X射线对转染Smac基因重组质粒的MCF-7细胞表达及增殖和凋亡的影响

Effect of X-rays on expression,proliferation and apoptosis in MCF-7 cells transfected with Smac gene recombination plasmid

目的探讨Egr-1启动子介导Smac基因的重组质粒pshuttle-Egr1-hSmac的辐射诱导特性,以及联合X射线照射后对MCF-7细胞增殖和凋亡的影响。方法将构建完全正确的pshuttle-Egr-1-hSmac质粒转染到人乳腺癌MCF-7细胞中,24h后给予X射线照射,24h后提取总RNA和蛋白,分别采用RT-PCR和Western印迹法检测细胞中SmacmRNA和蛋白的表达;分别采用MTT法和流式细胞术检测细胞增殖和凋亡的变化。结果未照射组及转染和空载体pshuttle组未见SmacmRNA和蛋白表达;而转染pshuttle-Egr-1-hSmac的MCF-7细胞在0.5Gy照射后未见其mRNA和蛋白表达;1、2和5Gy照射后明显增高,尤其以5Gy表达最高;2Gy照射后4h未见表达,而8～48h则逐渐增加,尤其以48h最高。MTT结果显示,转染了pshutlle质粒后存活率稍有降低,而凋亡未见明显变化;转染pshuttle-Egr-1-hSmac质粒后细胞存活率未见明显降低,而凋亡百分率稍有增加(P<0.05);经过2Gy照射后各组存活率降低,凋亡百分数明显增加(P<0.05,P<0.01),尤其以转染pshuttle-Egr-1-hSmac质粒更明显。结论X射线照射联合pshuttle-Egr1-hSmac能通过提高细胞内Smac的表达,并有效抑制MCF-7细胞增殖,促进细胞凋亡,提示Smac基因联合放射治疗可能成为肿瘤治疗的新策略。

Objective To study the radiation-induced properties of the recombination plasmid pshuttle-Egr1-hSmac of Smac gene mediated by Egr-1 promoter and its effect in combination with X-ray irradiation on the proliferation and apoptosis in MCF-7 cells.Methods The human breast cancer cells MCF-7 was irradiated with X-ray 24 h after transfecting the pshuttle-Egr-1-hSmac into the cells.Then RNA and protein from the cells were extracted 24 h after irradiation.The expressions of Smac mRNA and protein were detected by RT-PCR and Western blot respectively.The changes of proliferation and apoptosis in the cells were detected by MTT and flow cytometry respectively.Results There were no Smac mRNA and protein expressions in the control group and pshuttle group after X-ray irradiation.Also there were no Smac mRNA and protein expressions in the cells which were transfected with pshuttle-Egr-1-hSmac after irradiation with 0.5 Gy,but the Smac mRNA and protein expressions were increased significantly after irradiation with 1,2 and 5 Gy,especially with 5 Gy.There were no the expressions 4 h after irradiation with 2 Gy and the expressions increased gradually at 8 ~ 48 h,especially at 48 h.The survival rate of MCF-7 cells transfected with pshuttle was decreased slightly,while the percentage of their apoptosis did not change significantly.Meanwhile the survival rate of the cells transfected with pshuttle-Egr-1-hSmac did not decrease significantly and the percentage of their apoptosis was increased significantly（P0.05）.The survival rates of the cells in each group after X-ray irradiation with 2 Gy were lower and the percentage of their apoptosis increased（P0.05,P0.01）and especially significantly in the pshuttle-Egr-1-hSmac plasmid group.Conclusions The X-ray irradiation combined with pshuttle-Egr1-hSmac could increase the expression of Smac,inhabit the proliferation and accelerate the apoptosis of MCF-7 cells,suggesting that therapeutic method of X-ray irradiation combined with pshuttle-Egr1-hSmac may become a new strategy for cancer therapy.