靶向血管内皮生长因子siRNA对K562细胞凋亡及survivin表达的影响

目的 应用腺病毒介导的血管内皮生长因子(VEGF) siRNA沉默K562细胞中VEGF的表达,观察其对K562细胞凋亡及凋亡抑制基因survivin表达的影响.方法 应用成功构建的携带特异性VEGF siRNA的重组腺病毒(Ad5-VEGF siRNA)感染K562细胞,实验分为3组:实验组(K562/Ad5-VEGF siRNA组)、空载体组(K562/Ad5组)、对照组(K562组).通过RT-PCR法检测细胞内VEGF及survivin mRNA的表达,ELISA法检测细胞培养上清中VEGF蛋白表达,Western blot法检测细胞survivin蛋白表达,流式细胞术检测细胞凋亡.结果 实验组细胞VEGF及survivin mRNA表达水平较对照组均有明显降低(P＜0.01),且细胞培养上清中VEGF蛋白表达水平[(1121±15)pg/ml]低于空载体组[(1290±28)pg/ml]和对照组[(1303±28)pg/ml](P＜0.01).Western blot法检测结果显示,实验组survivin蛋白表达水平(0.26 ± 0.11)较对照组(0.74±0.10)也显著降低(P＜0.01).实验组细胞凋亡率与对照组比较明显增加(P＜0.01).结论 应用RNA干扰沉默K562细胞中VEGF基因表达后,survivin的表达也随之降低,同时细胞凋亡率相应增加.VEGF可诱导K562细胞中survivin基因表达,可能是survivin基因表达的上游调控因子.

Effect of siRNA targeting VEGF on cell apoptosis and the expression of survivin in K562 cells

Objective To study the effect of adenovirus vector-mediated siRNA targeting vascular endothelial growth factor (VEGF) on apoptosis and the expression of survivin iu K562 cells.Methods K562 cells were infected with recombinant adenovirus Ad5-VEGFsi for 72 hours as experimental group (K562/Ad5-VEGFsi),and empty vector group (K562/Ads) and blank control group (K562) as controls.VEGF mRNA and survivin mRNA expression were determined by RT-PCR.The protein levels of VEGF and survivin were measured by ELISA and Western blot,respectively.The apoptosis of K562 cells wss detected by flow cytometry.Results The levels of VEGF and survivin mRNA expression in experimental group cells were signitlcanfly decreased (P＜0.01).The protein concentration of VEGF in experimental group supernatant was (1121±15)pg/ml,being lower than that in empty vector group[(1290±28)pg/ml] and black control group [(1303±28)pg/ml] (P＜0.01),and the level of survivin protein in experimental group (0.26±0.11) was significantly reduced compared with that in blank control group(0.74±0.10)(P＜0.01).The apoptosis rate of K562/Ad5-VEGFsi cells(16.45±0.14)% was higher than those of K562/Ad5 cells (3.54±0.17)% and K562 cells(2.56±0.20)% (P＜0.01).Conclusions VEGF can up-regulate the expression of survivin.After inhibition of VEGF by RNAi,the expression of survivin is decreased subsequently and the rates of cell apoptosis are increased.