摘要目的 观察缺氧状态下人视网膜色素上皮(hRPE)细胞中音猥因子(Shh)与血管内皮生长因子(VEGF)表达的关系.方法 传代培养hRPE细胞,取对数生长期3～6代细胞用于实验.将hRPE细胞分为正常对照组、缺氧实验组及环巴明抑制组.缺氧实验组加入100 μmol/L CoCl2,环巴明抑制组于制作缺氧状态前lh加入20μnnol/L的环巴明进行预处理.3组细胞培养4、8、12、24 h.采用荧光定量聚合酶链反应检测3组hRPE细胞中Shh、VEGF mRNA表达.采用酶联免疫吸附法检测正常对照组、缺氧实验组细胞培养上清液中Shh、VEGF蛋白表达.采用Pearson相关性分析法分析缺氧实验组hRPE细胞中Shh、VEGF mRNA表达与蛋白表达之间的关系.结果 正常对照组可见少量Shh、VEGF mRNA及蛋白表达.缺氧实验组Shh、VEGFmRNA及蛋白表达较正常对照组明显增加,差异有统计学意义(F=178.364、183.732、77.456、91.572,P＜0.01).环巴明抑制组Shh、VEGF mRNA表达较缺氧实验组明显降低,差异有统计学意义(F=68.745、121.834,P＜0.01).相关性分析结果显示,缺氧实验组hRPE细胞中Shh蛋白表达与VEGF蛋白表达呈正相关(r=0.942,P＜0.05);Shh mRNA表达与VEGF mRNA表达呈正相关(r=0.970,P＜0.01).环巴明抑制组hRPE细胞中Shh mRNA表达与VEGF mRNA表达无相关性(r=0.915,P＞0.05).结论 缺氧状态下hRPE细胞中Shh与VEGF表达呈正相关.

abstractsObjective To study the relationship between the expression of sonic hedgehog (Shh) and vascular endothelial growth factor (VEGF) in hypoxic human retinal pigment epithelial (hRPE) cells.Methods Cultured hRPE-19 cells (3rd-6th generations) were used in this experiment.hRPE-19 cells were divided into three groups including the control group,the hypoxia experimental group (100 μmol/L CoCl2) and the inhibition group (pretreatment with 20 μmol/L cyclopamine 1 hour before hypoxia).After culturing for 4,8,12 and 24 hours,the mRNA level of Shh and VEGF genes in these cells were measured by fluorescence quantitative polymerase chain reaction,and the protein level of Shh and VEGF in the supematants were measure by enzymelinked immunosorbent assay.The relationship between the expression of Shh and VEGF was analyzed by Pearson correlation analysis.Results The control group expressed low levels of Shh and VEGF mRNA/protein.The expression of Shh and VEGF mRNA/protein in the hypoxia experimental group was significantly higher than that in the control group (F=178.364,183.732,77.456,91.572;P＜0.01).The expression of Shh and VEGF mRNA in the inhibition group was significantly lower than that in the hypoxia experimental group (F=68.745,121.834;P＜0.01).In the hypoxia experimental group,the expression of VEGF protein was positively correlated with the expression of Shh protein (r=0.942,P＜ 0.05);and the expression of VEGF and Shh mRNA was positively correlated (r=0.970,P＜0.01).However,there was no significant correlation in the expression of VEGF and Shh mRNA in the inhibition group (r=0.915,P＞ 0.05).Conclusion There is a positive correlation between the expression of Shh and VEGF in hypoxic hRPE cells.