摘要：
目的 通过体外实验探讨蛋白激酶Cα(PKCα)对人视网膜色素上皮(RPE)细胞移行的影响.方法 实验研究.培养正常人PKCαRPE细胞,将实验分为Control组、Thymeleatoxin (PKCα激动剂)组、siRNA-PKCα(PKCα抑制剂)组和Nonsilencing siRNA (Non-siRNA)组,应用RT-PCR检测RPE细胞内PKCαmRNA的表达.细胞划痕实验观察RPE细胞的移行速度,Transwell实验观察RPE细胞的跨膜细胞数,活细胞动态实验观察RPE细胞的细胞活力.组间数据比较采用独立样本t检验.结果 通过光镜和免疫荧光方法计数成功培养获得RPE细胞.Thymeleatoxin组的RPE细胞内PKCαmRNA的含量明显高于Control组(t=3.712,P=.034),siRNA-PKCα组的PKCαmRNA的含量明显低于Non-siRNA组(t=3.274,P=0.031).Thymeleatoxin组RPE细胞的移行速度、跨膜细胞数、细胞活力等均明显优于Control组(t=9.743、5.616、2.300,均P＜0.05),siRNA-PKCα组的移行速度、跨膜细胞数、细胞活力等均明显弱于Non-siRNA组(t=3.643,P＜0.001;t=7.202,P=0.002;t=2.418,P=0.042).结论 PKCα对人RPE细胞的移行有上调作用,提示PKCα可为增生性玻璃体视网膜病变的防治提供新的药物靶点.%Objective To determine if protein kinase Cα (PKCα) affects the migration of retinal pigment epithelium (RPE) cells.Methods In this experimental study,primary human RPE cells were obtained from normal human eyes (cornea donors).The human RPE cells were divided into four groups:control,thymeleatoxin,nonsilencing siRNA-PKCα,and siRNA-PKCα.PKCα mRNA was analyzed by reverse transcriptase-polymerase chain reaction.Cell migration was analyzed by wound healing,transwell chamber assays,and live-cell imaging.Independent samples t tests were used for comparison between pairs of the four groups.Results Human RPE cells were successfully cultured and identified in vitro.Microscopy and staining tests demonstrated that each cell culture consisted of a pure population of epithelial cells.Thymeleatoxin,an activator of PCK,significantly increased the mRNA levels of PKCα compared to the control group (t=3.712,P=0.034).siRNA-PKCα significantly reduced the mRNA levels of PKCα compared to the nonsilencing siRNA group (t=3.274,P=0.031).The wound healed faster in the thymeleatoxin group than in the control group (t=9.743,P＜0.001),and it healed slower in the siRNA-PKCα group compared to the nonsilencing siRNA group (t=3.643,P＜0.001).A similar tendency among the four groups was supported by cell number counts in the transwell chamber assays and cell migration distances in the 12-hour tracking test.Conclusion PKCα-mediated signal transduction plays a crucial role in RPE cell migration and may be used as a potential therapeutic target against RPE cell migration and proliferative vitreoretinopathy disease.
