抑癌基因PTEN真核表达质粒的构建及对乳腺癌MDA468细胞的影响  

Construction of PTEN eukaryotic expression plasmid and its effects on breast carcinoma cell line MDA468

作　　者：陈庆永[1] 陈道达[2] 王春友[2] 周友生[1] 

机构地区：[1]华中科技大学同济医学院附属协和医院急诊外科,武汉430022 [2]华中科技大学同济医学院附属协和医院普外科,武汉430022

出　　处：《中华肿瘤杂志》2005年第4期216-219,共4页Chinese Journal of Oncology

摘　　要：目的　研究外源性PTEN基因稳定转染对人乳腺癌MDA4 6 8细胞体外生长的影响。方法　先构建PTEN基因的真核表达质粒pcDNA3.1 - PTEN ,应用重组质粒pcDNA3.1 - PTEN和pcDNA3.1(- )空载体质粒,以脂质体转染法转染体外培养的人乳腺癌细胞株MDA4 6 8,以未转染组为对照。应用RT PCR、免疫组化和免疫印迹方法分析目的基因及其蛋白表达,MTT法分析细胞生长抑制作用,AnnexinV FITC和PI双染流式细胞术检测细胞凋亡。结果　稳定转染PTEN基因的细胞株有外源目的基因的整合和相应mRNA及其蛋白的高表达。MTT检测表明,pcDNA3.1 - PTEN转染组活细胞数低于未转染组和pcDNA3.1 - MDA4 6 8细胞转染组。流式细胞术显示,pcDNA3.1 - PTEN转染组凋亡率高于未转染组和pcDNA3.1 -MDA4 6 8细胞转染组。结论　外源性PTEN基因稳定转染可抑制人乳腺癌细胞的恶性表型。Objective To investigate the effects of exogenous wild PTEN gene stably transfection on growth of breast cancer cells in vitro. Methods At first, a recombinant eukaryotic expression plasmid pcDNA3.1-PTEN was constructed. Human breast cancer cell line MDA468 was transfected with pcDNA3.1-PTEN or mock transfected plasmid pcDNA3.1(-) with lipofectamine. RT-PCR, immunohistochemical staining and Western blot were used to determine target gene expression. Cell viability was tested by MTT assay. Apoptosis was determined by flow cytometry with a double-staining method using FITC-conjugated annexin V and PI. Results The PTEN stably transfected cells demonstrated the integration of the exogenous target gene and corresponding mRNA and protein over-expression. There was a significant decline in cell viability of pcDNA3.1-PTEN transfected MDA468 cells in comparison with the mock-transfected ones(P<0.01). The PTEN-trasfected MDA468 cells also showed an increase in the rate of apoptosis, compared with parental and mock-trasfected cells (P<0.001). ConclusionStable expression of exogenous PTEN can suppress the malignant phenotypes of the human breast cancer cell line MDA468.

关 键 词：真核表达质粒 抑癌基因PTEN 外源性PTEN基因 pcDNA3 人乳腺癌细胞株 Annexin 流式细胞术检测 细胞体外生长 稳定转染 脂质体转染法 RT-PCR 生长抑制作用 目的基因 细胞转染 MTT检测 载体质粒 重组质粒 体外培养 蛋白表达 

分 类 号：R737.9[医药卫生—肿瘤]