碱基错配对短发夹RNA逆转白血病细胞耐药作用的影响  

Effect of mismatched shRNA on reversing the multidrug resistance of leukemia cell

作　　者：顾玲[1] 刘霆[1] 龚玉萍[1] 孟文彤[1] 

机构地区：[1]四川大学华西医院血液科,成都610041

出　　处：《中华血液学杂志》2006年第11期736-738,共3页Chinese Journal of Hematology

基　　金：国家自然科学基金资助项目(30100074)

摘　　要：目的探讨碱基错配对短发夹RNA(shRNA)逆转白血病细胞耐药作用的影响。方法针对mdr1基因mRNA合成一条序列正确的shRNA及正义链少1个碱基的对应错配shRNA,构建一对抗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。采用实时荧光定量RT- PCR检测mdr1 mRNA表达;柔红霉素泵出实验检测P-gp外排泵功能;MTT法检测细胞对阿霉素的敏感性。结果序列正确和对应错配shRNA真核表达载体均明显下调mdr1 mRNA的表达,降低细胞膜P-gp外泵功能,60 min柔红霉素泵出率分别为6％和10％。显著低于对照组的45％;细胞内柔红霉素平均荧光强度分别为9．51和7．09,明显高于对照组的2．80;对阿霉素药物敏感性的相对逆转率为90％和87％。结论正义链碱基错配shRNA对RNA干扰功能无明显影响,与序列正确的shRNA一样可有效逆转mdr1所致的耐药。Objective To explore effect of mismatched short hairpin RNA （shRNA） on reversing the multidrug resistance of leukemia cell. Methods Eukaryotic expression vectors of shRNA targeting mdrl mRNA sequence and its corresponding shRNA with one base mismatch in sense strand were cloned and transfected into drug resistance cell line K562/A02 by liposome. The mdrl mRNA was identified by quantitive RT- PCR, the function of P-gp by daunorubicin （DNR） efflux experiment and the sensitivity of the cell line to doxorubicin （ADM） by MTT test. Results Both mdrl shRNA and its counterpart could almost completely down-regulate mdrl mRNA expression. The DNR efflux ratio at 60 min was 6% and 10% respectively, being significantly lower than that of the 45% of the control. The intracellular DNR was increased from 2.80 MF1 to 9.51 MFI and 7.07 MFI respectively. MTT test demonstrated the relative reversing efficiency to doxoruhicin was 90% and 87%. Conclusion mdrl shRNA with one base mismatch in sense strand does not interfere with RNAi function and can effectively reverse the transfeeted cells muhidrug resistance.

关 键 词：RNA干扰 碱基错配 基因 mdrl 抗药性 多药 

分 类 号：R733.7[医药卫生—肿瘤]