【摘要】 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、Ca²⁺-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Ca²⁺-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Ca²⁺-ATP蛋白表达量高于NC-OE组,KD组Ca²⁺-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Ca²⁺-ATP蛋白表达量高于NC-OE组,KD组Ca²⁺-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Ca²⁺-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。更多还原

【Abstract】 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 Ca²⁺-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.Ca²⁺-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 Ca²⁺-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.更多还原