目的：研究沉默血管内皮生长因子激酶功能区受体（ kinase-domain insert containing receptor ， KDR）基因对人肺癌A549细胞增殖以及对化疗药物多西他赛敏感性的影响。方法设计合成KDR的小干扰RNA（ small interfering RNA ，siRNA）序列，Lipofectamine TM 2000转染入A549细胞。通过反转录-聚合酶链反应和Western Blot检测KDR基因沉默后KDR mRNA及蛋白的表达情况，利用流式细胞仪检测A549细胞的周期变化。采用四甲基偶氮唑盐比色法及细胞克隆形成实验观察，沉默KDR基因后A549细胞对多西他赛的敏感性。结果 KDR基因经48 h沉默后，A549细胞的KDR基因和蛋白的表达出现较明显的下降（ P＜0.05）。A549细胞的周期在G0／G1期阻滞，S期细胞数目减低（P＜0.05）。在KDR基因沉默组，A549细胞对多西他赛的敏感性有明显的增强（P＜0.05）。结论 KDR-siRNA能够明显沉默A549细胞KDR基因和蛋白的表达，并能抑制A549细胞的增殖，增强其对多西他赛的敏感性。%Objective This study investigated the effect of small interfering RNA-mediated kinase-domain insert containing receptor ( KDR) knock-down on proliferation of human lung cancer cell line A549 and their sensitivity to docetaxel .Methods The small interfering RNA ( siRNA) against KDR was constructed and transfected into A 549 cells with Lipofectamine TM 2000.The expre-ssion of KDR was detected by reverse transcription-polymerase chain reaction and Western Blot . Flow cytometry was used to detect the cell cycle .Sensitivity to docetaxel after transfection were exa-mined by methyl thiazolyl tetrazolium assay and clonogenic assay .Results In A549 cells, the pro-tein and mRNA levels of KDR were decreased significantly after transfection ,and reduction of proli-feration was related to an increase in the fraction of G 0/G1 phase .The sensitivity of A 549 cells to docetaxel was increased significantly after transfection .Conclusion The KDR special siRNA si-lenced KDR ,decreased A 549 cells proliferation and enhanced their sensitivity to docetaxel .
展开▼
