摘要
目的:探讨血管壁组织因子小干扰核糖核酸(siRNA)对大鼠血管平滑肌细胞组织因子基因沉默作用的可行性和有效性。方法:设计并合成针对大鼠组织因子的25 bp双链siRNA分子。使用组织因子siRNA、对照siRNA转染大鼠血管平滑肌细胞,同时设立空白对照,实验共分6组(各组n=5)。荧光显微镜观察转染效率。血小板源性生长因子-BB刺激血管平滑肌细胞后逆转录多聚酶链反应检测组织因子信使核糖核酸表达,免疫印迹和免疫组化检测组织因子蛋白表达。结果:siRNA成功转染到血管平滑肌细胞,转染效率约(90.0±2.3)%。3对候选siRNA筛选出基因抑制效率最高一对,与阴性对照组相比,转染24 h组织因子信使核糖核酸表达减少(86.0±0.6)%,转染48 h蛋白质印迹分析组织因子蛋白表达减少(92.0±1.0)%,同时免疫组化检测组织因子表达明显减弱。结论:RNA干扰能明显下调大鼠血管平滑肌细胞组织因子表达。
Objective：To investigate the feasibility and efficacy of tissue factor（TF） gene silencing in rats＇ vascular smooth muscle cells （VSMCs） by using chemically synthesized small interfering RNA（ siRNA）.
Methods ： Double-stranded 25 bp-RNA molecules targeted at sequences within the rats＇ TF gene were designed and construc- ted. Primary rats＇ VSMCs were cultured with either TF siRNA or control siRNA. Transfection efficacy was determined by fluores- cence microscopy,the mRNA expression of TF was analyzed by RT-PCR,and the protein expression was examined by Western blot and immunohistochemistry after stimulation with platelet-derived growth factor（PDGF）-BB.
Result ：Successful siRNA transfection of VSMCs was confirmed by fluorescence microscopy, and the transfection efficiency was about 90 ± 2. 3%. Cells transfected with the highest effective siRNA from one of three candidate sequences showed a reduction of 86 ± 0. 6% in TF mRNA expression at 24h after transfection, and a reduction of 92 ±1.0% in TF protein expression at 48h after transfection compared with untransfected ceils, at the same time ,TF positive staining was markedly decreased after transfection.
Conclusion：TF expression could be effectively silenced by RNA interfering in VSMCs from rat aorta.
出处
《中国循环杂志》
CSCD
北大核心
2009年第4期304-307,共4页
Chinese Circulation Journal
基金
湖北省卫生厅科研基金(JX2B16)
