线粒体加工肽酶干扰RNA真核表达载体转染PC12细胞系的构建及鉴定  

Establishment and identification of PC12 cell line transfected by eukaryotic expression vectors of RNA interference specific for mitochondrial processing peptidases

作　　者：张艳梅[1] 马飞煜[2] 王芳[3] 王佳烈[1] 李兰根[1] 呼格吉乐[4] 

机构地区：[1]内蒙古自治区医院神经内科,内蒙古呼和浩特市010010 [2]汕头市中心医院,广东省汕头市515031 [3]江汉大学医学院,湖北省武汉市430056 [4]内蒙古医学院生化教研室,内蒙古呼和浩特市010010

出　　处：《国际神经病学神经外科学杂志》2010年第2期99-102,共4页Journal of International Neurology and Neurosurgery

基　　金：国家自然基金(30960520);内蒙古自然科学基金(20080404MS1114)

摘　　要：目的利用干扰RNA技术构建线粒体加工肽酶(MPPs)的干扰RNA真核表达载体,稳定转染大鼠PC12细胞来抑制MPPs基因的表达,为研究MPPs蛋白的功能提供实验基础。方法脂质体转染已经构建好的4个MPPs干扰RNA真核表达载体和阴性对照干扰RNA真核表达载体到大鼠PC12细胞株,G418筛选阳性转染细胞克隆,制备稳定转染5个MPPs干扰RNA真核表达载体的PC12细胞系。用RT-PCR检测MPPs基因表达。结果获得稳定转染5个MPPs干扰RNA真核表达载体的PC12细胞系,RT-PCR证实所有4个干扰组(siRNA1,siRNA2,siRNA3 and siRNA4)MPPs-mRNA表达均下调,分别达到28%、38%、26%、16%。结论成功建立稳定转染MPPs干扰RNA的PC12细胞系。通过RT-PCR等相关方法证实MPPs干扰RNA真核表达载体转染PC12细胞成功。Objective To provide the experimental basis for studying the function of mitochondrial processing peptidases（MMPs）protein,we transfected PC12 cells in mice to inhibit the expression of MMPs gene by constructing MPPs eukaryotic expression vector of interference RNA.Methods PC12 cell line was transfected with four MPPs RNAi vectors and a negative control vector via oligofectamine.Stable transfected PC12 cell line was obtained by G418 selection.The gene expression of MPPs was examined by RT-PCR.Results PC12 cell line with stably transfected 5 MPPs RNAi eukaryotic expression vectors was obtained.The expression of MPPs-mRNA was reduced by 28%,38%,26% and 16% in siRNA1,siRNA2,siRNA3 and siRNA4 vectors groups,respectively.Conclusions PC12 cell line stably transfected by MPPs RNAi eukaryotic expression vectors was established and was verified by RT-PCR.

关 键 词：干扰RNA 线粒体加工肽酶 转染 细胞系 大鼠 

分 类 号：R346[医药卫生—基础医学]