靶向抑制肝癌耐药细胞多药耐药基因表达的实验研究

目的：构建含多药耐药基因MDR1的短发夹状RNA（shorthairp in RNA,shRNA）表达质粒,并观察其对肝癌耐药细胞Bel-7402/R的MDR1 mRNA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-7402/R,RT-PCR分析MDR1mRNA的表达,流式细胞仪检测细胞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;P-gp的表达阳性率降低了57.3%,P〈0.05。结论：构建的pshRNA-MDR1表达质粒能靶向抵制肝癌耐药细胞MDR1mRNA和P-gp的表达。

INHIBITION OF MDR1 EXPRESSION IN HEPATOCELLULAR CARCINOMA CELL BEL-7402/R BY VECTOR-MEDIATED RNA INTERFERENCE

Objective：To construct a recombinant plasmids generating short hairpin RNA contain of multi-drug resistance gene MDR1 in mammalian cells in order to investigate suppression of MDR1 mRNA in hepatocellular carcinoma cells Bel-7402/R.Methods： Using molecular cloning technique,double-stranded DNA which contained MDR1 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,we determined the expression of P-gp by flow cytometry（FCM）.Results： The size of the PCR product was 669bp.DNA sequencing showed the sequence of recombinant vector pshRNA-MDR1 was successfully constructed,and suppressed MDR1 mRNA expression;positive rate of P-gp receded from 77.7% to 20.4%,P0.05.Conclusion： The result showed that the recombinant plasmid constructed by the authors can suppress the expression of MDR1 mRNA and P-gp in hepatocellular carcinoma cells Bel-7402/R.