实验动物与比较医学2012，Vol.32Issue(2)：105-110,6.DOI:10.3969/j.issn.1674-5817.2012.02.005
西藏小型猪骨髓间充质干细胞分离培养及慢病毒载体介导EGFP标记间充质干细胞
Isolation and Culture of Bone Marrow Mesenchymal Stem Cells (MSCs) of Tibetan miniature pig, and Lentivirus-mediated EGFP Gene Transfer into MSCs
摘要
Abstract
Objective To establish a simple and effective method used to isolate and purify bone marrow mesenchymal stem cells (BMSCs) of Tibetan miniature pigs, and further set up the lentivirus-mediated in vitro gene delivery system for BMSCs of Tibetan miniature pigs. Methods ① MSCs of Tibetan miniature pigs were initially isolated by density gradient centrifugation, followed by purifying MSCs using differential attachment method, and further passage 3 BMSCs of Tibetan miniature pigs were characterized by CD45, CD90 and CD 105.② According to the standard protocol from Invitrogen, lentiviruses carrying EGFP gene were produced, and then infected MSCs, followed by EGFP assay under fluorescent stereo microscope and fluorescence activated cell sorting (FACS) for determining the infecting efficiency. Results BMSCs of Tibetan miniature pigs obtained by the combined use of density gradient centrifugation and differential attachment method were shown to enjoy the characteristics of MSCs. Additionally, the BMSCs of Tibetan miniature pigs were successfully and efficiently infected by lentivirus supernatant, and the infecting efficiency was 84.1%. Conclusion High purified BMSCs of Tibetan miniature pigs can be obtained by the combined use of density gradient centrifugation and differential attachment method, and lentivirus-mediated in vitro gene delivery systems for BMSCs of Tibetan miniature pigs were successfully established.关键词
西藏小型猪/骨髓间充质干细胞(BMSCs)/慢病毒/增强型绿色荧光蛋白(EGFP)Key words
Tibetan miniature pigs/ Bone marrow mesenchymal stem cells (BMSCs)/ Lentivirus/ Enhanced green fluorescent protein (EGFP)分类
生物科学引用本文复制引用
唐华,顾为望,毕文浩,艾威,李德胜,潘光辉,史俊文,刘薇,黄威,肖东..西藏小型猪骨髓间充质干细胞分离培养及慢病毒载体介导EGFP标记间充质干细胞[J].实验动物与比较医学,2012,32(2):105-110,6.基金项目
广东省医学科学技术研究基金(A2007359)、南方医科大学优秀中青年科技人才库科研资助金和广州地区科学仪器协作共用网专用基金(2006176)、广东省科技计划项目(2009B060300009)、广东省科研条件建设项目(1021010200004)、广东省科技计划项目(2010A011200003)、广东省科技计划项目(2011B060300028) （A2007359）
