急性氧化应激状态下人晶状体上皮细胞株SRA01/04中衰老标记蛋白30、Caspase-3、Ca2+-ATP表达的变化

Effect of acute oxidative stress on expression of senescence marker protein-30,Caspase-3,Ca2+-ATP in human lens epithelial cell line SRA01/04

目的:探讨在急性氧化应激状态下人晶状体上皮细胞株SRA01/04内衰老标记蛋白30(SMP30)、Caspase-3、Ca^2+-ATP表达的变化。方法:通过慢病毒转染建立SMP30过表达(OE)组、过表达空载对照(NC-OE)组、SMP30沉默(KD)组及沉默空载(NC-KD)组SRA01/04细胞,RT-PCR和Western blotting检测SMP30含量鉴定细胞是否转染成功;用300μmol/L H2O2 作用2 h,构建急性氧化应激状态时,PCR检测各组细胞SMP30基因的表达情况,Western blotting检测各组细胞SMP30、Caspase-30和Ca^2+-ATP蛋白的表达量。结果:在正常状态时,OE组SMP30基因及其蛋白表达量高于NC-OE组(P<0.05),KD组的SMP30基因及其蛋白表达量低于NC-KD组(P<0.05)。OE组及KD组细胞SMP30基因表达量均出现增加(P<0.05),与正常状态时相比,急性氧化应激状态时OE组细胞SMP30蛋白表达上调(P<0.05);正常状态时,OE组Caspase-3蛋白表达量低于NC-OE组,KD组Caspase-3蛋白表达量高于NC-KD组(P<0.05)。急性氧化应激状态时,OE组Caspase-3蛋白表达量低于NC-OE组,KD组Caspase-3蛋白表达量低于NC-KD组(P<0.05)。在急性氧化应激状态时OE组Caspase-3蛋白表达量低于正常状态时;KD组的Caspase-3蛋白表达量高于正常状态时KD组(P<0.05)。在正常状态时,OE组Ca^2+-ATP蛋白表达量高于NC-OE组,KD组Ca^2+-ATP蛋白表达量低于NC-KD组(P<0.05)。在急性氧化应激状态时,OE组Ca^2+-ATP蛋白表达量高于NC-OE组,KD组Ca^2+-ATP蛋白表达量低于NC-KD组(P<0.05)。在急性氧化应激状态时OE组的Ca^2+-ATP蛋白表达量高于正常状态时(P<0.05)。结论:在急性氧化应激状态时,SMP30含量出现反应性增加,过表达SMP30可能对人晶状体上皮细胞株SRA01/04具有抗氧化、抗凋亡、增加钙调节活性的作用。

Objective:To investigate the effect of acute oxidative stress on expression of senescence marker protein-30(SMP30),Caspase-3 and Ca2+-ATP in human lens epithelial cell(HLEC) line SRA01/04.Methods:SMP30 over-expression group and knock-down group and corresponding negative control(NC-OE and NC-KD) groups were established by lentivirus transfection,RT-PCR and Wester Blotting were used to detect whether cells were successfully transfected.Acute oxidative stress was established by using 300 μmol/L H2O2 for 2 h,and the expression of SMP30,Caspase-3 and Ca^2+-ATP in each group were detected by Western Blotting.Results:Under acute oxidative stress condition,Caspase-3 was down-regulated in both OE group and KD group compared with corresponding NC-OE and NC-KD groups.Ca^2+-ATP content was up-regulated in OE group and down-regulated in KD group(P<0.05).Compared with normal culture condition,expression of SMP30 in each group increased under acute oxidative stress.Caspase-3 expression was down-regulated in OE group and up-regulated in KD group.The activity of Ca^2+-ATP was up-regulated in OE group(P<0.05).Conclusion:The expression of SMP30 increased in response to acute oxidative stress,and over-expression of SMP30 may have antioxidant,anti-apoptotic and calcium-regulation activities on SRA01/04.