CORNEAL ENDOTHELIAL WOUND REPAIR IN THE MAMMALIAN EYE

这项建议的长期目标是调查机制， 在体内创伤修复过程中参与角膜内皮细胞迁移 和体外培养。 具体来说，微丝的分布， 中间丝和微管，所有的细胞骨架结构， 用荧光抗体在非损伤和损伤组织中进行检查 平片标本免疫细胞化学。 利用器官培养 模型，额外的实验将采用适当的细胞骨架 抑制剂，如秋水仙碱和细胞松弛素，以检查 在伤口修复过程中，每个细丝网络的贡献。 的 后弹力膜对内皮损伤修复影响将是 考察 光学和电子显微镜，使用荧光和过氧化物酶 抗体免疫细胞化学，将被用来研究的作用， 创伤愈合过程中的细胞外基质蛋白层粘连蛋白和纤连蛋白 in vivo. 超微结构分析采用钌组织化学染色 红色和阿尔新蓝，突出微纤维将被用来检查 细胞/基质粘附。 戊二醛-鞣酸-鬼笔菌素固定 将用于对比和稳定F-肌动蛋白，以研究 微丝在正常基质粘附中的作用和损伤过程中的变化 修复. 不同基质对内皮细胞骨架的影响 将通过在涂有IV型的玻璃盖玻片上生长细胞进行检查 胶原蛋白、纤连蛋白和层粘连蛋白。 偶联凝集素将 可用于确定细胞表面糖蛋白的变化， 损伤修复并将其与细胞骨架改变相关联。 领导 将使用透射和扫描检查迁移细胞的边缘 电子显微镜来确定为什么细胞迁移如此明显 德斯密氏膜向损伤中心移动。 因为人类角膜 内皮细胞增殖能力差，迁移是其主要的表达方式。 修复. 这些调查的结果预计将提高我们的 了解内皮细胞运动，并可为 促进人内皮的修复。

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.