PUMA介导氧化应激诱导的人晶状体上皮细胞凋亡

目的探讨PUMA在氧化应激诱导的人晶状体上皮细胞(human lens epithelialcell,HLEC)凋亡中的作用。方法体外培养的HLEC-B3,用50μmol·L-1H2O2刺激不同的时间后,Hoechst33258染色,凋亡细胞呈核固缩、碎裂形态,计数细胞凋亡率;采用West-ern blot方法检测PUMA及Caspase-3蛋白的表达水平。采用RT-PCR方法检测PUMAmRNA表达水平;设计合成针对人PUMA基因的小干扰片段(siPUMA1、siPUMA2)和一条阴性对照,用lipofectamine2000转染细胞后,验证干扰有效性,观察其对氧化应激诱导的HLEC凋亡的影响。结果 H2O2刺激HLEC4h、8h、12h后,细胞凋亡率分别为3.30%±0.25%、27.16%±0.31%、48.14%±0.57%,凋亡相关蛋白Caspase-3发生时间依赖的表达上调。H2O2可诱导PUMA mRNA及蛋白质表达上调。干扰PUMA的表达能减少H2O2诱导的细胞凋亡,H2O2处理HLEC-B312h后细胞凋亡率为48.24%±0.84%,而敲除PU-MA的表达后细胞凋亡率显著降低为13.72%±1.06%(siPUMA1)和17.56%±0.51%(siPUMA2)。结论 PUMA介导了H2O2诱导的HLEC凋亡,可能在白内障的发生发展中发挥重要作用。

Effect of PUMA on oxidative stress induced apoptosis of human lens epithelial cells

Objective To investigate the effect of PUMA in apoptosis of human lens epithelial cells(HLEC) induced by oxidative stress.Methods After HLEC-B3 cultured in vitro were treated with 50 μmolL-1 H2O2,Hoechst 33258 staining was performed,the apoptotic rate was calculated.PUMA and caspase-3 protein expression were detected with Western blot.RT-PCR was applied to detect the mRNA level of PUMA.Two different siRNA(siPUMA1,siPUMA2) targeting against human PUMA gene and one negative control siRNA were designed,HLEC were transfected with siRNA using lipofectamine 2000,the effect of siRNA on oxidative stress induced apoptosis of HLEC was observed after validating its effectiveness.Results After treated with H2O2 for 4 hours,12 hours and 8 hours,the apoptotic rates were 3.30%±0.25%,27.16%±0.31%,48.14%±0.57% of the cells,the expression of apoptosis related protein caspase-3 increased with the time increasing.Up-regulation of PUMA mRNA and protein was induced by H2O2 in a time-dependent manner.Interfering the PUMA expression can decrease the apoptosis induced by H2O2,the apoptotic rate after treated with H2O2 for 12 hours were 48.24% ±0.84%,which after RNAi-mediated PUMA knock-down decreased to 13.72%±1.06%(siPUMA1) and 17.56%±0.51%(siPUMA2).Conclusion PUMA is involved in the apoptosis of HLEC induced by H2O2,and may be play an important role in the development of cataract.