碱基错配对短发夹RNA逆转白血病细胞耐药作用的影响

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mdrl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mdrlshRNA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mdrlmRNA表达；柔红霉素泵出实验检测P．gp外排泵功能；M1rr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mdrlmRNA的表达，降低细胞膜P-gP外泵功能，60min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mdrl所致的耐药。

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

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 mdr1 mRNA sequence and its corresponding shRNA with one base mismatch in sense strand were cloned and trans-fected 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 MFI to 9.51 MFI and 7.07 MFI respectively. MTT test demonstrated the relative reversing efficiency to doxorubicin 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 transfected cells multidrug resistance.