猪骨髓间充质干细胞分离培养及生物学特性  

Isolation,culture and biological properties analysis of mesenchymal stem cells from porcine bone marrow

作　　者：高健[1] 梁桂金[1] 朴英姬[1] 李方正[1] 张文平[1] 宋学雄[1] 

机构地区：[1]青岛农业大学动物科技学院,山东青岛266109

出　　处：《中国兽医学报》2013年第5期788-794,共7页Chinese Journal of Veterinary Science

基　　金：国家转基因生物新品种培育重大专项课题(2009ZX08006-007B)

摘　　要：采集健康猪股骨骨髓,利用percoll梯度离心法分离单个核细胞,进行体外原代和传代培养,并用不同代数细胞进行了细胞生长能力、膜表面抗原(CD105、CD90、CD45)及诱导分化能力的检测。结果显示分离培养的单个核细胞贴壁生长,形态呈成纤维细胞样及涡旋状克隆团;细胞传代至第17代生长状况仍然良好;用F 3代细胞进行膜表面抗原标记显示,CD90和CD105阳性表达率分别为(97.4±1.8)%和(99.6±0.9)%,而CD45阳性表达率仅为(1.8±0.55)%,证实这些细胞具有猪骨髓间充质干细胞(Mesenchymal stem Cells,MSCs)表面抗原;经地塞米松、维生素C和β-磷酸甘油等诱导F 3代细胞,21d后出现钙物质沉积细胞群,用茜素红和Von kossa染色呈阳性,证实这些细胞已分化形成成骨细胞;经地塞米松、IBMX、胰岛素及吲哚美辛等诱导F 3代细胞,21d后细胞质出现脂滴样结构,用油红O染色呈阳性,证实已分化形成成脂细胞;冷冻-解冻细胞的膜表面抗原及多能分化能力与未冻存细胞的检测结果基本一致。结果证实,从猪骨髓中分离培养及经传代扩增获得的贴壁细胞是纯化的MSCs。To isolate and culture, and identify the mesenchymal stem bone marrow for laying the foundation for directional differentiation transgenic donor somatic cell. The bone marrow was collected from mononuclear cells by percoll gradient centrifugation. After primary a lar cells in vitro ,the cell growth curve and the membrane antigen （C differentiation ability were evaluated with several different generati（ that the morphology of isolated mononucle ning groups after primary cultured, and the the F17 generation. The labeled membrane cells （MSCs） from porcine of pig MSCs and providing and isolated the monocuar cells exhibited-fibroblast-like cells and vortex clo- cell growth was still well when the cell subcuhured to surface antigen of CD90 and CD105 positive cell were （97.4±1. 8）% and （99. 6±0. 9）% respectively, but the CD45 positive cell was only （1. 8±0.55）% in F3 generation cell, indicating that these cells have MSCs surface antigen. When the cells were induced in vitro by dexamethasone, L-ascorbicacid-2-phosphate and [3-glycerophos- phate,calcium material deposition cell group was displayed at 21 d post culture, and showed positive when stained by alizarin red and Von kossa,indicating that these cells were differentiated to osteoblasts. When the cells were induced in vitro by dexamethasone, IBMX, insulin and indometacin, lipid drops structure appeared in cytoplasm and showed positive when stained by Oil red O at 21 d, indicating that these cells were differentiated to adipocytes. The detection results of Frozen-thawed cells membrane antigen and multiple differentiation potential were the same as the non-Frozen cells. The research demonstrated that the adherent cells from isolated and cultured of porcine bone marrow were the purified MSCs.

关 键 词：猪 骨髓间充质干细胞 膜表面抗原 诱导分化 

分 类 号：S828.89[农业科学—畜牧学] S852.1[农业科学—畜牧兽医]