缺氧及小干扰RNA转染对人RPE细胞增生及血小板源性生长因子-BB表达的影响

Effect of hypoxia and PDGF-BB siRNA transfected on the production of PDGF-BB and proliferation in cultured human RPE cells

背景 增生性玻璃体视网膜病变（PVR）为视网膜表面发生无血管、纤维细胞性增生膜,其发生和发展的具体机制尚未完全阐明.人视网膜色素上皮（hRPE）及血小板源性生长因子（PDGF）在PVR发展中的作用是近几年研究的热点. 目的 探讨缺氧对体外培养hRPE细胞PDGF-BB表达及增生的影响. 方法 hRPE细胞在6孔板中培养,实验组用10、15、20、30、40 μmol/L CoCl2模拟体外培养hRPE细胞的缺氧环境,对照组用未加CoCl2的培养液培养hRPE细胞,采用逆转录PCR（RT-PCR）与ELISA法检测PDGF-BB mRNA和蛋白的表达,采用MTT法检测hRPE细胞增生率.依据转染siRNA的不同将细胞分为PDGF-BB siRNA1组、PDGF-BB siRNA2组、PDGF-BB siRNA3组（为针对PDGF-BB的3条不同的siRNA,其中有一条为有效siRNA）、β-actin siRNA组、无关siRNA组和只加Lip2000的空白对照组.转染4～6h后,除空白对照组外,其余各组加入对细胞PDGF-BB mRNA和蛋白及对hRPE细胞增生影响最明显的15 μmol/L CoCl2模拟细胞缺氧环境24 h,检测PDGF-BB mRNA和蛋白及hRPE细胞增生率. 结果 未加CoCl2的对照组未检测出PDGF-BBmRNA和蛋白的表达,不同浓度CoCl2培养hRPE细胞PDGF-BB mRNA和蛋白的表达量不同,差异均有统计学意义（F=43.737,P＜0.01;F=54.612,P＜0.05）,15μmol/L CoCl2组PDGF-BB的表达量最多,与其他各组比较差异均有统计学意义（P＜0.05）.MTT检测结果显示,不同浓度CoCl2处理后PDGF-BB蛋白表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=95.379,P＜0.01;F=63.375,P＜0.05）,15 μmol/L CoCl2组hRPE细胞增生率明显高于其他浓度组,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达量与hRPE细胞增生率呈线性正相关（r=0.994,P＜0.05）.各细胞转染组hRPE细胞中PDGF-BB mRNA及蛋白的表达整体比较,差异均有统计学意义（F=156.330、125.650,P＜0.01）,且PDGF-BB siRNA2组PDGF-BB mRNA较其他各组明显下降,差异均有统计学意义（P＜0.01）.MTT检测结果显示,各细胞转染组PDGF-BB蛋白的表达及hRPE细胞增生率明显不同,差异均有统计学意义（F=73.131、98.564,P＜0.01）,PDGF-BB siRNA2组PDGF-BB蛋白的表达及细胞增生率与其他各组比较明显下降,差异均有统计学意义（P＜0.05）,PDGF-BB蛋白表达与hRPE细胞增生率呈线性正相关（r=0.996,P＜0.05）.结论 缺氧能够促进PDGF-BB的表达,PDGF-BB的表达上调可显著促进hRPE细胞的增生.在转染靶向PDGF-BB siRNA后,PDGF-BB的表达受到抑制,可有效降低hRPE细胞的增生率.

Background Proliferative vitreoretinopathy （PVR）：no blood vessels,fibrous membrane cell proliferation occurs retinal surface.The development of specific mechanisms has not been completely clarified,but the retinal pigment epithelium（RPE） and platelet-derived growth factor（PDGF） for the past few years are the research hotspot.Objective To explore the effects of hypoxia on the production of PDGF-BB and proliferation in cultured human RPE （hRPE） cells.Methods In the experimental group,10,15,20,30,40 μmol/L CoCl2 were used to mimic hypoxia environment of hRPE cells,the control group did not add CoCl2 on cultured hRPE cells.By using reverse transcriptive PCR （RT-PCR） and enzyme linked immunosorbent assay（ELISA）,the expression of PDGF-BB mRNA and protein was detected,and detected proliferation of hRPE cells by MTT.The cells were divided into 6 groups,different siRNAs were transfected into PDGF-BB siRNA1 group,PDGF-BB siRNA2 group,PDGF-BB siRNA3 group （because PDGF-BB has three different siRNAs,one of which is a valid siRNA）,β-actin siRNA group,unrelated sequence siRNA group,only Lip2000 was plused in the blank control group.After transfection of 4-6 hours,except for blank control group,the other five groups were added to PDGF-BB mRNA and protein and its effect on the proliferation of hRPE cells obviously CoCl2 concentrations （15 μmol/L） simulated hypoxia environment of 24 hours,the detection of PDGF-BB mRNA and protein and hRPE cell proliferation rate.Results Neither PDGF-BB mRNA or protein was found in the blank control group.The production of PDGF-BB mRNA and protein in hRPE cells cultured by different concentrations were different,with significant differences among them （F=43.737,P〈0.01;F=54.612,P〈0.05）,and the expressions of PDGF-BB mRNA and protein of 15 μmol/L CoCl2 group were more than other groups,with significant differences between the two groups （all at P〈0.05）.MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different concentions of CoCl2 groups were different,with significant differences among them（F=95.379,P〈0.01 ; F =63.375,P〈0.05）,the expression of PDGF-BB protein was more and hRPE cell proliferation rate was higher in 15 μmol/L CoCl2group than other groups,with significant differences between the two groups （all at P〈0.05）,postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate （r =0.994,P〈0.05）.The production of PDGF-BB mRNA and protein in the different siRNA transfected groups were different,with significant differences among them （F =156.330,125.650,both at P〈0.01）,and the expressions of PDGF-BB mRNA and protein of PDGF-BB siRNA2 group were more than other groups,with significant differences between them（all at P〈0.05）.MTT showed that PDGF-BB protein and hRPE cell proliferation rate in different siRNA transfectedgroups were different,with significant differences among them （F =73.131,98.564,both at P〈 0.01）.The expression of PDGF-BB protein was less and hRPE cell proliferation rate was lower in PDGF-BB siRNA2 group than other groups,with significant differences between them （all at P 〈 0.05）,postive correlation was found between PDGF-BB protein and hRPE cell proliferation rate （r =0.996,P〈0.05）.Conclusions Proper hypoxia can induce the expression of PDGF-BB,PDGF-BB expression can significantly promote the proliferation of hRPE cells.In the transfection targeting PDGF-BB siRNA,PDGF-BB expression is suppressed,can effectively reduce the value of hRPE cell hyperplasia.