体外瞬时转染野生型PTEN过表达对乳腺癌MCF-7细胞的影响

Effects of overexpression of transiently transfected wild PTEN on breast cancer MCF-7 cells

目的:研究野生型PTEN基因过表达对乳腺癌MCF-7细胞的生长抑制作用。方法:应用携带野生型PTEN的真核表达载体pEGFP-C1-PTEN,以脂质体介导的方法体外瞬时转染乳腺癌MCF-7细胞, 利用流式细胞术检测抗凋亡蛋白bcl-2的表达,通过相差显微镜、细胞生长曲线、MTT、DNA琼脂糖凝胶电泳等方法,观察PTEN基因过表达对MCF-7细胞生物学特性的影响。结果:荧光显微镜观察、免疫细胞化学和RT-PCR检测证实,转染后MCF-7细胞中PTEN呈高水平表达;相差显微镜观察到MCF-7/PTEN 细胞出现典型的凋亡形态学改变;细胞生长曲线低平;流式细胞检测显示,G1期细胞比例比空载体组增加14.79%,并出现凋亡峰;DNA琼脂糖凝胶电泳可见梯状条带;同时还检测到转染PTEN后细胞bcl-2 表达下降。结论:外源性PTEN基因过表达可显著抑制MCF-7细胞的周期进程并诱导细胞凋亡。

Objective: To investigate the effects of wild PTEN overexpression on human breast cancer MCF-7 cells. Methods: The eukaryotic expression plasmid pEGFP-C1-PTEN containing wild-type PTEN was transfected into MCF-7 cells by Lipofectamine 2000. The growth suppression was observed through phase-microscope, growth curve, MTT, flow cytometry and DNA agarose gel electrophoresis. The expression of anti-apoptosis protein bcl-2 was also evaluated by flow cytometry. Results: High levels of PTEN protein and mRNA expression in MCF-7/PTEN cells were confirmed through fluorescence microscope, immunocytochemistry and RT-PCR analysis. Typical apoptosis cells were observed under phase-microscope. The cell growth curve of MCF-7/PTEN was significantly lower than that of MCF-7/pEGFP. Flow cytometry showed increase of G1 cell population(14.79%) and apoptosis cell ratio(11.03%). DNA ladder was observed through DNA agarose gel electrophoresis and a decreased level of bcl-2 protein was observed through flow cytometry. Conclusion: These findings demonstrate that PTEN exerts its tumor-suppressive effect on MCF-7 cells through blocking cell cycle progression and inducing cell apoptosis.