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目的 构建在哺乳动物细胞中表达MDR1短发夹RNA(shorthairpinRNA ,shRNA)的表达质粒 ,并初步探讨其对耐药肝癌细胞MDR1mRNA的抑制作用及抗肿瘤药物耐药性的影响。方法 根据Genbank中MDR1mRNA设计的两条多聚核苷酸序列 ,退火形成双链DNA ,再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细胞株BEL 74 0 2 /ADM ,RT PCR分析MDR1mRNA的表达 ,MTT法检测阿霉素对细胞的半数抑制浓度 (IC50 )。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BEL 74 0 2 /ADMMDR1mRNA的表达 ,对阿霉素的耐药指数降低了17.5倍 ( 2 99.2 / 17.1)。结论 构建的 pshRNA MDR1表达质粒能有效地抑制转染细胞MDR1mRNA ,从而提高肿瘤细胞的药物敏感性。
Objective To construct a short hairpin RNA (shRNA) expression vector expressing MDR1 in mammalian cells and investigate its inhibitory effect on the MDR1 mRNA expression in drug-resistant hepatocellular carcinoma cells and its anti-tumor drug resistance. Methods Two polynucleotide sequences designed by MDR1 mRNA in Genbank were annealed to form double-stranded DNA. The double-stranded DNA was ligated with PGE 1 vector to construct pshRNA MDR1 recombinant plasmid. The hepatocellular carcinoma cell line BEL 74 0 2 / ADM was stained with RT-PCR and the expression of MDR1 mRNA was detected by RT-PCR. The half inhibitory concentration (IC50) of doxorubicin on cells was detected by MTT assay. Results PCR and DNA sequencing confirmed that the expression plasmid was successfully constructed and the expression of BEL 74 0 2 / ADMMDR1 mRNA was significantly inhibited. The resistance index to doxorubicin was 17.5-fold (2 99.2 / 17.1) lower than that of control. Conclusion The constructed pshRNA MDR1 expression plasmid can effectively inhibit the MDR1 mRNA transfection, thereby enhancing the drug sensitivity of tumor cells.