VP3的细胞定位及其诱导乳腺癌细胞凋亡效应研究

摘要

目的:研究鸡传染性贫血病毒VP3基因在正常细胞和乳腺癌细胞中不同时间段的定位及其诱导细胞凋亡效应的变化.方法:用PCR方法扩增鸡贫血病毒的VP3基因,克隆至绿色荧光载体pEGFP-C1,构建含VP3的基因重组体,利用FuGENE(R)6 Reagent将pEGFP-C1-VP3分别转染乳腺癌MCF-7和成纤维细胞L929,观察VP3转染24、48、72 h后的细胞定位,并用流式细胞术检测MCF-7不同时间段的细胞凋亡率.结果:VP3融合蛋白定位于乳腺癌细胞MCF-7核内,并呈现细胞凋亡不同阶段的典型核改变,VP3融合蛋白在L929细胞中经历了由核内迁移到细胞质的变化过程,不引起L929细胞凋亡.结论:VP3在乳腺癌细胞MCF-7中定位于细胞核,并能以诱导凋亡的方式引起癌细胞的死亡,并且凋亡率高于对照组且具有时间依赖性;VP3在正常细胞中定位于细胞质,且不引起凋亡效应.%Objective:To observe the localization of chicken infectious anemia virus VP3 gene in normal cells and breast cancer cells in different times and its apoptosis-inducing effect.Methods: The fundamental cloning method,inserting the VP3 gene of chicken anemia virus into the eukaryotic expression vector pEGFP-C1 was used.Then,the positive recombinant containing VP3 gene pEGFP-C1-VP3 was transfected into human breast cancer cell line MCF-7 and mouse fibroblasts L929 by FuGENE(R)6 transfection reagent in vitro respectively.After 24 hours,48 hours and 72 hours,fluorescence microscope was used to observe the distribution of VP3 in cells and the rate of apoptosis was studied on the treated MCF-7 cells by FCM(Flow Cytometry).Results: The recombinant plasmid pEGFP-C1-VP3 could be localized in the nuclei of breast cancer cells,which showed the typical nuclear changes in different stages of apoptosis.In L929 cells,pEGFP-C1-VP3 underwent a process of migration from the nucleus to the cytoplasm,which didn′t induce apoptosis of L929 cells.Conclusion: VP3 located in the nucleus of MCF-7 breast cancer cells,which can led to cancer cell death by inducing apoptosis,and the apoptosis rate was higher than the control group with time dependence.VP3 located in the cytoplasm of normal cells,and didn′t induce apoptosis.