人玻璃体液对培养的视网膜色素上皮细胞形态及骨架蛋白表达的影响

目的 观察人视网膜色素上皮(retinal pigment epithelium, RPE)细胞体外培养时的形态及相关细胞骨架蛋白变化规律，进而研究人玻璃体液对RPE细胞形态及细胞骨架蛋白表达的影响，探讨玻璃体液在增生性玻璃体视网膜病变(proliferative vitreoretinopathy,PVR)中的调控作用。 方法 行人RPE细胞原代培养并传代扩增，采用Western blot法，分别检测角蛋白18(cytokeratin 18, CK18)、平滑肌动蛋白(α-smooth muscle actin,α-SMA)在第2、5、8代细胞中的表达情况。用1∶4玻璃体液培养RPE细胞6 d后，观察细胞形态及超微结构改变，采用免疫细胞化学及Western blot法，检测CK18、α-SMA表达变化。 结果 人RPE细胞在体外培养时，会逐渐丧失上皮样形态，向成纤维样转变，CK18在2代表达量中等，5代最高，而在8代明显下降，而α-SMA逐渐升高。与对照组相比，人玻璃体液促进RPE细胞在形态上向成纤维样细胞转变。同时细胞内CK18表达低于对照组，差异具有显著性的意义(P＜0.05)；α-SMA表达高于对照组，差异具有显著性的意义(P＜0.01)。 结论 人RPE细胞在玻璃体液与血清的作用下向具有收缩性能的间质细胞转变，同时细胞移行能力可能发生改变，结果提示玻璃体液可能促进RPE细胞参与的PVR的发展。 (中华眼底病杂志, 2002, 18: 289-292)

Effect of human vitreous fluid on the morphology and expression of cytoskeleton of cultured retinal pigment epithelial cells

Objective To describe cultured human retinal pigment epithelial (RPE) cells transdifferentiation and investigate the effects of human vitreous fluid on the morphologic and cytoskeleton changes of RPE cells in vitro. Methods Cytoskeleton characteristics in the 2nd, 5th, 8th passage of RPE cells in normal culture, which included cytokeratin 18 (CK18) and α-smooth muscle actin (α-SMA) were analyzed by Western blot. RPE cells were cultured in human vitreous-conditioned medium (VCM) at the concentration of 1∶4 for 6 days, morphologic changes were examined by light and electron microscopy, and cytoskeleton characteristics were analyzed by imunocytochemistry and Western blot. Results During culture in vitro, RPE cells lost epithelial characteristics and aquired fibroblast-like phenotype. The expression of CK18 was the highest at the 5th passage, and it decreased in the following passage, but α-SMA increased gradually. The morphologic transdifferentiation from epithelial to fibroblast-like cells of RPE was accelerated by VCM. Ultrastructural changes such as decreased microvilli and gradually increased rough endoplasmic reticulum and Golgi complex were found during the cultivation. CK18 produced by RPE cells decreased in VMC (P＜0.05), and α-SMA increased (P＜0.01). Conclusion Morphologic changes in epithelial mesenchymal transdifferenetiation of RPE cells are stimulated by VCM and accomplied by the shift of cytoskeleton proteins, The results imply that cells migration may be decreased and contraction may be enhanced in VCM. It may suggest that vitreous accelerates the pathogenesis of PVR and RPE cells play an important role. (Chin J Ocul Fundus Dis, 2002, 18: 289-292)