中文摘要：

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。

英文摘要：

Objective： To screen effective sequences of small interfering RNA targeting human multidrug associated- protein gene（ mrp1）. Methods： Four siRNAs（ mrp1- si251,mrp1- si480,mrp1- si795,mrp1- si1016） targeting mrp1 genes were designed and synthesized by in vitro transcription. The siRNA duplexes were used to transfected into the human hepatocellular carcinoma HepG2 / mrp1 cells,which transfected with human mrp1 gene and obtained MDR phenomenon. The expression level of mrp1 mRNA was detected by RT- PCR. The multidru resistance- associated protein and accumulation of intracellular daunorubicin（ DNR） were examined by flow cytometry,respectively. The cell sensitivity to adriamycin（ ADM） was demonstrated by MTT. Results： The HepG2 / mrp1 cells treated with 4 siRNAs led to reversal effectively on multidrug resistance to different extents. Among the HepG2 / mrp1 cells treated by siRNAs for 7 2 h,the expression level of mrp1 mRNA in cells of mrp1- si1016 or mrp1- si795 groups[（ 85. 54± 1. 04） % or（ 86. 36 ± 2. 26） %]was more decreased than that in cells of mrp1- si2 5 1 or mrp1- si4 8 0groups（ P〈0. 05）. The accumulation of DNR in cells of mrp1- si795 group was the most. In cells of mrp1- si1016 group,more,in cells of mrp1- si480 group,lower,and in cells of mrp1- si251 group,the lowest（ P〈0. 05）. The relative reversal efficiency of cells of mrp1- si1016 and mrp1- si795 groups to ADR was higher than in the cells of mrp1- si251 and mrp1- si480 groups significantly（ P〈0. 05）. The expression level of multidrug resistance- associated protein in cells of mrp1- si1016 and mrp1- si795 groups was lowest among the HepG2 / mrp1 cells treated by siRNAs for 72 h. Conclusion： The mrp1- si795 with most,mrp1- si1016 with more,mrp1- si480 with less and mrp1- si251 with least reversal effects on mrp1 gene mediated multidrug resistance were found in the human hepatocellular carcinoma HepG2 / mrp1 cells.