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骨架 迁移 通过定义细胞骨架结构的变化 发生在细胞迁移过程中， 增殖性玻璃体视网膜病变的机制可以更好地 了解，导致更有效的治疗， 条件 本研究的具体目的是确定 细胞内环-3，5 '-AMP（cAMP）介导的 收缩蛋白F-肌动蛋白的频率和分布， 膜相关蛋白黏着斑蛋白 细胞内的微丝束 迁移和趋化性。 细胞内cAMP的介导剂具有 已被证明可抑制人类的迁移和趋化性 视网膜色素上皮细胞，也已被证明可以促进形成和 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 介质既抑制RPE迁移，又影响 成纤维细胞中的F-肌动蛋白排列是确定 与细胞迁移抑制相关的细胞骨架变化 人类RPE 在体外人RPE细胞的粘附和铺展之后， 与测试试剂（二丁酰-cAMP、毛喉素、L- 肾上腺素、L-去甲肾上腺素、L-异丙肾上腺素和咖啡因）将 进行，然后进行黏着斑蛋白的免疫荧光标记 使用小鼠单克隆抗鸡黏着斑蛋白抗体， 荧光素缀合的山羊抗小鼠IgG抗体。 双 用F-肌动蛋白荧光探针罗丹明-鬼笔环肽标记， 将被执行。 肌动蛋白的频率和分布 和黏着斑蛋白以及它们与微丝束的关系将 通过荧光显微镜记录，并与 对照 如果肾上腺素能药物诱导细胞骨架 变更，将使用药物阻断（噻吗洛尔） 确定β肾上腺素能受体在介导 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.