中文摘要：

目的构建针对PeroxiredoxinⅠ（PrxⅠ）基因的短发夹RNA（short hairpin RNA,shRNA）真核表达载体,观察下调PrxⅠ基因表达后对乳腺癌MCF-7细胞生物学功能的影响。方法构建针对PrxⅠ基因的shRNA真核表达载体,经酶切和测序鉴定正确后,转染入乳腺癌MCF-7细胞。流式细胞术检测细胞转染效率,RT-PCR及Western blot分别检测干扰前后PrxⅠmRNA和蛋白表达,MTT法及流式细胞术检测干扰前后乳腺癌MCF-7细胞体外的增殖情况及细胞周期和细胞凋亡。结果成功构建真核表达载体pGPU6-HK、pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pGPU6-Prx4,并转染入乳腺癌MCF-7细胞中,转染率为80%左右,RT-PCR及Westernblot分析表明转染pGPU6-Prx1、pGPU6-Prx2、pGPU6-Prx3、pG-PU6-Prx4后的细胞EGFR mRNA和蛋白表达均不同程度地受抑,以pGPU6-Prx3为甚,其mRNA和蛋白表达抑制率分别为82.6%和80.5%。与未转染组和pGPU6-HK组相比,pGPU6-Prx3组的细胞生长明显延迟（P〈0.05）,细胞凋亡率显著增加,G1期细胞明显增多,而S期细胞减少（P〈0.05）。结论靶向PrxⅠ基因的shRNA真核表达载体构建成功,能够特异性下调乳腺癌MCF-7细胞的PrxⅠ基因的表达,并明显抑制乳腺癌细胞增殖,促进细胞凋亡,调控细胞周期再分布。

英文摘要：

Objective To construct short hairpin RNA（shRNA） eukaryotic expression vectors targeting peroxiredoxinⅠ（PrxⅠ） gene and investigate the effect of down-regulating PrxⅠ expression on the biological functions of breast carcinoma MCF-7 cells.Methods The eukaryotic expression vectors targeting PrxⅠ gene were constructed and then transfected into MCF-7 cells.Transfection efficiency was evaluated by flow cytometry.The mRNA and protein expressions of PrxⅠ in MCF-7 cells were detected by RT-PCR and Western blotting.The proliferation of MCF-7 cells was determined by MTT assay.The cell cycle and apoptosis were evaluated by flow cytometry.Results The eukaryotic expression vectors pGPU6-HK（negative control）,pGPU6-Prx1,pGPU6-Prx2,pGPU6-Prx3 and pGPU6-Prx4 were successfully constructed and then transfected into MCF-7 cells respectively.Transfection efficiency was about 80%.The expression of PrxⅠ in MCF-7 cells transfected with 4 Prx vectors were inhibited significantly at both mRNA and protein levels,especially in the cells with pGPU6-Prx3,whose inhibitory rates were 82.6% and 80.5% respectively at mRNA and protein levels.When pGPU6-Prx3 group was compared with untransfected group and the group transfected with pGPU6-HK,the cell proliferation was markedly delayed（P0.05）,the cell apoptosis rate was significantly increased,and the cells were arrested in G1 phase and S phase were significantly decreased（P0.05）.Conclusion Eukaryotic expression vectors of shRNA PrxⅠis successfully constructed,and specifically down-regulate the PrxⅠ expression at mRNA and protein levels.Transfection of pGPU6-Prx3 vector significantly inhibits cell proliferation,induces cell apoptosis,and regulates cell phase redistribution in MCF-7 cells.