视网膜下液致视网膜色素上皮细胞和神经胶质细胞增殖过程中PKC活性的变化(英文)  

The changes of protein kinase C for human retinal pigment epithelium and retinal glial cells proliferation induced by the subretinal fluid

作　　者：孙兆义[1] 洪晶[1] 

机构地区：[1]中国医科大学附属一院眼科,中国辽宁省沈阳市110001

出　　处：《国际眼科杂志》2006年第3期513-518,共6页International Eye Science

基　　金：中国国家自然科学基金资助项 目(N o.30471865)~~

摘　　要：目的:研究视网膜下液(SRF)能否引起体外培养的人视网膜色素上皮(RPE)和神经胶质(RG)细胞发生增殖及在其增殖过程中是否出现蛋白激酶 C 的激活和转位, 来探讨RPE 和 RG 细胞增殖与其细胞中 PKC 信号系统变化的关系以及 PKC 抑制剂的作用。方法:实验对象为体外培养的 RPE 和 RG 细胞;刺激因素为提取 PVR 分级是 B、C 级两级患者的 SRF 和来自角膜移植后的供体眼球所提供的正常玻璃体成分;PKC 特异激活剂佛波酯(PMA)为阳性对照;DMEM 培养液作为空白对照。用 3H- 胸腺嘧啶脱氧核苷(3H- TdR)掺入法测定各自的RPE 和 RG 细胞增殖情况。用 B、C 级 SRF、正常玻璃体成分、PMA、DMEM 培养液分别在不同的时间刺激 RPE 和 RG细胞, 通过细胞裂解和离心获取细胞质和细胞膜蛋白粗提液,用同位素 32P 标记和液体闪烁计数法检测细胞质和细胞膜 PKC 活性水平。选用 PKC 抑制剂地喹氯铵预处理各组细胞后,再分别观察各组 RPE 和 RG 细胞中 PKC 活性表达水平及增殖情况。结果:用 B、C 级 SRF 和 PMA 处理过的 RPE 细胞出现高增殖;SRF 和 PMA 都可以激活 RPE 和 RG 细胞质中的 PKC,并使其由胞质向胞膜转位,但 SRF 作用于 RPE 和 RG 细胞时,胞膜上 PKC 活性峰值出现的时间较 PMA 明显延长。其中,B 级 SRF 作用于 RPE 和 RG 细胞时,胞膜上 PKC 活性峰值出现的时间较 C 级长且峰值低,增殖程度也低;正常玻璃体成分和 DMEM 培养液组没有出现 PKC 活性变化和高增殖。用 PKC 特异抑制剂预处理各组细胞后,没有出现PKC 活性和细胞增殖的改变,组间无显著性差异,P>0.05。结论:SRF 可促进 RPE 和 RG 细胞增殖,RPE 和 RG 细胞中的 PKC 是以激活和转位方式参与细胞增殖的过程;使用PKC 特异抑制剂可阻止此过程发生。AIM： To study the effect of the subretinal fluid （SRF） on proliferation of retinal pigment epithelium （RPE） cells and retinal glial （RG） cells and associated activation and translocation of protein kinase C （PKC） as well as the application of PKC inhibitor. MTEHODS： RPE and RG cells were disintegrated to obtain PKC activity of cytoplasm and cellular membrane after being treated by the subretinal fluid （SRF） from the different stages of PVR patients （grade B and C） or being treated with PKC specific activator [phorbol-12-myris-tate-13-acetate （PMA）] or normal vitreous or DMEM culture medium. PKC activity in cytoplasm and cellular membrane was measured using radioactive isotope 32P labeling in a specific reaction of phosphorylation on PKC substrate. In addition, the PKC inhibitor, dequalinium chloride, was used to pretreat the RPE and RG cells before the cells exposed to SRF or PMA or normal vitreous. 3H-TdR （tritiated thymidine） was used to measure the levels of proliferation of RPE and RG cells with or without the activation and translocation. RESULTS： SRF and PMA promoted the proliferation of RPE and RG cells. SRF and PMA activated PKC in the cytoplasm of RPE and RG cells and the activated cytoplasm PKC translocated to the cellular membrane of RPE or RG cells. The cell proliferation or PKC activation or translocation were not equally active in RPE as in RG cells. However, PKC inhibitor which attenuated the cell proliferation did not show significant difference on inhibition of RPE and RG cell proliferation. （P 〉0.05）. CONCLUSION： SRF can lead to the activation and translocation of PKC in RPE and RG cells, which promote the proliferation of RPE and RG cells. Dequalinium chloride can inhibit PKC activation and translocation hence slow down the cells proliferation.

关 键 词：视网膜下液 蛋白激酶C 视网膜色素上皮细胞 神经胶质细胞 激活和转位 增殖 

分 类 号：R77[医药卫生—眼科]