【摘要】 目的:应用RNA干扰(RNAi)技术特异地干扰HAX-1在乳腺癌细胞MCF-7中的表达,研究其在过氧化氢诱导的细胞凋亡中的作用。方法:应用载体pSR-GFP/Neo构建针对HAX-1基因的小干扰RNA(siRNA)表达质粒;转染MCF-7细胞,G418筛选稳定细胞系,Westernblot鉴定筛选的细胞克隆;用流式细胞仪检测筛选的细胞系在过氧化氢条件下的凋亡率。结果:电泳和测序证实合成的siRNA序列正确并准确克隆到pSR-GFP/Neo载体中;Westernblot证实获得MCF-7/HAX-1siRNA稳定细胞株,其HAX-1蛋白水平降低95%左右;用1mmol/L过氧化氢处理获得的稳定细胞株8h,细胞凋亡率明显高于对照组。结论:HAX-1能够保护乳腺癌细胞MCF-7免于过氧化氢诱导的细胞凋亡。更多还原

【Abstract】 Objective:To silence the expression of HS1-associated protein X-1(HAX-1) gene in MCF-7 human breast cancer cells using RNA interference technique and to investigate the effect of the HAX-1 on cell apoptosis induced by hydrogen peroxide(H2O2).Methods:The HAX-1 small interfering RNA(siRNA) fragment was cloned into plasmid pSR-GFP /Neo,and the resulting plasmid was transfected into MCF-7 cells.The cell plaques which were selected out with G418 were subjected to Western blot to identify HAX-1 protein level.The selected cell strains and wild type MCF-7 were treated for 8 h with H2O2,and the apoptosis rate were measured by FACS.Results:Electrophoresis and DNA sequencing confirmed that the correct HAX-1 specific siRNA sequence was cloned into the expression vector pSR-GFP /Neo.The selected MCF-7 /HAX-1 siRNA cell strain under G418 showed the HAX-1 protein level was knocked down 95%,and their apoptosis rate increased significantly under 1 mmol /L H2O2 treatment.Conclusion:HAX-1 can protect the MCF-7 cells form apoptosis induced by H2O2.更多还原