摘要
目的:利用核糖核酸干扰(RNA interference,RNAi)技术沉默抗凋亡基因survivin,观察其对人肺腺癌细胞A549增殖以及顺铂药物敏感性的影响。方法:将靶向survivin的基因片段插入带有绿色荧光蛋白基因的载体后构建重组质粒,将其导入A549细胞,RT-PCR分析转染前后survivin mRNA的表达情况,免疫荧光及Western blot分析转染前后survivin蛋白的表达情况,TUNEL法检测细胞凋亡情况,MTT法检测转染后A549细胞对顺铂的敏感性变化。结果:成功构建pGenesil1。1-survivin重组质粒。与对照组相比,转染重组质粒后,survivin mRNA和蛋白的表达明显降低,抑制率分别为65%和71%;细胞凋亡率增加;转染前顺铂对A549细胞的IC50为转染后的7.68倍,两者比较,差异有统计学意义(P<0.05)。结论:靶向survivin的RNA干扰表达载体能下调survivin基因表达,增强A549细胞对顺铂药物敏感性。
Objective：Inhibit expression of survivin with RNA intrference technique, to observe its effect on proliferation of A549 cells and sensitivity of A549 cells to cisplatin. Methods：The DNA sequence targeting human survivin was inserted into the plasmid which contain green fluorescent protein gene, and the recombinant plasmid were constructed. The recombinant plas- mids were transfected into A549 cells. The levels of survivin mRNA and survivin protein were detected by RT-PCR and immu- nofluorescence staining and Western blot before and after transfection. Ceils apoptosis were detected by TUNEL method. The sensitivity of A549 cells to cisplatin was detected by MTT after transfection. Results： The recombinant plasmid pGenesill. 1- survivin were successfully constructed. The A549 cells transfected with recombinant plasmid showed a lower mRNA and protein expression of survivincompared with control group, the inhibition rate were 65% and 71%,respectively. The apoptosis rate of A549 cells was increased after transfection. The IC50 of cisplatin to A549 cells before transfection was 7.68 fold compared with those after transfection. There was significant difference between the two groups（P〈0.05）. Conclusion：The RNA inter- ference expression vectors targeting human survivin could down-regulate the expression of survivin, enhance the sensitivity of A549 cells to cisplatin.
出处
《中国临床医学》
2009年第5期665-668,共4页
Chinese Journal of Clinical Medicine
基金
山东省自然科学基金青年基金(NoQ2006D08)
