慢病毒介导的FoxA1高表达对乳腺癌MCF-7细胞株增殖凋亡探究  

Overexpression of Fox A1 by an adenoviral vector and its effects on cell cycle and apoptosis in MCF-7 human breast cancer cells

作　　者：郑璐[1] 汤铜[1] 钱波[1] 周连帮[1] 万圣云[1] 张磊[1] 史加宁[1] 李佳[1] 

机构地区：[1]安徽医科大学第二附属医院普外科,合肥230601

出　　处：《安徽医科大学学报》2016年第6期822-827,共6页Acta Universitatis Medicinalis Anhui

基　　金：安徽省自然科学基金(编号:1308085QH152)

摘　　要：目的利用慢病毒载体,构建乳腺癌MCF-7高表达Fox A1细胞株,并初步探究Fox A1对乳腺癌MCF-7细胞株增殖凋亡的影响。方法将Fox A1基因重组构建慢病毒载体穿梭质粒EX-Z1010-LV201,经PCR和测序后在脂质体Lipofectamine2000介导下转染293T细胞包装慢病毒,嘌呤霉素筛选出稳定转染的细胞,Real-Time PCR法检测稳定转染MCF-7细胞株中Fox A1基因的表达水平,MTT及流式细胞术对比高表达Fox A1细胞株与对照细胞株增殖凋亡差异。结果 PCR扩增和测序结果证实成功构建Fox A1慢病毒载体并包装慢病毒,成功筛选出Fox A1高表达的乳腺癌MCF-7细胞系。MTT及流式细胞术结果显示:与对照组比较,上调Fox A1乳腺癌MCF-7细胞株增殖下降,凋亡率增加。结论成功构建了重组慢病毒载体,并筛选出稳定高表达Fox A1的MCF-7细胞株,Fox A1可能参与了乳腺癌MCF-7细胞株增殖凋亡调控,为下一步进行其机制探讨提供了依据。Objective To investigate the effects of Fox A1 overexpression on proliferation and apoptosis of humanbreast cancer cell line MCF-7 by constructing high-level expression vetor Fox A1 for MCF-7 cell line. Methods Fox A1 gene was amplified by PCR to construct the recombinant shuttle plasmid EX-Z1010-LV201. After DNA sequence analysis,the recombinant lentiviral package plasmid was transfected into 293 T cells by Lipofectamine 2000 to construct packed lentivirus. Stable transfected cell lines were selected out by puromycin. The Fox A1 protein expression level of stable MCF-7 cell line was detected by real-time PCR method. Differences of cell proliferation and cell apoptosis between Fox A1 overexpressed cell line and empty vector cell lines were determined by MTT assay and FCM. Results The recombinant Fox A1 lentiviral vectors and packed lentivirus were confirmed by PCR and DNA sequencing,and MCF-7 cell lines transfected by packed lentivirus which stably overexpress Fox A1 were successfully selected. MTT and FCM results presented a significant decrease of cell proliferation in human breast cancer MCF-7 cell lines with up-regulated Fox A1. Conversely,more obvious apoptosis was presented in MCF-7 cell lines with up-regulated Fox A1 than the control group. Conclusion Fox A1 is successfully selected and MCF-7 cell lines are transfected by packed lentivirus which are stably overexpressed. Fox A1 is involved in some important intracellular process of breast cancer cell line MCF-7,such as cell proliferation and apoptosis. This research provides a valid evidence for further investigation on its mechanism.

关 键 词：FOX A1 慢病毒载体 MCF-7 增殖凋亡 

分 类 号：R346[医药卫生—基础医学]