RNA干扰逆转白血病细胞耐药性的研究  

Study on RNA Interference Reversing the Multidrug Resistance of Leukemia Cell

作　　者：顾玲[1] 刘霆[1] 龚玉萍[1] 席亚明[1] 

机构地区：[1]四川大学华西医院血液科,成都610041

出　　处：《四川大学学报（医学版）》2007年第1期45-48,共4页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金(批准号30100074)资助

摘　　要：目的用RNA干扰技术下调白血病耐药细胞系K 562/A 02多药耐药基因m d r1的表达以逆转白血病对化学药物的耐药性。方法针对m d r1基因已知mRNA序列不同位点,选择两条靶序列,构建靶向m d r1 shRNA真核表达载体,脂质体介导转染白血病耐药细胞株K 562/A 02。实时荧光定量RT-PCR检测mRNA表达,W esternb lot检测细胞膜P-gp表达,柔红霉素泵出试验检测P-gp外排泵功能,M TT法检测细胞对阿霉素的敏感性。结果构建了二条针对m d r1基因的shRNA真核表达载体,分别下调m d r1 mRNA的表达89.74%和87.18%,降低细胞膜P-gp表达,使膜外泵功能明显下降,柔红霉素在细胞内贮留明显增多,60m in时柔红泵出率为13.16%、22.02%,对照组为40.44%、45.31%,对阿霉素药物敏感性的相对逆转率为84.36%和76.69%。结论RNA干扰技术可有效逆转m d r1所致耐药。Objective To downregulate the expression of mdr1 gene in K562/A02 eell line by RNA interference. Methods The eukaryotic expression vectors of shRNA aiming at two mdr1 mRNA target sequences were constructed and used to transfect the drug resistance cell line K562/A02 with liposome-induced gene transfeetion. The mRNA of mdr1 gene was identified by Q-PCR. The P-gp expression of was deteeted by Western blot. The funetion of P-gp was measured by daunorubiein （DNR） efflux experiment and the sensitivity of eell lines to doxorubiein （ADM） was deteeted by MTT test. Results Two shRNA plasmids targeting mdr1 mRNA were eonstrueted and eloned. Two mdr1-targeted shRNA eould down-regulate mdr1 mRNA expressions with 89. 74% and 87.18% respectively, almost completely down-regulate the P-gp expression. The intracellular DNR increased after RNAi treating. The daunorubiein efflux ratio at 60 rain were 13. 16% and 22.02%, eompared with eontrol 40. 44%, 45. 31%, P〈0. 05. MTT test demonstrated the relative reversing efficiencies to doxorubicin were 84.36% and 76. 69% respeetively. Conclusion RNA interferenee ean effeetively reverse multidrug resistanee caused by mdr1.

关 键 词：RNA干扰 短发夹RNA MDR1基因 多药耐药 

分 类 号：R733.7[医药卫生—肿瘤]