EFFECT OF CYCLIC AMP MEDIATORS ON THE CYTOSKELETON

这项研究的长期目标是确定 视网膜色素上皮细胞中的细胞骨架 迁移。通过定义细胞骨架构型的变化 发生在细胞迁移过程中，病理生理学 增殖性玻璃体视网膜病变的机制可以更好地 理解，从而导致更有效的治疗方法 条件。这项研究的具体目的是确定 细胞内环-3，‘5’-环磷酸腺苷(CAMP)的介导物 收缩蛋白F-肌动蛋白和肌动蛋白的频率和分布 膜相关蛋白纽蛋白与 与细胞有关的微丝束 迁移和趋化作用。细胞内cAMP的介体有 已被证明能抑制人体内的迁移和趋化 RPE细胞，并已被证明促进形成和 抑制应力纤维(微丝束)的收缩 主要由F-肌动蛋白组成)。因此，这些 既抑制RPE迁移又影响RPE迁移的介质 F-肌动蛋白在成纤维细胞中的排列是确定 与细胞迁移抑制相关的细胞骨架改变 人类的RPE。 随着人RPE细胞在体外的黏附和扩散， 与试验试剂(二丁酰环磷腺苷、福斯可林、L)孵育 肾上腺素、L-去甲肾上腺素、L-异丙肾上腺素和咖啡因)将 然后进行纽蛋白的免疫荧光标记 用小鼠抗鸡纽蛋白单抗， 荧光素标记的羊抗鼠抗体。双倍 用F-肌动蛋白荧光探针罗丹明-鬼臼糖苷标记， 然后就会被执行。肌动蛋白的频率和分布 和纽蛋白及其与微丝束的关系将 被荧光显微镜记录下来，并与 控制。在肾上腺素能药物诱导细胞骨架的情况下 变化，药物阻滞剂(噻吗洛尔)将用于 确定β-肾上腺素能受体在中介中的作用 这样的变化。

The long-term objective of the study is to determine the role of the cytoskeleton in retinal pigment epithelial (RPE) cell migration. By defining changes in the cytoskeletal configuration which occur during cell migration, the pathophysiologic mechanisms of proliferative vitreoretinopathy can be better understood, leading to more effective treatments of this condition. The specific aim of this study is to define the effect of mediators of intracellular cyclic-3, '5'-AMP (cAMP) on the frequency and distribution of the contractile protein F-actin and the membrane-associated protein vinculin with respect to microfilament bundles which have been implicated in cell migration and chemotaxis. Mediators of intracellular cAMP have been demonstrated to inhibit migration and chemotaxis in human RPE cells, and have also been shown to promote formation and inhibit contraction of stress fibers (microfilament bundles consisting primarily of F-actin) in fibroblasts. Thus, these mediators which are both inhibitory of RPE migration and affect F-actin arrangements in fibroblasts are ideal agents to determine cytoskeletal changes associated with inhibition of cell migration of human RPE. Following adherence and spreading of human RPE cells in vitro, incubation with test reagents (dibutyryl-cAMP, forskolin, L- epinephrine, L-norepinephrine, L-isoproterenol, and caffeine) will be performed, followed by immunofluorescent labeling of vinculin using mouse monoclonal anti-chicken vinculin antibody and fluorescein-conjugated goat anti-mouse IgG antibody. Double labeling with the F-actin fluorescent probe, rhodamine-phalloidin, will then be performed. The frequency and distribution of actin and vinculin and their relationship to microfilament bundles will be documented by fluorescence microscopy and compared with controls. In the event that adrenergic agents induce cytoskeletal changes, pharmacologic blockade (timolol) will be used to determine the role of beta adrenergic receptors in the mediation of such changes.