【摘要】 目的:探讨自噬基因Beclin1通过影响血管生成素的表达对SKOV3/DDP细胞增殖抑制的可能机制。方法:脂质体包裹重组构建pcDNA3.1-Beclin1和pSUPER-Beclin1质粒后,分别体外转染SKOV3/DDP细胞,并筛选稳定表达株,通过实时荧光定量RT-PCR检测不同细胞株中Beclin1、Ang-1、Ang-2及Tie-2受体mRNA表达,蛋白质印迹法检测Beclin1、Ang-1、Ang-2及Tie-2蛋白水平变化,MTT法测定细胞生长增殖情况。结果:pcDNA3.1-Bec组Beclin1mRNA定量为31.39±3.16,明显高于未转染对照组的1.03±0.08,P<0.001;而pSUPER-Bec组为0.29±0.05,较对照组明显降低,P=0.003。pcDNA3.1-Bec组Ang-1mRNA定量为6.13±0.29,明显低于对照组的9.28±0.53,P=0.023。pSUPER-Bec组Ang-2mRNA定量为7.33±0.35,较对照组的11.36±0.59明显降低,P=0.021。pSUPER-Bec组Tie-2mRNA定量为7.96±0.38,与空白对照组的13.23±0.61相比亦明显降低,P=0.018。MTT法检测结果提示,pcDNA3.1-Bec组转染48h吸光度值为1.13±0.06,72h为1.21±0.05,96h为1.39±0.06;与未转染对照组的1.24±0.05、1.49±0.05及1.93±0.06比较,细胞增殖均明显减慢,F值分别为11.136、20.827和41.633,P值分别为0.029、0.011和0.005。pSUPER-Bec组转染48h为1.52±0.06,72h为1.88±0.06,96h为2.16±0.08,与对照组比较,细胞增殖均明显增快,F值分别为18.976、33.819和15.265,P值分别为0.012、0.007和0.016。结论:Beclin1过度表达能抑制SKOV3/DDP细胞增殖,其机制可能与Ang及其Tie-2受体的异常表达和平衡失调有关。更多还原

【Abstract】 OBJECTIVE:To investigate the effect of Beclin1 gene on inhibition proliferation of human ovarian cancer SKOV3/DDP cells by angiopoietin and its possible mechanism.METHODS:Beclin 1overexpression(pcDNA3.1-Beclin1)and knockdown(pSUPER-Beclin1)plasmids were independently transfected into SKOV3/DDP cells,and stably transfected cells were selected.Real-time fluorescence quantitative PCR and Western blot analyses were performed to detect the mRNA and protein expression of Beclin 1,Ang-1,Ang-2,and Tie-2.MTT assays were employed to determine cell proliferation rates.RESULTS:In the cells transfected with pcDNA3.1-Beclin1,the expression of Beclin1mRNA(31.39±3.16)was markedly increased as compared with those of the non-transfectant cells(1.03±0.08),but expression of Ang-1mRNA in pcDNA3.1-Beclin1(6.13±0.29)was dramatically reduced than that in non-transfectant cells(9.28±0.53).In the SKOV3/DDP cells transfected with pSUPER-Beclin1,the expression of Beclin 1mRNA(0.29±0.05),Ang-2mRNA(7.33±0.35),and Tie-2mRNA(7.96±0.38)were dramatically reduced than that in non-transfectant cells(1.03±0.08,11.36±0.59,13.23±0.61),all P<0.05.After 48 h,72hand 96 htransfected with different plasmids,MTT assays revealed that the proliferation of pcDNA3.1-Beclin1(1.13±0.06,1.21±0.05,1.39±0.06)were significantly suppressed as compared with those of the non-transfectant cells(1.24±0.05,1.49±0.05,1.93±0.06);while those of pSUPER-Beclinl were 1.52±0.06,1.88±0.06 and 2.16±0.08,which were suppressed compared with control group,all P<0.05.CONCLUSION:Overexpression of Beclin 1can inhibit the proliferation of SKOV3/DDP cells,which may be attributed to an imbalance among the expression of Ang-1,Ang-2,and Tie-2.更多还原