目的：探讨通过RNA干扰技术沉默mdr1基因对人白血病HT9细胞和胃癌SGC7901/ADM细胞多药耐药的逆转效果。方法：针对mdr1 mRNA序列设计合成3对编码shRNA的DNA模板序列，分别定向克隆到含有U6和H1启动子的质粒载体pSilencer 2.1-U6 neo 和pSilencer 3.1-H1 neo上，构建6种表达shRNA的重组载体，电转染HT9细胞和SGC7901/ADM细胞，经G418筛选后制备转染细胞单克隆，经PCR扩增基因组DNA鉴定后扩大培养。通过RT-PCR和荧光定量PCR检测mdr1-mRNA的表达水平，Western blot和免疫组化检测P-糖蛋白的表达水平，Rho123外排实验检测P-糖蛋白的转运功能，通过MTT法测定药物作用后的细胞存活率和在光学显微镜、荧光显微镜、透射电镜下，以及琼脂糖凝胶电泳时，药物作用后细胞呈现的形态结构和生化特征，从而检测细胞耐药逆转效果。结果：1. 用pSilencer 2.1-U6 neo 和pSilencer 3.1-H1 neo质粒成功构建了6种表达shRNA的重组载体，即p2.1-1、p2.1-2、p2.1-3、p3.1-1、p3.1-2、p3.1-3；2.重组载体成功转染HT9细胞和SGC7901/ADM细胞，获得了稳定转染的细胞单克隆; 3.RT-PCR、荧光定量PCR、Western blot和免疫组化结果显示，3对干涉片段（6种重组载体）均能不同程度地抑制HT9细胞和SGC7901/ADM细胞mdr1基因表达，干涉片段3的抑制作用优于干涉片段2和干涉片段1，U6启动子优于H1启动子；4. Rho123外排实验结果显示，6种重组载体转染的HT9细胞和SGC7901/ADM细胞P-糖蛋白的外排转运功能均有不同程度的降低； 5.MTT检测结果显示，转染6种重组质粒的HT9细胞和SGC7901/ADM细胞对三尖杉酯碱、阿霉素、紫杉醇、姜黄素的敏感性均有不同程度的增强，耐药性均有不同程度逆转，其中，转染p2.1-3、p3.1-3重组质粒（带有干涉片段3）的细胞对药物的敏感性最强。6.在光学显微镜、荧光显微镜、透射电镜下，以及琼脂糖凝胶电泳时，用三尖杉酯碱、阿霉素、紫杉醇、姜黄素作用后，转染p2.1-3、p3.1-3重组质粒载体（带有干涉片段3）的HT9细胞和SGC7901/ADM细胞呈现明显的凋亡特征。结论：本研究设计的3对编码shRNA的DNA干扰片段所构建的重组载体均能转染HT9细胞和SGC7901/ADM细胞，并能不同程度抑制其mdr1基因的表达，逆转其耐药性。

Objective: To explore the reversal effect of mdr1 gene silence with RNAi on multidrug resistance in leukemia cells HT9 and gastric cancer cells SGC7901/ADM.Methord: The three pairs of DNA sequence coding shRNA targeting mdr1 mRNA were designed and Synthesized, they were cloned into pSilencer 2.1 U6-neo and pSilencer 3.1 H1-neo plasmids respectively to construct six shRNA-recombinant plasmids. The recombinant plasmids were transfected into HT9 cells and SGC7901/ADM cells by electroporation. Made monoclonal cell when the transfected cells were screened by G418 and the clones were identified by PCR of genomic DNA.The expression of mdr1-mRNA was evaluated with RT-PCR and real-time PCR; the expression of P-gp was detected by western blot and immmunohistochemistry; the transport function of P-glycoprotein was detected by Rho123 efflux experiment; cell survival rate was analyzed by MTT methord, the morphologic changes of the cells which treated with drugs were observed under optical microscope, fluorescence microsopet and electron microscope, respectively, biochemical characteristics were detected by agarose gel electrophoresis.Results: 1.Three shRNA recombinant vectors mediated by pSilencer 2.1-U6 neo and pSilencer 3.1-H1 neo plasmids were constructed successfully, they were designated as p2.1-1、p2.1-2、p2.1-3、p3.1-1、p3.1-2、p3.1-3; 2.The six recombinant plasmids were transfected into HT9 cells and SGC7901/ADM cells successfully, the stable transfective monoclonal cells were prepared; 3.The results of RT-PCR、fluorescence quantitative PCR 、Western blot and immmunohistochemistry indicated that the three shRNA interference fragements(six recombinant vectors) can inhibit the expression of mdr1 gene in HT9 cells and SGC7901/ADM cells to different extents.The inhibition effects of interference fragment 3 were better than interference fragment 2 and interference fragment 1, U6 promotor was better than H1 promotor.; 4.Rho123 efflux experiment showed that the transport function of HT9 cells and SGC7901/ADM cells transfected by recombinant plasmids has decreased in different degree; 5.MTT assay showed that chemosensitivity of transfected HT9 cells and SGC7901/ADM cells to adriamycin、harringtonine、paclitaxel、 curcumin increased in different degree, and the drug resistance was reversed to some extent. The HT9 and SGC7901/ADM cells transfected by p2.1-3、p3.1-3 recombinant plasmids (with interference fragement 3) were most sensitive to drugs; 6. The results of optical microscope、fluorescence microsopet 、electron microscope, agarose gel electrophoresis showed that HT9 cells and SGC7901/ADM cells transfected by p2.1-3、p3.1-3 recombinant plasmids (with interference fragement 3) presented typical characteristics of apoptosis.Conclusion: The recombinant plasmids coding three pairs of shRNA can be transfected into leukemia cells HT9 and gastric cancer cells SGC7901/ADM，they inhibited the expression of mdr1 gene and reversed the multidrug resistance.