论文部分内容阅读
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.
In order to reverse the tolerance of tumor cells to various chemotherapeutic drugs mediated by tumor multidrug resistance gene (MDR1) product P-gp protein, a hammer that can cleave the GUC sequence of MDR1 mRNA codon 196 was designed and synthesized. Ribozlyme was cloned and directionally cloned into the BamH I site of the transcriptional viral vector pDOR-neo. After being packaged with PA317, the virus-encapsulated cells were infected with human hepatocellular carcinoma multidrug resistant cell line BEL-7402/DOX and screened with G418 for stability. The transformed cell line Northem Blot hybridization confirmed the high expression of the virus in the packaging cell PA317 and transformed BEL-7402/DOX cells. RT-PCR confirmed that the MDR1 mRNA in the transformed cells was significantly reduced or even not amplified compared to the untransformed cells. The expression of P-gp in transformed cells detected by flow cytometry was reduced to 8.2 to 14.6% compared with 93.4 to 97.5% of non-transformed cells. MTT assay confirmed that transformed cells regenerated with higher sensitivity to various chemotherapeutic drugs. The results show that retrovirus vectors expressing Ribozyme can effectively inhibit the expression and translation of MDR1 after transfection of hepatocellular carcinoma multidrug resistance cell BEL-7402/DOX, and reverse the multidrug resistance phenotypes of drug-resistant tumor cells. .