We hypothesize that the stationary phenotype and orderly matrix deposition characteristic of corneal fibroblasts will be correlated with a precise organization of integrin heterodimers. This will be analyzed using immunoblotting, immunoprecipitation, various immunolocalization techniques, and flow cytometry. We have documented spatial and temporal differences in matrix deposition during the initiation of migration and differentiation of cornea fibroblasts/precursors. We hypothesize that these changes will correlate with changes in integrin expression, which will be analyzed using the same techniques. Non-integrin receptors may also be important during corneal development. Next, we will characterize the expression of non-integrin matrix receptors: NG2, which binds type VI collagen, and the hyaluronate receptor. Type VI collagen and hyaluronate exhibit changes in distribution during corneal development. Thus, we hypothesize that receptors for both these ligands will be important at this time. It will then be determined what in vitro culture conditions maintain expression of matrix receptors. Use of freshly isolated cells will enable us to do short term functional essays, but we hypothesize that by adjusting culture conditions we will be able to maintain expression for longer term studies. Finally, functional studies will determine which receptor is actually binding a particular ligand. We hypothesize that cells from different developmental stages will exhibit differential attachment, spreading and migration which can be correlated with matrix receptor expression. Antibody inhibition assays will assess the role of various integrin subunits/heterodimers and non-integrins in these processes. Characterization of cell-matrix interactions during corneal development will provide information concerning the fundamental processes regulating cornea transparency as well as those involved in wound repair.