慢病毒介导RNA干扰血管内皮生长因子基因增强K562细胞对STI571敏感性

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

目的:探讨慢病毒载体介导RNA干扰(RNAi)抑制血管内皮生长因子(VEGF)基因表达对白血病细胞系K562增殖和凋亡的影响。方法:构建靶向VEGF基因的短发夹状RNA(shRNA)慢病毒载体,与慢病毒包装质粒通过脂质体转染至293T细胞,包装产生的病毒液感染K562细胞。采用实时荧光定量PCR、Western blotting、ELISA等方法检测RNAi对K562细胞VEGF mRNA和蛋白表达的影响;台盼蓝拒染法和MTT法分析转导VEGF-shRNA对K562细胞增殖的影响;流式细胞术检测经STI571(甲磺酸伊马替尼)作用后细胞凋亡变化情况。结果:构建VEGF基因shRNA慢病毒载体(pRNAT-shRNA),并成功将其导入K562细胞。与K562和K562-con(转导空载体)组细胞相比,转导pRNAT-shRNA的K562-shVEGF细胞VEGF mRNA和蛋白表达均显著下降;生长曲线提示K562-shVEGF细胞增殖速度减慢;在STI571作用下,K562-shVEGF细胞凋亡率较K562和K562-con组细胞明显增高(P<0.05)。结论:慢病毒载体介导的VEGF基因RNA干扰可有效抑制K562细胞增殖,并能够增强细胞对STI571的敏感性。

AIM： To investigate the effects of RNA interference （RNAi） inhibiting the expression of vascular endothelial growth factor （VEGF） gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS ： A lentiviral vector containing short hairpin RNA （shRNA） targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real - time quantitative RT - PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTr assay. STI571 （imatinib mesylate） -induced apoptosis was analyzed by flow cytometry. RESULTS： The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562 - sh- VEGF cells transfected with pRNAT - shRNA were significantly inhibited when compared with those of K562 and K562 - con cells （mock transduction）. The proliferation rate of K562 -shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562 - shVEGF cells increased more significantly than those of K562 and K562 - con cells （P 〈 0. 05）. CONCLUSION ： Inhibition of VEGF by lentivirus - mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.