多药耐药基因反义寡核苷酸逆转肝癌细胞耐药的研究

Reversal of multidrug resistance gene MDRl and MRP of drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM with antisense phosphorothioate oligonucleotides

目的 观察反义硫代磷酸酯寡核昔酸(AsODN)联合逆转耐药肝癌细胞多药耐药基因1(MDR1)和多药耐药相关蛋白基因(MRP)的作用。 方法 用人工合成互补于MDR1基因及MRP基因的反义20聚硫代磷酸寡核苷酸,以脂质体作载体,转染入耐阿霉素(ADM)肝癌细胞SMMC-7721/ADM,四甲基偶氮唑蓝法测定细胞对化学疗法药物的敏感性,流式细胞仪分析细胞相对荧光强度,激光扫描共聚焦显微镜测定细胞内Rhdaming123(Rh123)及柔红霉素(DNR)潴留以反映蛋白质p170和p190功能。 结果 ASODN/MDR1+MRP联合转染SMMC-7721/ADM细胞,能更大程度增加细胞对ADM(47.8倍)和DNR(21.6倍)的敏感性。ASODN/MDR1+MRP联合转染SMMC-7721/ADM细胞,与单独任一种ASODN转染相比,对p170或p190表达的抑制并不增加(q值分别为3.23、3.24,P>0.05)。 结论 针对MDR1+MRP的ASODN联合转染SMMC-7721/ADM细胞,能更大程度逆转肝癌细胞的耐药性。

Objective To investigate the reversal effect of gene MDR1 and MRP with combinational antisense phosphorothioate oligonucleotide on Drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM. Methods SMMC-7721/ADM was transfected with synthetic antisense phosphorothioate oligonucleotides complementary to gene MDR1 and MRP mediated by Lipofectamine. Drug sensitivity was measured by MTT assay, Fluorescence intensity of cells was determined by flow cytometric analysis, RH123 and DNR retention was assayed by confocal scanning laser microscopy. Results ASODN of MDR1+MRP increased the sensitivity of SMMC-7721/ADM to chemotherapeutic drug more significantly than that any of MDR1 and MRP did separately. But they did not enhance the inhibition expression of protein of p190 or p170. Conclusion Drug-resistance could be reversed significantly when antisense phosphorothioate oligonucleotide of MDR1+MRP were transfected into drug-resistant human hepatocellular carcinoma cells SMMC-7721/ADM together.