PTEN基因转染对卵巢癌A2780细胞周期和增殖能力影响的研究

Effect of transfected PTEN gene on cell cycle and proliferation of human ovarian cancer cell line A2780

目的:研究外源性PTEN基因稳定转染对人卵巢癌A2780细胞周期和增殖能力的影响。方法:构建PTEN基因的野生型真核表达质粒pEGFP-C1-WT-PTEN和突变型表达质粒pEG-FP-C1-C124A-PTEN,以脂质体介导法转染体外培养的人卵巢癌细胞株A2780,同时转染空载体pEGFP-C1质粒,以未转染细胞为对照(转染成功后分别命名为WT-PTEN/A2780组、C124A-PTEN/A2780组、pEGFP-C1/A2-780组和A2780组。应用RT-PCR、Western blot方法分析目的基因及其蛋白表达,并采用MTT法和流式细胞术检测细胞增殖和细胞周期。结果:与对照细胞相比,WT-PTEN/A2780组和C124A-PTEN/A2780组PTEN mRNA及PTEN蛋白出现明显的高表达,与对照组细胞相比差异有统计学意义(t=5.300,P=0.034;t=11.963,P=0.007;t=7.869,P=0.016;t=22.421,P=0.002);WT-PTEN/A2780组细胞生长速度明显慢于未转染A2780细胞,然而C124A-PTEN/A2780组和pEGFP-C1/A2780组细胞生长速度无明显变化。流式细胞术显示WT-PTEN/A2780细胞G1期细胞数增加到(71.18±4.34)%,S期细胞数变为(17.48±1.96)%,与对照组相比差异有统计学意义(t=19.508,P=0.003;t=25.354,P=0.002),提示从G1期到S期发生抑制。结论:野生型PTEN可依赖其磷酸酶活性使A2780细胞在G1/S期发生细胞周期阻滞,并抑制A2780细胞增殖。

OBJECTIVE： To investigate the effects of exogenous wild PTEN gene stably transfection on the cell cycle and proliferation in human ovarian cancer cell line A2780. METHODS： Wild-type PTEN and mutant PTEN recombinant eukaryotic expression plasmids were constructed, then they and the empty vector were transfected into A2780 cells by lipofectamine. The cell non transfected was considered as the control （four groups were named as WT-PTEN/A2780, C124A-PTEN/A2780, pEGFP-C1/A2780 and A2780 ）. RT-PCR and Western blot were used to determine the target gene and protein expression, the cell viability was tested by MTT assay, and the cell cycle was determined by flow cytometry. RESULTS.. Compared with the control, WT PTEN/A2780 group and C124A-PTEN/A2780 group demonstrated high expression of PTEN mRNA and protein （t=5.300,P=0.034; t= 11. 963 , P= 0. 007 ; t=7.869,P= 0.016; t：22.421,P：0.002）. The proliferation speed of WT-PTEN/A2780 was obviously slower than those of nontransfected cells. The proliferation speed did not change obriously in C124A PTEN/A2780 group and pEGFP-C1/A2780 group. Flow cytometry showed that in WT-PTEN/A2780 group, the cells of G1 phase was （71.18±4.34）%, the cells of S phase was （17.48±1.96）%, and were significantly dif- ferent from those of control group （t= 19. 508, P= 0. 003; t=25. 354, P=0. 002）. The progression of cell cycle was ar rested from G1 to S phases. CONCLUSION.. The growth of A2780 cells could be arrested at G1/S phase and be suppressed by phosphatase activity of exogenous PTEN gene.