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目的探讨视网膜下液(SRF)能否引起体外培养的视网膜色素上皮(RPE)细胞增殖及在其增殖过程中是否出现蛋白激酶C(PKC)的激活和转位,进一步明确RPE细胞增殖与RPE细胞中PKC信号系统变化的关系及PKC抑制剂的作用。方法实验对象为体外培养的RPE细胞;刺激因素为提取的增生性玻璃体视网膜病变(PVR)为B、C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特异抑制剂N,N-二甲基鞘氨醇预处理各组细胞后,再分别观察各组RPE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细胞质中的PKC,并使其由胞质向胞膜转位,但SRF作用于RPE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培养液组未出现PKC活性变化和高增殖。用PKC特异抑制剂预处理各组细胞后,未出现PKC活性和细胞增殖的改变,组间比较差异无统计学意义(P>0．05)。结论SRF可促进RPE细胞增殖,RPE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 retinal pigment epithelial (RPE) cells cultured in vitro and the activation and translocation of protein kinase C (PKC) in the process of proliferation, further clarify the relationship between RPE cell proliferation and RPE cells In PKC signaling system changes and the role of PKC inhibitors. Methods The experimental subjects were RPE cells cultured in vitro. The stimulating factors were the extracted vitreoretinopathy (PVR), the SRF from patients with grade B and C and the normal vitreous component from the donor eye after corneal transplantation. PKC was specifically activated Agent phorbol ester (PMA) as a positive control; DMEM culture medium as a blank control. Proliferation of RPE cells was determined by 3H-thymidine incorporation (3H-TdR). RPE cells were stimulated with B, C grade SRF, normal vitreous body composition, PMA and DMEM culture medium at different times. Cytoplasm and plasma membrane protein crude extracts were obtained by cell lysis and centrifugation. Cytoplasm was detected by isotope 32P labeling and liquid scintillation counting And cell membrane PKC activity levels. PKC specific inhibitor N, N-dimethyl sphingosine were used to pretreat the cells of each group, and then the expression level and proliferation of PKC in RPE cells were observed. Results Both RPE cells treated with B and C SRF and PMA showed high proliferation. Both SRF and PMA activated PKC in the cytoplasm of RPE and translocated from the cytoplasm to the cytoplasm. However, when SRF and RPA were applied to RPE cells, The peak of PKC activity on the membrane was significantly longer than PMA. The peak of PKC activity appeared on the membrane when the B grade SRF was applied to RPE cells. The peak value of PKC activity was lower than that of the C grade and the proliferation was also low. The normal vitreous body composition and DMEM culture medium showed no change of PKC activity and high proliferation. After PKC-specific inhibitor pretreatment of each group of cells, there was no change of PKC activity and cell proliferation. There was no significant difference between the two groups (P> 0.05). Conclusion SRF can promote the proliferation of RPE cells. PKC in RPE cells is involved in the process of cell proliferation by activation and translocation; PKC-specific inhibitors can prevent this process.