稳定过表达人SIRT1基因的HEK293细胞系的建立  

Establishment of stable over-expressing human SIRT1 HEK293 cell line

作　　者：林蓉[1] 张凯帆[1] 王维蓉[1] 林琴琴[1] 杨莉娜[1] 任峰[1] 张建丰[1] 

机构地区：[1]西安交通大学医学院药理系,710061

出　　处：《实用医学杂志》2013年第1期18-20,共3页The Journal of Practical Medicine

基　　金：国家自然科学基金资助项目(编号:81072643)

摘　　要：目的:构建人沉默信息调节因子2同源蛋白1(SIRT1)基因的真核表达载体,建立稳定过表达人SIRT1的HEK293细胞系。方法:将含有SIRT1的克隆载体pCR Ⅱ-TOPO-SIRT1双酶切后,连接至真核表达载体pcDNA3.1(+)中,连接产物经酶切鉴定后进行测序。构建的重组真核表达质粒pcDNA3.1(+)-SIRT1转染HEK293细胞,G418进行筛选,Real-time PCR和Western Blot分别从mRNA和蛋白水平检测SIRT1的表达。结果:酶切分析和测序结果证实,SIRT1基因成功插入真核表达质粒pcDNA3.1(+)中。Real-time PCR和Western Blot结果显示稳定转染pc DNA3.1(+)-SIRT1的HEK293细胞SIRT1的表达水平明显高于未转染细胞(P﹤0.01)。结论:成功构建了真核表达载体pcDNA3.1(+)-SIRT1,并建立了稳定过表达SIRT1基因的HEK293细胞系,为进一步研究SIRT1基因在心血管疾病中的作用及其机制奠定了基础。Objective To construct a plasmid expressing human SIRT1 and to establish HEK293 cell line with stable over-expressing human SIRT1. Methods Full length of human SIRTI gene cDNA was ligated into an expressing vector pcDNA3.1 （＋）. After confirmed by restriction analysis and sequencing, the recombinant plasmid was transfected into HEK293 cells mediated with liposome. G418-resistant clones of HEK293 cells were then detected by real-time PCR and Western blot for the expression level of SIRT1. Results The accuracy of constructed and selected plasmids was confirmed by restriction enzymatic analyses and DNA sequencing. As compared with untransfected HEK293 cells, the levels of SIRTI mRNA and SIRT1 protein of transfected HEK293 cells were significantly increased （P 〈 0.01）. Conclusions The pcDNA3.1 （＋）-SIRT1 plasmid is successfully constructed. HEK293 cell line with stable over-expressing SIRT1 is established, which provides a useful tool for further study on the effect of SIRT1 on cardiovascular diseases.

关 键 词：心血管疾病 SIRT1 稳定过表达 HEK293细胞 

分 类 号：R363[医药卫生—病理学]