摘要
目的:重组构建抑制多药耐药MDR1基因表达的shRNA真核表达载体,研究其对肝癌耐药细胞MDR1基因表达的抑制作用,以及对P-糖蛋白(P-gp)表达和功能的抑制作用.方法:根据MDR1基因序列,设计两段21个碱基的MDR1特异性靶序列作为shRNA目标序列(sh-MDR1-1,sh-MDR1-2),重组构建psh-MDR1表达质粒.采用脂质体介导转染肝癌耐药细胞SMMC-7721/R,用MTT法测定细胞对化疗药物阿霉素(ADM)的敏感性,流式细胞仪检测细胞膜表面P-糖蛋白(P-gp)表达和细胞内Rhdaming123(Rh123)的潴留.结果:PCR和DNA测序证实了psh-MDR1-1和psh-MDR1-2重组质粒的成功构建,均能有效地抑制细胞P-gp表达;转染细胞后均能够一定程度地恢复耐药细胞对化疗药物ADM敏感性,P-gp表达水平明显降低,细胞内的Rh123稳态积累量均明显增高;第1条序列更能有效的抑制MDR1基因表达.结论:体外完成shRNA RNAi载体的构建,能有效地逆转肝癌耐药细胞MDR1基因过度表达,抑制P-gp介导的多药耐药.从DNA载体产生的shRNA在肝癌耐药细胞内能诱导RNAi,能够序列特异性地抑制MDR1基因表达.
AIM ： To construct a recombinant plasmid generating short hairpin RNA（shRNA） containing multi-drug resistance gene MDR1 segment in mammalian cells and to investigate its suppression on MDR1 mRNA and P-glycoprotein （P-gp） expressions in hepatocellular carcinoma cells. METHODS ： Based on the design of two fragments of oligonucleotides for shRNA expression which targeted MDR1 gene （sh-MDRI-1, sh-MDR1-2）, RNAi plasmids （psh-MDR1）were constructed and transfected into SMMC-7721/ R cells by LyoVecTM. Drug sensitivity was measured by MTT assay, P-gp expression and intracellular Rh123 accumulation were determined by flow cytometry （FCM）. RESULTS ： Recombinant psh-MDR1 vectors were identified by PCR and confirmed by sequencing analysis. They could suppress P-gp expression in hepatocellular carcinoma cells. Drug sensitivity was increased significantly after pshRNA-MDR1 was transfected in SMMC-7721/R cells. The level of P-gp expression was reduced significantly. The intracellular accumulation of Rh123 was increased greatly after psh-MDR1 treatment in SMMC-7721/R cells. The sh-MDRI-1 was more effective in the suppression of MDR1. CONCLUSION： Recombinant psh-MDR1 vector can suppress the expressions of MDR1 mRNA and P-gp in SMMC-7721/R cells. The inhibitory effect of the shRNA generated from the DNA vector is highly related to the target sites and to the different cell lines.
出处
《第四军医大学学报》
北大核心
2007年第18期1639-1642,共4页
Journal of the Fourth Military Medical University
基金
湖北省教育厅资助(2004D006)
关键词
RNA干扰
抗药性
多药
短发夹RNA
重组
遗传
质粒
基因表达
RNA interference
drug resistance, multiple
short hairpin RNA
recombination, genetic
plasmids
gene expression
