外源性PTEN基因转染对炎性乳腺癌MDA-MB-468细胞株体外生物学行为的影响

Effects of Exogenous PTEN Gene Transfection on the Biological Behaviors of MDA-MB-468 Inflammatory Breast Cancer Cell Line

目的研究外源性PTEN(phosphatase and tensin homolog deleted on chromosome 10)基因表达对炎性乳腺癌MDA-MB-468细胞株体外生物学行为的影响。方法应用基因重组技术构建并鉴定pcDNA3.1-PTEN真核表达质粒,重组质粒被BglⅡ酶切线性化后运用脂质体介导基因转染法,将外源性PTEN基因导入乳腺癌细胞系MDA-MB-468中,经G418筛选阳性克隆,空载体pcDNA3.1(-)转染入细胞作为对照,逆转录-聚合酶链反应(RT-PCR)、免疫印迹法(Western blot)分析目的基因及蛋白表达,膜联蛋白Ⅴ-FITC(FITC-AnnexinⅤ)和碘化丙啶(PI)双染流式细胞术检测细胞凋亡。结果重组真核表达质粒pcDNA3.1-PTEN经限制性内切酶XbaⅠ和BamHⅠ双酶切,电泳后显示约1.4kb的PTEN目的片段和5.4 kb的pcDNA3.1(-)载体片段,证明重组质粒连接正确。RT-PCR、Western blot均显示pcDNA3.1-PTEN转染组有PTEN mRNA及其蛋白表达,流式细胞分析显示恢复PTEN蛋白表达后其凋亡率较其它两组细胞凋亡率增高(均P<0.01)。结论成功地构建了pcDNA3.1-PTEN真核表达质粒,外源性PTEN基因可通过诱导细胞凋亡而抑制乳腺癌细胞的增殖。

Objective To study in vitro effects of exogenous PTEN gene expression on the biological behavior of a human inflammatory breast cancer cell line MDA-MB-468. Methods The recombinant eukaryotic expression plasmid pcDNA3. 1- PTEN was constructed by using recombinant DNA technique, then linearized by BRl Ⅱ restriction enzyme and transfected into an inflammatory breast cancer cell line MDA-MB-468 by using lipofectamine 2000. Positive clones were selected by G418 and expanded in DMEM culture medium. Cells were also stably transfected with the vector alone, selected with G418 and used as a control. The expression of target genes was detected by RT-PCR and Western blotting. The apoptosis of the MDA-MB-468 cells was determined by flow cytometry with the double-staining method using FITC-conjugated Annexin V and propidium iodide. Results The recombinant eukaryotic expression plasmid for PTEN was digested with Xba Ⅰ and BamH Ⅰ , and the electrophoresis of the digested products showed two fragments of 1.4 kb and 5.4 kb. In the transfected MDA-MB-468 cells, the expression of PTEN mRNA and protein was detected by RT-PCR and Western blotting. Flow cytometric analyses indicated that the expression of PTEN protein caused an increase in the rate of apoptosis in the pcDNA3.1-PTEN transfection group as compared with other groups （P〈0.01）. Conclusion The recombinant eukaryotic expression plasmid pcDNA3. 1-PTEN was constructed correctly. Exogenous PTEN gene could inhibit the proliferation of breast cancer cells by inducing cell apoptosis.