兔骨髓间充质干细胞的体外分离培养和生物学特性观察  

The Culture Method and Biological Characteristics of Rabbit Mesenchymal Stem Cells Derived from Bone Marrow in Vitro

作　　者：张磊 张琦[2] 钱济先[2] 文艳华[2] 裘秀春[2] 马保安[2] 范清宇[2] 

机构地区：[1]解放军一一八医院,浙江温州325000 [2]第四军医大学唐都医院全军骨肿瘤研究所,陕西西安710038

出　　处：《现代生物医学进展》2010年第17期3239-3243,共5页Progress in Modern Biomedicine

摘　　要：目的:探讨兔骨髓间充质干细胞体外分离、培养和鉴定方法,观察其生物学特性。方法:采集兔股骨及胫骨骨髓组织,采用密度梯度离心法结合贴壁培养法体外分离、培养和扩增兔骨髓间充质干细胞,倒置相差显微镜观察细胞形态,绘制原代、第1、3、8代细胞生长曲线,流式细胞术检测细胞表面标志物,成骨和成脂肪诱导培养鉴定,观察细胞生物学特性。结果:培养的BMSCs呈纺锤形、长梭形,旋涡状排列、放射性生长,增值活跃。各代细胞生长曲线呈S型,细胞增值活跃。细胞表面标志物CD44分子阳性,CD34和CD45分子阴性。经成骨和成脂肪诱导后细胞碱性磷酸酶染色和油红O染色阳性。结论:成功建立了兔BMSCs体外分离、培养的有效方法,扩增的BMSCs仍保留多向分化潜能,是理想的组织工程种子细胞。Objective：To investigate the effective approach of isolation and cultivation of rabbit bone marrow mesenchymal stem cells（BMSCs）in Vitro and to observe their biological characteristics.Methods：Bone Marrow aspirate was harvested from femurs and tibias of the New Zealand white rabbits and BMSCs were isolated and cultured by using density gradient centrifugation and adherent culture ways.The morphological changes of the cells were observed under inverted phase contrast microscope and the growth curves of primary and passage 1,3,8 cells were drawn.The BMSCs were induced into osteogenetic and adipose cells and detected cell surface markers by flow cytometer.Results：By observing the morphology of the BMSCs,we found that the cells were fusiformate and the proliferation ability declined with time of the culture.The cells that were induced into osteogenetic cells were positive for the ALP staining and the cells were positive for the oil red O which were induced into adipose cells.What＇s more,CD44 expression was positive but CD34 and CD45 was negative.Conclusion：The combination of density gradient centrifugation and adhere culture is an effective method to isolate BMSCs from bone marrow aspirate and the cells were still reserved multi-directional differentiation potentiality which were cultured in Vitro.

关 键 词：骨髓间充质干细胞 细胞培养 细胞分化 组织工程 

分 类 号：Q95-3[生物学—动物学] R329.24[医药卫生—人体解剖和组织胚胎学]