蛋白酶体β5亚单位过表达对氧化环境下人晶状体上皮细胞的影响_医学论文.docVIP

蛋白酶体β5亚单位过表达对氧化环境下人晶状体上皮细胞的影响_医学论文 蛋白酶体β5亚单位过表达对氧化环境下人晶状体上皮细胞的影响_医学论文 作者：张铁英， 柳夏林， 吴明星， 刘奕志 【摘要】 【目的】 探讨蛋白酶体β5亚单位(PSMB5)基因过表达对氧化条件下晶状体上皮细胞的影响。【方法】构建重组质粒pcDNA3.1－PSMB5并将其转染入人晶状体上皮细胞株SRA01/04中，形成稳定表达，同时设pcDNA3.1空载体为对照组。RT-PCR法及Western blot法分别检测PSMB5基因及蛋白的表达情况。H2O2分别作用PSMB5转染细胞及空载体转染细胞，MTT法检测细胞增殖能力的变化。【结果】 转染后用G418筛选3周后，获得PSMB5转染及空载体转染的G418抗性的细胞克隆。转染PSMB5基因的细胞PSMB5 mRNA表达强度明显增高，而空载体转染细胞与未转染细胞无显著差异；转染PSMB5基因的细胞的PSMB5蛋白表达也显著高于空载体转染细胞。低浓度H2O2作用后，转染PSMB5基因的细胞增殖能力高于空载体转染细胞。【结论】低浓度氧化环境下蛋白酶体β5亚单位PSMB5过表达能对人晶状体上皮细胞具有保护作用。 【关键词】 过表达； 蛋白酶体； PSMB5； 晶状体； 增殖 Abstract: 【Objective】 To explore the influence of proteasome subunit β5 (PSMB5) gene overexpression in human lens epithelial cells under the oxidative condition. 【Methods】 The recombinant plasmid pcDNA3.1-PSMB5 was constructed and transfected into human lens epithelial cell line SRA01/04 to form stable transfection, and empty pcDNA3.1 was also transfected into lens epithelial cells at the same time as control. RT-PCR and Western blot analysis were performed to detect the PSMB5 gene and protein expression. Cell viability assay was performed using 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide substrate (MTT) after the above cells were treated by H2O2. 【Results】 The G-418-resistant cell clones, which had been transfected with PSMB5 or empty vector were obtained by screening continually with G-418 for 3 w after transfection. There was stronger expression of PSMB5 mRNA in the cells transfected with PSMB5 gene than in those transfected with empty vector or non-transfected cells the expression of PSMB5 protein was also significantly upregulated in PSMB5 transfected cells than in empty vector transfected cells. Cell viability of PSMB5 transfected cells was statistically higher than that of empty-transfected cells under the low H2O2 concentration.【Conclusion】Overexpression of the proteasome β5 catalytic subunit can play protective role to human lens epithelial cells under the low oxidative environment. Key words: overex