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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。
Objective: To investigate whether subretinal fluid (SRF) can induce the proliferation of human retinal pigment epithelium (RPE) and glial cells (RG) cultured in vitro and whether protein kinase C activation and translocation occurs during its proliferation To investigate the relationship between the proliferation of RPE and RG cells and the changes of PKC signaling system in their cells and the role of PKC inhibitors. Methods: The experimental subjects were RPE and RG cells cultured in vitro. The stimulus factors were SRF extracted from patients with PVR grade B and C grade and normal vitreous body from donor eye after corneal transplantation. PKC-specific activator PMA was positive control; DMEM culture medium served as a blank control. Proliferation of RPE and RG cells was determined by incorporation of 3H-thymidine (3H-TdR). RPE and RG cells were stimulated with B, C grade SRF, normal vitreous body composition, PMA and DMEM culture medium at different times, and cytoplasm and plasma membrane protein crude extracts were obtained by cell lysis and centrifugation. Isotope 32P labeling and liquid scintillation counting The level of cytoplasmic and cellular membrane PKC activity was measured. PKC inhibitor of quinolium chloride pretreatment cells of each group, and then observed the expression of RPE and RG cells in each group of PKC activity and proliferation. RESULTS: RPE cells treated with SRF and PMA were highly proliferated. Both SRF and PMA activated PKC in RPE and RG cytoplasm and translocated from the cytoplasm to the plasma membrane, but SRF acted on RPE And RG cells, the peak time of PKC activity on the membrane was significantly longer than PMA. Among them, the peak of PKC activity appeared on the membrane when the B-class SRF was applied to RPE and RG cells, the peak value of PKC activity was longer than that of the C-stage, and the proliferation was also low. No changes of PKC activity were found in the normal vitreous body and DMEM culture medium proliferation. No significant difference in PKC activity and cell proliferation occurred after pretreatment of cells with PKC specific inhibitor. There was no significant difference between the two groups (P> 0.05). CONCLUSIONS: SRF can promote the proliferation of RPE and RG cells. PKC in RPE and RG cells is involved in cell proliferation through activation and translocation; PKC-specific inhibitors can prevent this process.