人耐药白血病细胞多药耐药基因-1小干扰RNA的构建及功能研究  

Construction of Multidrug Resistance Gene-1 shRNA Vector and Research of Its Function for Human Resistance Leukemia Cells

作　　者：格根通拉嘎[1] 马玉珍[2] 石玉涛[2] 张继英[1] 刘洋[1] 扈廷茂[1] 

机构地区：[1]内蒙古大学,内蒙古呼和浩特010021 [2]内蒙古自治区医院,内蒙古呼和浩特010017

出　　处：《现代生物医学进展》2009年第19期3647-3650,共4页Progress in Modern Biomedicine

基　　金：内蒙古自然科学基金(200508010912);内蒙古卫生厅医疗卫生科研项目(2006004)

摘　　要：目的:构建人耐药白血病细胞多药耐药基因-1小干扰RNA并研究其功能。方法:人工合成编码mdr1小发夹状双链RNA的DNA片段,与pSilencer4.1-CMV质粒连接构建RNAi真核表达载体,采用脂质体介导法转染人耐药白血病细胞K562/A,经潮霉素B筛选转基因阳性克隆细胞,RT-PCR和Western Blotting检测转基因细胞中mdr1基因的表达量,MTT法检测转基因细胞对阿霉素的敏感性。结果:RT-PCR结果显示,与未转基因组和阴性对照组比较,RNA干扰组mdr1基因在mRNA水平上表达量降低43.55%;Western Blotting检测显示,RNA干扰组mdr1基因在蛋白水平上表达量降低69.46%;MTT法检测显示mdr1干扰细胞对化疗药物柔红霉素的敏感性提高23倍。结论:mdr1小发夹状RNA可显著抑制K562/A细胞中的mdr1基因的表达,提高白血病细胞对化疗药物的敏感性,对白血病多药耐药性的逆转和白血病的治疗具有重要理论和实际意义。Objective：Construction of Multidrug resistance gene-1 shRNA vector and research of its function for human resistance leukemia cells.Methods： Artificial synthesized DNA oligonucleotide encoding the small hairpin RNA of mdr1 gene was recombined into pSilencer4.1-CMV.The expression vector obtained eukaryotic RNAi was introduced into K562/A cells with LipofectamineTM2000,and positive colonies were screened by Hygromycin B;The expression of mdr1 in K562/A cells before and after transfection were detected by RT-PCR, Western Blotting, and MTT method. Results： The mRNA expression of mdrl in K562/A group was decreased by 43.55% with control group by the above small hairpin RNA, and the protein expression was decreased by 69.46%. The mulfidrug reversable resistance was 23 times by MTT method. Conclusions： The specific small hairpin RNA could inhibit the expression of mdrl gene. This study of multidrug reversible resistance and treatment of leukemia has theoretical and practical significance.

关 键 词：RNA干扰 SHRNA mdr1 K562/A 

分 类 号：R733.7[医药卫生—肿瘤]