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目的:筛选靶向人多药耐药相关蛋白基因(mrp1)特异性小分子干扰RNA(siRNA)的有效序列。方法:设计并体外转录合成靶向mrp1的4条siRNA(mrp1-si251,mrp1-si480,mrp1-si795,mrp1-si1016和空白对照si-阴性),转染转基因单因素肝癌多药耐药细胞Hep G2/mrp1。用RT-PCR检测mrp1 mRNA表达,流式细胞仪检测多药耐药相关蛋白表达、细胞内柔红霉素(DNR)蓄积,四甲基偶氮唑蓝(MTT)法检测细胞对阿霉素(ADM)的敏感性。结果:4条siRNA均能不同程度逆转Hep G2/mrp1细胞由mrp1介导的多药耐药。转染72h后,mrp1-si795组和mrp1-si1016组的mrp1 mRNA表达水平分别分别下调了(86.36±2.26)%和(85.54±1.04)%,较mrp1-si251组和mrp1-si480组明显下降(P<0.05);细胞内柔红霉素蓄积由多到少依次为mrp1-si795组最多,mrp1-si1016组次之,mrp1-si480组较少,mrp1-si251组最少(P<0.05);mrp1-si795组对ADR耐药的相对逆转率(86.36%)最高,mrp1-si1016组(85.54%)次之,较mrp1-si251组(60.93%)和mrp1-si480组(70.29%)有明显差异(P<0.05);mrp1-si1016组和mrp1-si795组多药耐药相关蛋白表达明显下调。结论:实验设计的靶向mrp1 mRNA的siRNA序列能够不同程度逆转多药耐药相关蛋白介导的人肝癌耐药细胞HepG 2/mrp1的多药耐药性,mrp1-si1016和mrp1-si795效果最好,mrp1-si480较差,mrp1-si251最差。mrp1-si1016和mrp1-si795可以作为进一步实验的靶序列。
Objective: To screen the effective sequence of small interfering RNA (siRNA) targeting human multidrug resistance-associated protein gene (mrp1). Methods: Four siRNAs targeting mrp1 were designed and synthesized in vitro. Transfection of multi-drug resistant HepG2 cells G2 / mrp1. The mRNA expression of mrp1 was detected by RT-PCR, the expression of multidrug resistance-related protein was detected by flow cytometry, the accumulation of intracellular daunorubicin (DNR) and the proliferation of doxorubicin (ADM) sensitivity. Results: All four siRNAs could reverse the multidrug resistance mediated by mrp1 in Hep G2 / mrp1 cells to varying degrees. The expression of mrp1 mRNA in mrp1-si795 group and mrp1-si1016 group decreased by (86.36 ± 2.26)% and (85.54 ± 1.04)%, respectively, 72h after transfection compared with those in mrp1-si251 group and mrp1-si480 group (P <0.05). The accumulation of daunorubicin in the mrp1-si795 group was the largest among the groups, followed by mrp1-si1016 group, followed by mrp1-si480 group and mrp1-si251 group (P <0.05) Compared with the mrp1-si251 group (60.93%) and the mrp1-si480 group (70.29%), the relative reverse rate (86.36%) of the siRNA-si795 group was the highest, followed by the mrp1-si1016 group (85.54% P <0.05). The expression of multidrug resistance-related protein in mrp1-si1016 group and mrp1-si795 group was significantly down-regulated. CONCLUSIONS: The siRNA sequences targeting mrp1 mRNA designed by the experiment can reverse the multidrug resistance of HepG 2 / mrp1 cells induced by multidrug resistance-related protein to varying degrees, and mrp1-si1016 and mrp1-si795 are most effective Well, mrp1-si480 is poor, mrp1-si251 is the worst. mrp1-si1016 and mrp1-si795 can serve as target sequences for further experiments.