【摘要】 目的构建shRNA-Survivin慢病毒载体,对肺腺癌A549细胞中Survivin基因表达进行干扰,探讨其对肿瘤细胞增殖、迁移和凋亡的影响。方法将A549细胞分成3组,即空白对照组,空白病毒载体对照组和shRNA干扰Survivin慢病毒载体组。采用慢病毒载体构建的方法,采用RT-qPCR和Western-Blot检测凋亡因子Caspase-3和Caspase-9的表达量,采用CCK-8试剂盒检测肺癌细胞的增殖,Transwell小室检测肺癌细胞的迁移,AV-PI凋亡试剂盒检测肺癌细胞的凋亡。结果 (1)慢病毒转染效率较高,以肺癌细胞A549为靶点种子细胞,慢病毒转染率为90%;(2)空白载体病毒组的Caspase-3的mRNA的相对表达量(与空白对照组相比)为(1. 231±0. 112,n=3),shRNA-Survivin组的Caspase-3的mRNA的相对表达量为(0.121±0. 009,n=3);两组比较具有统计学差异(t=2. 097,P=0. 0219 <0. 05),Caspase-9的mRNA表达具有相同的趋势;(3) Caspase-3的蛋白相对表达量为(0. 173±0. 231,n=3),shRNA-Survivin组与空白载体病毒组相比,具有统计学差异(t=3.221,P=0.0241<0.05);(4)Transwell实验结果,CCK-8检测结果和AV-PI检测结果显示,空白对照组、空白载体病毒组、shRNA-Survivin组三组之间比较具有明显的统计学差异(P<0.05)。结论 shRNA干扰Survivin可以有效的抑制肺癌细胞的迁移和增殖,并且提高其凋亡率。更多还原

【Abstract】 Objective The study on the molecular mechanism of shRNA-survivin gene on proliferation,migration and apoptosis of lung cancer cells provides a new way for the treatment of lung cancer cells. Methods Construction building,this research adopts the lentivirus vectors shRNA-Survivin slow virus vector,RT-PCR,Western Blot were used to detect the apoptosis factor Caspase-3 and the expression of Caspase-9,CCK 8 kit to detect lung cancer cell proliferation,Transwell Chambers detection of lung cancer cell migration,AV-PI kit apoptosis detection of lung cancer cell apoptosis. Results( 1) Lentiviral transfection efficiency was high. The target cells were lung cancer cell A549,and the transfection rate was 90%. This indicates that lentivirus transfected cells have high transfection efficiency.( 2) The relative expression of Caspase-3 mRNA in the blank vector virus group( compared with the blank control group) was( 1.231+0.112,n = 3); the relative expression of Caspase-3 mRNA in the shRNA-Survivin group( 0.121+ 0.009,n = 3); and the difference between the two groups was statistically significant( t = 2.097,P = 0.0219< 0.05). These results indicated that the expression of Caspase-3 in the virus group of shRNA interfering Survivin vectors was lower than that in the virus group of blank vectors. The mRNA of Caspase-9 has the same trend.( 3) The relative expression of Caspase-3 protein was( 0. 173. 231,n = 3); there was significant difference between shRNASurvivin group and blank vector virus group( t = 3. 221,P = 0. 0241 < 0. 05). These results indicated that the expression of Caspase-3 in the virus group of shRNA interfering Survivin vectors was lower than that in the virus group of blank vectors.( 4) Transwell test results,CCK-8 test results and AV-PI test results showed that there were significant statistical differences among the blank control group,blank vector virus group and shRNASurvivin group( P<0.05). Conclusion shRNA Survivin can effectively inhibit the migration and proliferation of lung cancer cells and increase its apoptosis rate.更多还原