vegf shRNA对耐药白血病细胞化疗敏感性的增强作用  

vegf shRNA Enhances the Sensitivity of Multidrug-resistant Leukemia Cells to Anticancer Agent

作　　者：沈慧玲[1] 方莉莉[1] 陈琛[1] 房新建[1] 陈巧云[1] 李娟[1] 许文林[1] 

机构地区：[1]江苏大学附属人民医院中心实验室,江苏镇江212002

出　　处：《中国实验血液学杂志》2011年第1期34-39,共6页Journal of Experimental Hematology

基　　金：江苏省卫生重大课题基金资助项目;编号K2005017

摘　　要：本研究采用RNA干扰技术探讨血管内皮生长因子(VEGF)对耐药白血病细胞K562/A02药物敏感性的影响及其机制。针对人vegf基因合成3个特异性shRNA干扰片段,采用脂质体介导的方法将其导入K562/A02细胞,用RT-PCR检测vegf及mrp1的mRNA表达水平,用Western blot检测VEGF、MRP1、AKT及P-AKT的蛋白表达情况,用MTT法检测阿霉素对各组细胞的半数抑制浓度(IC50),用流式细胞术检测细胞凋亡、胞内罗丹明123(Rho123)积聚情况。结果表明:转染vegf shRNA后,K562/A02细胞vegf的mRNA表达下调,其中vegf shRNA2组和vegf shRNA3组与HK组比较有显著差异(p<0.05),以vegf shRNA3组最显著,VEGF蛋白表达也出现下调,与mRNA的表达基本一致;MTT检测结果显示阳性转染组对阿霉素的敏感性增加,其中vegf shRNA2组和vegfshRNA3组的半数抑制浓度(IC50)与HK组比较具有显著的统计学差异(p<0.05);阳性转染组胞内Rho123积聚增多,与HK组比较均有统计学差异(p<0.05);阳性转染组细胞凋亡增加,其中vegf shRNA2组和vegf shRNA3组与HK组比较有统计学意义(p<0.05);阳性转染组MRP1mRNA蛋白及表达均下调,以vegf shRNA3组下调最显著;阳性转染组P-AKT的表达水平低于对照组,以vegf shRNA3组最明显,而总的AKT无明显变化。结论:vegf shR-NA可以抑制vegf的基因的转录及翻译,从而增加耐药白血病细胞对阿霉素的敏感性,其机制可能是vegf shRNA通过抑制PI3K/AKT信号转导通路的活化而促进细胞凋亡及下调MRP1的表达。This study was aimed to explore the effect of vascular endothelial growth factor（VEGF） on sensitivity of leukemia cell line K562/A02 to doxorubicin by using RNA interference,and to investigate its mechanism.The 3 shRNA targeting human vegf gene were synthesized,then transfected into K562/A02 cells by lipofectamine 2000 reagent.RT-PCR was used to detect the expression of vegf and mrp1 at the mRNA level;Western blot was used to analyze the expression of VEGF,MRP1,AKT,P-AKT at the protein level;MTT was used to determine the IC50 value of transfected cells to doxorubicin（DOX）;flow cytometry was used to detect cell apoptosis and intracellular Rho123 retention.The results showed that after vegf shRNA were transfected into K562/A02 cells,the expression of vegf at the mRNA level decreased,and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was statistically significant（p0.05）,the greatest decrease was observed in the cells transfected with vegf shRNA3;and the protein level of VEGF was also down-regulated.The IC50 value of positively transfected group was lower than that of control groups,and the difference between vegf shRNA2 group or vegf shRNA3 group and HK group was significant（p0.05）.The retention of intracellular Rho123 was enhanced in three positively transfected groups（p0.05）.Cell apoptosis increased in positively transfected groups,and there was statistically difference between vegf shRNA2 group or vegf shRNA3 group and HK group（p0.05）.The expression of mrp1 at the mRNA level were decreased,and there were statistical difference between vegf shRNA3 group and HK group（p0.05）,and the protein level of mrp1 was also down-regulated;the expression of P-AKT at protein level decreased in positively transfected groups,and the greatest decrease was seen in vegf shRNA3 group.It is concluded that the transfection with exogenous vegf shRNA can inhibit the expression of vegf at both mRNA and protein levels,and enhance the sensitivity of K562/A02 cell to doxorubicin,the mech

关 键 词：VEGF SHRNA RNA干扰 白血病 K562/A02细胞 阿霉素 药物敏感性 MRP1 细胞凋亡 

分 类 号：R733.7[医药卫生—肿瘤] R979.1[医药卫生—临床医学]