为逆转肿瘤多药耐药基因(MDR1)产物P-gp蛋白所介导的肿瘤细胞对多种化疗药物的耐受性,设计合成了一种能切割MDR1 mRNA第196密码子GUC序列的锤头状核酶(Ribozlyme)并定向克隆于转录病毒载体pDOR-neo的BamH Ⅰ位点.经病毒包装细胞PA317包装后感染人肝癌多药耐药细胞株BEL-7402/DOX细胞,经G418筛选得到稳定的转化细胞株.Northem Blot杂交证实包装细胞PA317及转化的BEL-7402/DOX细胞中均有病毒的高表达,RT-PCR证实转化细胞中MDR1 mRNA与未转化细胞相比明显减少甚至不能扩增出来,流式细胞技术检测转化细胞P-gp的表达与非转化细胞的93.4～97.5%相比下降至8.2～14.6%.MTT法检测证实转化细胞对多种化疗药物重新产生较高的敏感性.结果表明,表达Ribozyme的逆转录病毒载体转化肝癌多药耐药细胞BEL-7402/DOX后能有效抑制MDR1的表达和翻译,使已产生耐药的肿瘤细胞的多药耐药表型发生逆转.

To reverse drug resistance mediated by the MDR1 gene product P-glycoprotein(P-gp) in tumor cells in a specific manner, a hammerhead ribozymes which can cleave the GUC sequence in codon 196 of MDR1 mRNA was designed and cloned into a recombinant retroviral vector pDOR-neo at BamH I restriction sites and packaged with packaging cell line PA317 cells.The viral supernatant was used to infect the multidrug-resistant human hepatocarcinoma cell line BEL-7402/DOX. After selection with G418,resistant colonies were obtained.Stable expression of retroviruses in both PA317 and infected BEL-7402/DOX cells was confirmed by Northern Blot hybridization. Down-regulation of P-gp and even of MDR1 mRNA was found in BEL-7402/DOX infected with ribozyme construct. The rate of BEL - 7402/DOX infected cell defected by flow cytometric analysis was 8.2 ~ 14.6% while in uninfected cell it was 93 .4 ~ 97.5% . The BEL-7402/ DOX cell infected with ribozyme construct was found back to drug-sensitivity to a series of drugs by MTT colorimetric assay . The results demonstrated that the recombinant retroviral vector expressing ribozyme transfecting human hepatocarcinoma BEL-7402/DOX could inhibit MDR1 gene expression and reverse tumor MDR phenotype back to drug-sensitive condition.