shRNA干扰VEGF表达对白血病细胞多药耐药影响.docVIP

shRNA干扰VEGF表达对白血病细胞多药耐药影响 　 作者：方莉莉， 许文林， 陈琛， 房新建， 陈巧云， 李娟 【摘要】 目的： 探讨血管内皮细胞生长因子（VEGF）与人白血病耐药细胞株K562/A02细胞多药耐药的关系及其机制。方法： 针对VEGF基因设计合成3条干扰片段shRNA1，2，3，采用脂质体2000分别将其转染入K562/A02细胞，RTPCR法检测VEGF和多药耐药相关基因MDR1，MRP1，topoⅡ，GST的mRNA水平；蛋白质印迹法检测VEGF蛋白水平；MTT法检测各组细胞对多柔比星的半数抑制浓度（IC50值）。结果： K562/A02细胞VEGF mRNA表达水平显著高于敏感株K562细胞，差异有统计学意义（Plt;0.01），且VEGF蛋白水平也显著高于K562细胞；转染shRNA后，K562/A02细胞中VEGF mRNA水平出现不同程度下调，其中shRNA2组和shRNA3组与转染随机片段组比较差异有统计学意义（Plt;0.05），以shRNA3组下调最显著，并且VEGF蛋白水平也出现相应的下调；转染48 h后K562/A02细胞中MDR1，MRP1及topoⅡ的mRNA水平均出现不同程度下调，以shRNA3组下调最显著，分别下调（39.7±1.21）％，（29.1±0.97）％，（28.2±1.04）％，GST基因表达则无明显变化；阳性转染组细胞对多柔比星的敏感性明显增加，其中以shRNA3组最显著。结论： VEGF shRNA可以抑制K562/A02细胞VEGF基因与蛋白的表达，不同干扰片段的沉默效果不同，并增强K562/A02细胞对多柔比星的敏感性，其机制可能与MDR1，MRP1及topo Ⅱ的表达下调有一定的关系。 【关键词】 血管内皮生长因子； RNA干扰； K562/A02细胞； 多药耐药 ［Abstract］ Objective: To study the relationship between vascular endothelial growth factor(VEGF) and multidrug resistance of human leukemia cell line K562/A02 by RNA interference, and then the mechanism was explored.Methods： The VEGF shRNA chain1,2 and 3 were designed,synthesized, then transfected into K562/A02 cells line by lipofectamine 2000 reagent.RTPCR was used to detect the expression of VEGF and MDRrelating genes MDR1,MRP1,topoⅡ,GST in mRNA level; Western blotting was used to analyzed the expression of VEGF in protein level; MTT was used to determine the IC50 of transfectional cells to doxorubicin. Results： Compared to K562 cell,the expression of VEGF in K562/A02 cell was even more(Plt;0.01),and after VEGF shRNAs were transfected into K562/A02 cells,the expression of VEGF at the mRNA level were decreased,and there were statistical difference between VEGF shRNA2 group or VEGF shRNA3 group and HK group(Plt;0.05),the greatest decrease was seen in cells transfected with VEGF shRNA3;and the protein level of VEGF were also downregulated. The MDR1,MRP1,topoⅡ mRNA of K562/A02 cell were significantly decreased; the greatest decrease was seen in VEGF shRNA3 group. The IC50 value of posit