目的:研究钙通道拮抗剂-维拉帕米(verapamil,Ver)诱导视网膜色素上皮(retinal pigment epithelium,RPE)细胞凋亡过程中钙离子及凋亡基因caspase-3变化。方法:应用80mg/L的Ver分别作用健康人眼RPE细胞12,24及48h诱导凋亡,设立对照组。逆转录聚合酶链反应(RT-PCR)检测凋亡基因caspase-3的表达,采用Fluo-3/AM负载技术,MetaFluo4.5/coolsnapfx/IX70细胞内钙离子荧光成像系统测定每组20个RPE细胞钙荧光值,并计算RPE细胞内钙浓度([Ca2+]i)。结果:对照组RPE细胞Ca2+荧光分布胞核最强,胞质次之。Ver作用12,24及48h后,细胞内[Ca2+]i明显降低(P<0.01)。对照组RPE细胞可见caspase-3的mRNA有少量的表达。Ver作用12h后,可见caspase-3的mRNA有较高的表达,与对照组比较,具有显著性差异(P<0.01)。随着Ver作用时间的延长,caspase-3的mRNA表达逐渐增强,在48h时有所下降。结论:Caspase-3基因表达上调及RPE细胞内钙离子稳态失调可能在Ver诱导RPE细胞凋亡中起关键作用。

AIM:To study caspase-3 gene expression and [Ca2+]i homeostasis in verapamil(Ver)-induced human retinal pigment epithelium(RPE)cells apoptosis.METHODS:Ver 80mg/L was applied in cultured human RPE cells for 12,24 and 48 hours to induce RPE cells apoptosis.The expression of apoptotic effector gene caspase-3 was assessed by reverse transcription polymerase chain reaction(RT-PCR).Single cell was measured using fluorescence indicator Fura-3/AM with MetaFluo4.5/coolsnapfx/IX70 intracellular Ca2+ fluorescence imaging system.RESULTS:High levels of expression of caspase-3 mRNA were observed in normal RPE cells and it significantly increased after co-cultured with Ver.The fluorescence in resting RPE cells was strong and distributed throughout the cells.The nucleus appeared more fluorescent than the cytoplasm.Calcium fluorescence of RPE cells attenuated after co-cultured with Ver.CONCLUSION:Up-regulation of caspase-3 gene expression and disturbance of [Ca2+]i homeostasis might play pivotal roles in Ver-induced RPE cells apoptosis.

中国国家自然科学基金资助项目（No.30471865）