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

目的建立一种简便、有效的西藏小型猪骨髓间充质干细胞（BMSCs）体外分离培养及纯化方法，并基于BMSCs建立慢病毒介导的外源基因EGFP体外投递系统。方法密度梯度离心法分离西藏小型猪BMSCs，差速贴壁法不断纯化，流式细胞仪检测第3代BMSCsCD45、CD90和CD105表达；此外，用携带EGFP基因的慢病毒转染BMSCs以获得EGFP标记的BMSCs，倒置荧光显微镜下观察感染情况及流式细胞术检测感染效率。结果①密度梯度分离法结合差速贴壁法所获得的细胞经鉴定符合MSCs的特性。②借助慢病毒高效率将EGFP导入BMSCs，感染效率为84．1％。结论利用密度梯度分离法结合差速贴壁法可获得高纯度的BMSCs，基于BMSCs建立了慢病毒介导的外源基因体外投递系统。

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 cen/rifugation, followed by purifying MSCs using differential attachment method, and further passage 3 BMSCs of Tibetan miniature pigs were characterized by CD45, CD90 and CD105. （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 gerle delivery systems for BMSCs of Tibetan miniature pigs were successfully established.