ANXA1的克隆,表达及对树突状细胞转染的方法  

The Cloning,Expression of ANXA1 and a Method of Transfection in Dendritic Cells

作　　者：闵彦[1] 田野苹[1] 

机构地区：[1]第二军医大学免疫研究所,上海200433

出　　处：《药物生物技术》2009年第5期404-408,共5页Pharmaceutical Biotechnology

基　　金：2007年国家自然科学基金面上项目(30771991)

摘　　要：膜联蛋白1(ANXA1)是一种抗炎因子,但是ANXA1对单核细胞来源树突状细胞(monocyte-de-rived dendritic cells,MoDCs)功能成熟的影响还未见报道。首先以pShuttle-CMV为载体,通过一系列分子克隆技术,构建了ANXA1高表达质粒pShuttle-CMV-ANXA1。笔者将pShuttle-CMV-ANXA1转染细胞株293a,用western-blot法验证ANXA1高表达情况。结果表明,pShuttle-CMV-ANXA1可引起293a表达ANXA1增高;表明,pShuttle-CMV-ANXA1构建是成功的。然后,用脂质体转染试剂GenePorterⅡ将荧光对照质粒pShuttle-CMV-VNG-IRES转染TNF--αMoDCs。通过镜下荧光可知,GenePorterⅡ将对照质粒转染MoDCs的效率>80%;表明,GenePorterⅡ是一种较好的转染MoDCs的方法。用GenePorterⅡ将pShuttle-CMV及pShuttle-CMV-ANXA1转染TNF--αMoDCs,通过western-blot法检测ANXA1在TNF--αMoDCs中的高表达情况。结果表明,与pShuttle-CMV相比,pShuttle-CMV-ANXA1可引起TNF--αMoDCs高表达ANXA1;成功将pShut-tle-CMV-ANXA1转入MoDCs,并得到表达。GenePorterⅡ转染树突状细胞的新方法,简单高效,为进一步研究ANXA1对MoDCs的生物学功能奠定了基础。获得的高表达ANXA1的树突状细胞,可望在自身免疫病及移植排斥反应中得到应用。Although ANXA1 has been reported to work as an anti-inflammatory factor, its role on the maturation of monocye-derived dendritic cells （MoDCs） has not been examined. In this study, we constructed ANXA1 over-expression plasmid and investigated the method for transfecting MoDCs. Firstly, ANXA1 over-expressing plasmid was constructed by a series of molecular cloning technigues. Then, the over-expression plasmid was transfected into 293a cell line and the ANXA1 expression was detected by Western-Blot. Compared with control plasmid, ANXA1 expression was up-regulated in 293a cell line which was transfected with over-expression plasmid. The results suggested that ANXA1 over-expressing plasmid was constructed successfully. Secondly, the fluorescent control plasmid was transfected into MoDCs using transfection reagent, GenePorter Ⅱ. The transfection efficacy under fluorescent microscope was more than 80 %. The results suggestsed that GenePorter Ⅱ was a useful reagent for MoDCs transfection. Finally, the plasmids, pShuttle-CMV and pShuttle-CMV-ANXA1 were transfected into TNF-α- MoDCs and the ANXA1 expression was detected by Western-Blot. The results showed that compared with pShuttle-CMV, ANXA1 was up-regulated in TNF-α-MoDCs which was transfected with pShuttle- CMV-ANXA1. The results suggested that ANXA1 was successfully transfected into and over-expresssed in TNF-α-MoDCs. The easy and effective method to transfect dendritic cell provides a basis for the further biological study of ANXA1 on MoDCs. The dendritic cells over-expressing ANXA1 could be used in the treatment of autoimmune disease and transplant reiection.

关 键 词：膜联蛋白1 分子克隆 转染 树突状细胞 

分 类 号：R52[医药卫生—内科学] R446.61[医药卫生—临床医学]