摘要
目的 利用核酶技术切割肿瘤多药耐药基因 (MDR1) ,逆转肿瘤抗药性。方法 按照“锤头结构”模型设计、合成了核酶 196MDR1(196RZ) ,并定向克隆入包含RNA多聚酶III启动子的逆转录病毒载体中。通过脂质体介导 ,将抗mdr1核酶表达质粒 (N2A +tRNAimet iMDR1 Rz)导入人肝癌耐药细胞株HepG2。分别采用RT PCR、荧光PCR、蛋白质印迹分析 (westernblot)、流式细胞仪检测术、罗丹明聚集及MTT方法 ,观察细胞的RZ、mdr1mRNA、P糖蛋白 (Pgp)表达和对化疗药物敏感性的变化。结果 经抗MDR1核酶处理的耐药HepG2细胞 ,RZ稳定表达 ,MDR1mRNA、Pgp明显降低 ,细胞内罗丹明聚集 ,对阿霉素的敏感性提高 2 0 0倍。结论 针对多药耐药基因mdr1的核酶可明显降解mdr1mRNA ,抑制Pgp的表达 ,具有强大的逆转肝癌耐药细胞HepG2多药耐药的作用。
Objective To reverse multidrug resistance(MDR) of HepG2 by anti-MDR1 hammerhead ribozyme. Methods We developed an anti-MDR1 hammerhead ribozyme and delivered it to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase III promoter. We detected the expression of MDR1/Pgp and Rz in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-tranduced cells by real-time RT-PCR, semi-quantitative RT-PCR and western blot methods. Moreover, MTT assay was tested to detect sensitivity of these ribozyme-tranduced cells, and Rhodamine123 (Rh123) applied to test the function of Pgp. Results The Rz-tranduced HepG2 cells became doxorubicin-sensitive, concomitant with the decreases in MDR1 expression, P-gp amounts and efflux pump function. Conclusions The approaches using either retrovirus or liposome-mediated transfer of anti-MDR1 ribozyme may be selectively applicable to the treatment of MDR cells.
出处
《中华外科杂志》
CAS
CSCD
北大核心
2004年第7期424-427,共4页
Chinese Journal of Surgery
基金
卫生部 2 0 0 1~ 2 0 0 3年临床学科重点项目
