急性氧化应激初期人晶状体上皮细胞株SRA01/04中衰老标记蛋白30抗氧化作用的研究

The anti-oxidation of senescence marker protein 30 onhuman lens epithelial cell in the early stage of acute oxidative stress

目的初步探讨衰老标记蛋白30(senescence marker protein 30,SMP30)的增加对处于急性氧化应激初期的人晶状体上皮细胞株SRA01/04的保护作用。方法将处于对数生长期的SRA01/04接种于96孔板,分别使用含150μmol·L^(-1)、200μmol·L^(-1)、250μmol·L^(-1)、300μmol·L^(-1)、350μmol·L^(-1)、400μmol·L^(-1)、450μmol·L^(-1)H2O2的培养基作用2 h,CCK-8法选取最佳H2O2浓度;使用慢病毒对SRA01/04进行转染建立SMP30高表达模型,实验分3组:SMP30过表达(overexpression,OE)组、SMP30过表达相应空载(negative control over expression,NCOE)组及对照(control,CON)组。通过超氧化物歧化酶(superoxide dismutase,SOD)定量检测试剂盒、氧化型谷胱甘肽(oxidized glutathione,GSSG)/总谷胱甘肽(total glutathi-one,T-GSH)测定试剂盒测量细胞内SOD活力及GSSG/T-GSH比值的变化。结果经分析,建立模型的最佳H2O2浓度为300μmol·L^(-1)。在此浓度下,与CON组(5. 783±0. 192) U·mg^(-1)及NCOE组(5. 837±0. 182) U·mg^(-1)相比,OE组细胞内SOD活力升高,为(10. 251±0. 110) U·mg^(-1);与CON组(29. 943±0. 241)及NCOE组(30. 037±0. 433)相比,OE组细胞内GSSG/T-GSH比值(20. 990±0. 342)降低;差异均有统计学意义(均为P <0. 05)。结论当SRA01/04处于H2O2诱导的急性氧化应激初期时,SMP30表达的增加可通过增加SOD活力和降低GSSG/T-GSH比值,加强SRA01/04的抗氧化能力。

Objective To investigate the protective effect of increased senescence marker protein 30(SMP30)on the human lens epithelial cell(HLEC)SRA01/04 in the early stage of acute oxidative stress preliminarily.Methods The SRA01/04 in the period of logarithmic phase were seeded into 96-well plates,treated with 150μmol·L^-1,200μmol·L^-1,250μmol·L^-1,300μmol·L^-1,350μmol·L^1,400μmol·L^-1,450μmol·L-1 hydrogen peroxide(H2O2)for 2 h,respectively,and CCK-8 assay was used to select the optimum concentration.Then the SRA01/04 were transfected with lentivirus to establish a high-expressed model of SMP30.There were three groups:over expression(OE)SMP30 group,negative control over expression(NCOE)group,control group(CON).The levels of intracellular superoxide dismutase(SOD)and total glutathione(T-GSH)/oxidized glutathione(GSSG)were determined by the SOD activity assay kit and T-GSH/GSSH assay kit.Results The optimum concentration of H2O2 in the acute oxidative stress cell model was 300μmol·L^-1.At this concentration,compared with the CON group(5.783±0.192)U·mg^-1 and the NCOE group(5.837±0.182)U·mg^-1,the intracellular SOD value of OE group(10.251±0.110)U·mg^-1 was increased.When compared with the CON group(29.943±0.241)and the NCOE group(30.037±0.433),the GSSG/T-GSH value in the OE group(20.990±0.342)was decreased,and the difference was statistically significant(all P<0.05).Conclusion During the early stage of acute oxidative stress induced by H 2O 2,the up-regulation of SMP30 expression can enhance the antioxidant capacity of SRA01/04,by means of increasing the activity of SOD and decreasing the GSSG/T-GSH ratio.