载体介导的RNA干扰技术抑制肝癌耐药细胞MDR1表达的研究

Inhibition of MDR1 expression in hepatocellular carcinoma cell Bel-7402/R by vector-mediated RNA interference

目的 :构建含多药耐药基因MDR1的短发夹状RNA(shorthairpinRNA ,shRNA)表达质粒 ,观察对肝癌耐药细胞Bel 740 2 /R的MDR1mRNA的抑制作用。方法 :利用分子克隆技术 ,将含MDR1的双链DNA ,与经双酶切后的载体PGE 1连接 ,构建pshRNA MDR1重组质粒 ,在脂质体的介导下转染肝癌耐药细胞株Bel 740 2 /R ;RT PCR分析MDR1mRNA的表达 ,MTT法检测细胞对药物的敏感性 ,流式细胞仪检测细胞内罗丹明 12 3 (Rh12 3 )的潴留和P gp的表达。结果 :PCR和DNA测序证实了表达质粒构建成功 ,并能明显地抑制MDR1mRNA的表达 ;对盐酸表柔比星和顺铂的半数抑制浓度IC50明显降低 ,P <0 0 5 ;P gp的表达阳性率降低了 5 7 3 % ;细胞内Rh12 3的浓度显著增高 ,P <0 0 5。结论 :构建的pshRNA MDR1表达质粒能有效地降低肝癌耐药细胞MDR1mRNA和P gp的表达。

OBJECTIVE:To construct a recombinant plasmids generating short hairpin RNA which contain multi-drug resistance gene MDR1 in mammalian cells, in order to investigate the suppression of MDR1 mRNA in hepatocellular carcinoma cells Bel-7402/R.METHODS: By using molecular cloning technique, double-stranded DNA containing MDR1 was cloned into PGE-1 vector digesting by two restricted endoenzymes according to its special orientation. Human hepatocellular carcinoma cells Bel-7402/R was transfected with recombinant plasmid by LyoVecTM, MDR1 mRNA was assessed by RT-PCR, drug sensitivity was measured by MTT assay in vitro. The effect of intracellular Rh123 accumulation and the expression of P-gp were determined by the flow cytometry (FCM). RESULTS: The size of the PCR product was 669 bp. DNA sequencing showed the sequence of recombinant vector pshRNA-MDR1 was successfully constructed, and MDR1 mRNA expression was suppressed;IC 50 of cells which transfected pshRNA-MDR1 reduced obviously, P<0.05. Positive rate of P-gp receded from 77.7% to 20.4%, and the concentration of Rh123 in cells which transfected pshRNA-MDR1 increased greatly,P<0.05.CONCLUSION: The recombinant plasmid constructed by the authors can suppress the expression of MDR1 mRNA and P-gp in hepatocellular carcinoma cells Bel-7402/R.