重组腺病毒介导MRP反义RNA逆转人肝癌细胞多药耐药表型的体外实验研究

目的探讨重组腺病毒介导的多药耐药相关蛋白（MRP）反义RNA对阿霉素诱导的人肝癌耐药细胞株SMMC-7721／ADM多药耐药表型的体外逆转作用。方法应用自行构建的携带反义MRP的重组腺病毒（Ad—Asmrp）,在体外转染人肝癌耐药细胞,测定转染后细胞对阿霉素（ADM）及柔红霉素（DNR）的半数致死量（IC50）并计算耐药倍数（RF值）;在转染后不同时间点用流式细胞仪动态测定细胞对DNR摄取的变化及细胞膜表面P。。表达、并用RT—PCR反映细胞MRP之mRNA水平的变化。结果携带MRP反义RNA的重组腺病毒转染后的人肝癌耐药细胞对ADM、DNR的IC50分别为0．487μg／ml、0．328μg／ml,RF值分别为97．4、109．3,RF值分别下降36．8和35．4。在转染后24h,MRPmRNA水平开始下降并呈持续下降趋势（P〈0．01）,48h后伴有P190蛋白表达的持续下降（P〈0．01）,二者趋势一致。转染后48h,经流式细胞仪测得细胞内DNR浓度明显上升（P〈0．01）。结论重组腺病毒介导的MRP反义RNA可有效封闭MRP基因表达,在体外实验中能增加人肝癌耐药细胞株对化疗药物的敏感性,对肝癌耐药细胞的MDR表型有较好的逆转作用。

Reversal of the multidrug resistance in human hepatocellular carcinoma cells by recombinant adenoviral delivery of the MRP antisense RNA in vitro

OBJECTIVE: To explore the effect of multidrug resistance-associated protein (MRP) antisense RNA mediated by recombinant adenoviruses on reversal of the multidrug resistance in human drug-resistant hepatocellular carcinoma (HCC) cells. METHODS: The HCC cells were first treated in vitro by the recombinant adenoviruses containing antisense MRP. Chemosensitivity of all of the cells to ADM and DNR was determined by MTT assay. The levels of the MRP mRNA and its product P190 were measured by RT-PCR and flow cytometry (FCM) respectively according to the time after transfection. The accumulation of the DNR in these cells was analyzed by FCM simultaneously. RESULTS: IC50 of the cells infected by Ad-Asmrp to ADM and DNR was 0.487 microg/ml and 0.328 microg/ml respectively, with the factors of resistance (RF) decreased by 36.8 and 35.4 respectively (P < 0.05). Continuous decrease in levels of MRP mRNA and P190 was observed 24 h and 48 h after infection respectively (P < 0.01). The intracellular DNR accumulation was increased simultaneously in the Ad-Asmrp transfectants (P < 0.001). CONCLUSIONS: MRP antisense RNA mediated by recombinant adenoviruses could increase chemosensitivity and partially reverse the MDR in HCC cells in vitro by inhibit expression of MRP gene. The study would provide experimental basis for the reversal of MDR in HCC by antisense RNA technology mediated by recombinant adenoviruses.