CORNEAL ENDOTHELIAL WOUND REPAIR IN THE MAMMALIAN EYE

这项提议的长期目标是研究 角膜内皮细胞在体内创伤修复过程中的迁移 而且是在体外。具体地说，微丝的分布， 中间丝和微管，所有的细胞骨架结构，将 用荧光抗体在非损伤组织和损伤组织中进行检测 平板贴装制剂的免疫细胞化学。利用器官培养作为一种 模型，其他实验将使用适当的细胞骨架 抑制剂，如秋水仙素和细胞松弛素，以检查 每个细丝网络在伤口修复过程中的贡献。这个 Descemet膜对内皮伤口修复的影响将是 检查过了。光学显微镜和电子显微镜，使用荧光和过氧化物酶 抗体免疫细胞化学，将被用来研究其作用 伤口愈合过程中的细胞外基质蛋白、层粘连蛋白和纤维连接蛋白 在活体内。用Ru组织化学染色进行超微结构分析 红色和阿尔新蓝，突出微纤维将进行检查 细胞/底物粘附性。戊二醛-单宁酸-蝴蝶兰素固定化 将用于对比和稳定F-肌动蛋白以研究 微丝在正常基质黏附中的作用及其在损伤过程中的变化 修理。不同底物对内皮细胞骨架的影响 将通过在涂有IV型玻璃盖的玻璃纸上培养细胞进行检查 胶原蛋白、纤维连接蛋白和层粘连蛋白。荧光偶联凝集素将 用来测定细胞表面糖蛋白的变化 损伤修复并将这些与细胞骨架的改变相关联。领先者 将使用透射法和扫描法检查迁移细胞的边缘 用电子显微镜来确定为什么细胞迁移如此明显 Descemet的膜朝向损伤中心。因为人类的角膜 内皮细胞增殖不良，迁移是其主要途径 修理。这些调查的结果可望加强我们的 了解内皮细胞的运动性，并可为 促进人体内皮细胞的修复。

The long-term objective of this proposal is to investigate the mechanisms involved in corneal endothelial cell migration during wound repair in vivo and in vitro. Specifically, the distribution of microfilaments, intermediate filaments and microtubules, all cytoskeletal structures, will be examined in noninjured and injured tissues using fluorescent antibody immunocytochemistry on flat mount preparations. Using organ culture as a model, additional experiments will employ appropriate cytoskeletal inhibitors, like colchicine and the cytochalasins, to examine the contributions made by each filament network during wound repair. The influence of Descemet's membrane on endothelial wound repair will be examined. Light and electron microscopy, using fluorescence and peroxidase antibody immunocytochemistry, will be used to study the role of the extracellular matrix proteins laminin and fibronectin during wound healing in vivo. Ultrastructural analysis using the histochemical stains ruthenium red and alcian blue, that highlight microfibrils will e done to examine cell/substrate adhesion. Glutaraldehyde-tannic acid-phallacidin fixation will be used to contrast and stabilize F-actin for investigating the microfilament role in normal substrate adhesion and changes during injury repair. The influence of various substrates on endothelial cytoskeleton will be examined by growing cells on glass coverslips coated with type IV collagen, fibronectin and laminin. Fluorescently conjugated lectins will be used to determine the changes in cell surface glycoproteins during injury repair and correlate these to cytoskeletal alterations. The leading edge of migrating cells will be examined using transmission and scanning electron microscopy to determine why cells migrate so distinctly on Descemet's membrane toward the injury center. Because human corneal endothelial cells proliferate poorly, migration is their major means of repair. The results of these investigations are expected to enhance our knowledge of endothelial cell motility and could provide a basis for facilitating repair in the human endothelium.