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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 effect of stable transfection of exogenous PTEN gene on the growth of human breast cancer cell line MDA4 6 8 in vitro. Methods The eukaryotic expression plasmid pcDNA3.1 - PTEN of PTEN gene was constructed and transfected into human breast cancer cells in vitro by liposome transfection method using recombinant plasmid pcDNA3.1 - PTEN and pcDNA3.1 (-) empty plasmid. Cell line MDA4 6 8, untransfected group as a control. The target gene and its protein expression were analyzed by RT PCR, immunohistochemistry and immunoblotting. Cell growth inhibition was analyzed by MTT assay. Apoptosis was detected by Annexin V FITC and PI double staining flow cytometry. Results The cell lines stably transfected with PTEN gene had the integration of exogenous gene and the high expression of corresponding mRNA and protein. MTT assay showed that the number of viable cells in pcDNA3.1 - PTEN transfected group was lower than that in untransfected cells and pcDNA3.1 - MDA468 cells. Flow cytometry showed that the apoptosis rate of pcDNA3.1 - PTEN transfection group was higher than that of non - transfection group and pcDNA3.1 - MDA468 cell transfection group. Conclusion Stable transfection of exogenous PTEN gene can inhibit the malignant phenotype of human breast cancer cells.