【摘要】 目的:探讨构建带有肿瘤特异性Survivin启动子的PUMA基因表达载体pUC57-Survivin-PUMA对乳腺癌MCF-7细胞的肿瘤特异性促凋亡作用。方法:化学合成PUMA基因片段克隆至pUC57质粒,构建重组质粒pUC57-PUMA;克隆Survivin启动子序列,取代pUC57-PUMA质粒原有的CMV启动子,构建重组质粒pUC57-Survivin-PUMA,测序鉴定。实验设置pUC57-PUMA组、pUC57-Survivin-PUMA组、阴性对照组(pUC57group)和空白对照组(blank group)。将以上各组分别转染人乳腺癌MCF-7细胞及人正常乳腺Hs 578Bst细胞。转染后48h,western blot技术检测各组细胞中PUMA蛋白表达情况,流式细胞仪检测细胞的凋亡率。结果:重组质粒经测序鉴定证实符合设计要求,构建成功;MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的PUMA蛋白表达均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中只有pUC57-PUMA组的PUMA蛋白表达较其他3组显著增高(P<0.05)。MCF-7细胞中pUC57-PUMA组和pUC57-Survivin-PUMA组的细胞凋亡率分别为29.02%和29.31%,均较对照组显著增高(P<0.05),而人正常乳腺Hs 578Bst细胞中仅pUC57-PUMA组的细胞凋亡率较其他3组显著增高(P<0.05)。结论:Survivin启动子调控的PUMA表达载体能显著增高乳腺癌MCF-7细胞中PUMA的表达,进而促进乳腺癌MCF-7细胞凋亡,而对正常乳腺Hs 578Bst细胞的凋亡不产生影响。更多还原

【Abstract】 Objective:To construct PUMA gene-targeted expression vector driven by tumor specific Survivin promoter(pUC57-Survivin-PUMA) and investigate its cell apoptosis effects in MCF-7 breast cancer cells.Methods:The PUMA gene sequence by chemical synthesis was cloned in the plasmid pUC57 to construct the recombinant plasmid pUC57-PUMA,the CMV promoter in which then were replaced respectively by the Survivin promoter,and the plasmid pUC57-Survivin-PUMA was constructed finally.Four experimental groups were set up,they were pUC57-PUMA group,pUC57-Survivin-PUMA group,pUC57 control group and blank control group.And then,the plasmids were transfected into two types of cells(human breast cancer MCF-7 cells,human normal breast Hs 578Bst cells),western blot was performed to evaluate the expression of PUMA gene,and the cell apoptosis rate was observed by means of flow cytometry(FCM) in MCF-7 cells and Hs 578Bst cells respectively.Results:Sequencing confirmed the recombinant plasmid was constructed successfully.The results of western blot showed that the expression of PUMA protein had been significantly increased in pUC57-PUMA and pUC57-Survivin-PUMA groups in MCF-7 cells(P<0.05),but the expression of PUMA protein had been significantly increased only in pUC57-PUMA group in Hs 578Bst cells(P<0.05).The results of FCM showed that the cell apoptosis rate was 29.02% and 29.31% in pUC57-PUMA and pUC57-Survivin-PUMA groups in MCF-7 cells respectively,there were significant differences compared with pUC57 control group and blank control group;but the cell apoptosis rate had been significantly increased only in pUC57-PUMA group in Hs 578Bst cells(P<0.05).Conclusion:PUMA gene-targeted expression vector driven by tumor specific Survivin promoter(pUC57-Survivin-PUMA) can make the expression of PUMA protein increase and promote cell apoptosis in human breast cancer MCF-7 cells,but cell apoptosis has no influence on human normal breast Hs 578Bst cells.更多还原