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目的构建survivin-siRNA重组腺病毒质粒,探讨不同浓度和时间点的survivin-siRNA重组腺病毒质粒pBAsi-survivin对结直肠癌SW480细胞的影响。方法将制备好的survivin-siRNA重组腺病毒载体pBAsi-survivin,按浓度梯度分别为50、100、150、200、250、300 nmol/L 6个浓度梯度分别转染SW480,并检测最适浓度下转染12、24、36、48、60、72 h 6个时间梯度,用RT-PCR检测各组细胞中survivin mRNA的表达水平,用Western印迹检测各组细胞中survivin蛋白的表达水平,用MTT法检测细胞的生长情况。结果结直肠癌SW480细胞对survivin-siRNA重组腺病毒pBAsi-survivin有浓度依赖性,浓度在100 nmol/L时survivin mRNA表达抑制率39.01%,蛋白表达抑制率为47.83%,细胞生长抑制率为(15.1±1.0)%;在转染48 h后转染效率最高,survivin mRNA和蛋白表达抑制率分别为67.19%、68.24%,细胞生长抑制率为(37.0±2.7)%。结论结直肠癌SW480细胞对survivin-siRNA重组腺病毒pBAsi-survivin有浓度和时间依赖性,pBAsi-survivin浓度100 nmol/L是体外抑制结直肠癌SW480细胞的最适宜浓度,转染48 h效率最高。
Objective To construct the survivin-siRNA recombinant adenovirus plasmid and investigate the effect of survivin-siRNA recombinant plasmid pBAsi-survivin on colorectal cancer SW480 cells at different concentrations and time points. Methods The survivin-siRNA recombinant adenovirus vector pBAsi-survivin was transfected into SW480 cells at concentration gradients of 50, 100, 150, 200, 250 and 300 nmol / L respectively, and the optimal concentration Transfection of 12,24,36,48,60,72 h 6 time gradient, the expression of survivin mRNA in each group was detected by RT-PCR, and the expression of survivin protein in each group was detected by Western blotting. MTT Method to test cell growth. Results The colorectal cancer SW480 cells had a concentration-dependent effect on survivin-siRNA recombinant adenovirus pBAsi-survivin. The inhibitory rate of survivin mRNA was 39.01% and the protein expression was 47.83% at a concentration of 100 nmol / L. The cell growth inhibition rate was ( 15.1 ± 1.0)%. The highest transfection efficiency was achieved 48 h after transfection. The inhibitory rates of survivin mRNA and protein expression were 67.19% and 68.24%, respectively. The cell growth inhibition rate was (37.0 ± 2.7)%. CONCLUSIONS: The colorectal cancer SW480 cells have a concentration-dependent and time-dependent effect on the survivin-siRNA recombinant adenovirus pBAsi-survivin. PBAsi-survivin concentration of 100 nmol / L is the most suitable concentration for inhibiting SW480 cells in vitro. .