中文摘要：

目的构建shRNA／mrp1的质粒表达载体并验证其体外表达效率。方法根据业已筛选出的mrp1基因RNAi靶序列，按照pSUPER的设计要求合成64bp的寡核苷酸序列，将其退火后形成双链并用双酶切法克隆到pSUPER得到质粒pSUPER-shRNA／mrp1，转染感受态大肠杆菌，筛选阳性克隆，经测序证实后扩增培养并以去内毒素试剂盒提取，然后转染HepG2／mrp1细胞，阴性载体为对照组，Real—time PCR、流式细胞术检测mrp1基因mRNA、MRP1表达、细胞耐药性等功能变化。结果成功构建质粒载体pSUPER—shRNA／mrp1，经基因测序证实靶序列插入正确；HepG2／mrp1组Ct值较GAPDH增加5．61，HepG2／mrp1—si组Ct值比GAPDH升高11．35，mrp1基因的表达水平是耐药株HepG2／mrp1的179分之一；实验组HepG2／mrp1细胞和阴性对照组HepG2／mrp1细胞的MRP表达率分别为11．2％和97．6％，差别有统计学意义（P〈0．05）；药物敏感试验显示HepG2／mrp1细胞耐药倍数从45．0下降到1．2，相对逆转效率99．62％；细胞内DNR累积量也明显增加，与对照组比较（78．58％vs38．44％，P〈0．05）。结论成功构建质粒载体pSUPER-shRNA／mrp1，在HepG2／mrp1内稳定表达，逆转其耐药性。

英文摘要：

Objective To construct the expressing vector of shRNA/mrp1 and study its expression in vitro. Methods 64bp oligonucleotides of pSUPER and the targeted senquence of siRNA/mrp1 were synthesized and annealed to form duplex strand, then were cloned into pSUPER to construct pSUPER-shRNA/mrp1 vector. Competeneed Ecoli was transfected by vector of pSUPER-shRNA/ mrp1 to screen the positive clones for sequencing and extracting plasmid. The plasmids extracted were used to transfected HepG2/mrp1 cells with a control groups by negative vectors. The expression of mrp1 mRNA and MRP1 was measured by real-time PCR and resistance of HepG2/mrp1 by flowcytometry. Results pSUPER-shRNA/mrp1 was established successfully and was sequenced to test its accuracy. Expression of mrp1 mRNA in HepG2/mrp1-si was lower than that in HepG2/mrp1 （1-fold w 179.76-fold, P〈0. 001）. Compared to HepG2/mrp1, the expression of MRP1 in HepG2/mrp1 si was lower （11.2% vs 97.6%, P〈0.05）. The sensitivity of HepG2/mrp1-si to adiramyein was higher than that of HepG2/mrp1（45.0-fold vs 1.2-fold, P〈0.01）. Meanwhile, the accumulation of DNR in HepG2/mrp1-si increased significantly as compared with the control （78.58% vs 38.44% ,P〈0.05）. Conclusion Vector of pSUPER-shRNA/mrp1 can be constructed by the technique of enzymatic incision. The multidrug resistance of HepG2/mrp1 can be reversed by RNA interference.