RNA干扰逆转白血病细胞耐药性的研究

目的 用RNA干扰技术下调白血病耐药细胞系K562/A02多药耐药基因mdr1的表达以逆转白血病对化学药物的耐药性.方法 针对mdr1基因已知mRNA序列不同位点,选择两条靶序列,构建靶向mdr1 shRNA真核表达载体,脂质体介导转染白血病耐药细胞株K562/A02.实时荧光定量RT-PCR检测mRNA表达,Western blot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,MTT法检测细胞对阿霉素的敏感性.结果 构建了二条针对mdr1基因的shRNA真核表达载体,分别下调mdr1 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,60min 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mdr1所致耐药.

Study on RNA Interference Reversing the Multidrug Resistance of Leukemia Cell

Objective To downregulate the expression of mdr1 gene in K562/A02 cell 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 transfection.The mRNA of mdr1 gene was identified by Q-PCR.The P-gp expression of was detected by Western blot.The function of P-gp was measured by daunorubicin(DNR) efflux experiment and the sensitivity of cell lines to doxorubicin(ADM) was detected by MTT test.Results Two shRNA plasmids targeting mdr1 mRNA were constructed and cloned.Two mdr1-targeted shRNA could 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 daunorubicin efflux ratio at 60 min were 13.16% and 22.02%,compared with control 40.44%,45.31%,P<0.05.MTT test demonstrated the relative reversing efficiencies to doxorubicin were 84.36% and 76.69% respectively.Conclusion RNA interference can effectively reverse multidrug resistance caused by mdr1.