序列特异性短发夹状RNA逆转白血病多药耐药性研究  

Reversal of Leukemia Multidrug Resistance by Sequence-Specific Short Hairpin RNA

作　　者：杜伟廷 刘斌[1] 顾东生 韩之波 刘鹏霞 徐洁 梁璐 赵海丰 卢士红 杨仁池 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院,实验血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2009年第3期563-567,共5页Journal of Experimental Hematology

基　　金：天津市应用基础研究计划重点项目(编号06YFJZJC01800);国家自然科学基金(项目编号:30670900);公益性卫生行业科研专项基金(项目编号:200802031)

摘　　要：本研究旨在构建并筛选MDR1序列特异性短发夹状RNA干扰载体,研究其对K562/A02细胞的影响。采用脂质体介导方法,将含mdr1-sh RNA质粒转入K562/A02细胞,经G418筛选得到稳定表达细胞株,用实时定量RT-PCR和Western blot分别检测干扰后mdr1 mRNA水平及蛋白水平的表达变化,通过CCK8测定干扰后细胞对化疗药的敏感性变化,Rhodamine123外排实验检测P-gp功能的变化。结果发现:与对照组相比,4种MDR1-sh RNA载体均能显著下调P-gp的表达,其中508-526靶位点的shRNA作用效果最好。结论:筛选得到的MDR1-sh RNA载体能够显著下调MDR1的表达,逆转耐药表型,为后续进行的动物实验研究创立了条件。This study was aimed to design and screen short hairpin RNA （shRNA）molecules targeting multidrug resistance gene（ mdrl ）, as well as to investigate the effects of shRNA expression vector on K562/A02 cells. Mdr1- shRNA expression vector was transfected into K562/A02 cells by lipofectamine 2000, and G418 was added to screen and establish the stable expression cell strain. The expressions of mdrl mRNA and protein were detected by real-time RTPCR and Western blot respectively. The sensitivity of cells to chemodrngs after inferference were tested by CCK8 assay. The function of p-glycoprotein was determined by Rhodamine 123 effiux experiment. The results showed that all of 4 mdr1-shRNA expression vectors could significantly knockdown the expression of p-glycoprotein as compared with control vector, moreover, the vector targeting 508 - 526 sites of mdr1 gene was the best one. It is concluded that the mdr1- shRNA expression vector gained by screening can significantly knockdown the expression of mdrl gene and reverse leukemia drug resistance, paving the way for the application of RNAi in the following animal experiments.

关 键 词：RNA干扰 白血病 多药耐药 SHRNA 

分 类 号：R733.7[医药卫生—肿瘤]