shRNA介导RNAi载体构建及对肝癌耐药细胞MDR1基因表达抑制.docVIP

shRNA介导的RNAi载体构建及对肝癌耐药细胞MDR1基因表达的抑制 作者：秦维超 张有顺 周新 胡礼仪 【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. 【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 　　【Abstract】 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MDR1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MDR1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 site