大鼠骨髓间充质干细胞体外培养条件的优化及生物学特性  

Optimization of culturing rat bone marrow mesenchymal stem cells and their biological behaviors

作　　者：赵玉金[1] 李瑶琛[2] 赖洁娟[3] 王海燕[1] 黄苏娜[1] 蒋建新[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所第四研究室、创伤、烧伤与复合伤国家重点实验室,重庆400042 [2]第三军医大学西南医院全军眼科中心,重庆400038 [3]第三军医大学西南医院全军肝胆外科研究所,重庆400038

出　　处：《第三军医大学学报》2010年第20期2163-2167,共5页Journal of Third Military Medical University

摘　　要：目的建立一种新的高效体外骨髓间充质干细胞纯化培养方法,并对所获得细胞进行生物学行为研究。方法采用全骨髓培养获得原代骨髓间充质干细胞,经体外成脂、成骨诱导分化并检测细胞表面分子表达对所获得干细胞进行鉴定,传代后低密度接种(3×103/cm2)结合机械擦除对所培养细胞进行纯化,并对未纯化及纯化细胞分别进行集落形成单位实验、生长曲线及细胞周期分析来研究其生物学特性。结果全骨髓贴壁法可以成功培养出骨髓间充质干细胞,细胞呈现干细胞特性生长;可在体外成功地进行成脂、成骨分化;阴性表面标记分子CD34、CD45表达率均小于2%,阳性表面标记CD73、CD90表达率均高于95%。经纯化后的细胞具有更优的生物学行为,表现在:克隆形成率达到(28.80±5.00)%(P<0.01);培养12d后细胞数量增加(26.18±4.80)倍(P<0.05);处于S+G2/M期的细胞比例达到(45.98±1.60)%(P<0.01)。结论低密度接种结合机械擦除方法可以获得高纯度骨髓间充质干细胞,且该细胞具有更佳的生物学特性。Objective To establish a new and effective method of purifying rat bone marrow mesenchymal stem cells（BMSCs）in vitro and to study the biological behaviors of the cultured cells.MethodsBMSCs were cultured by whole bone marrow culture method.To identify BMSCs,the experiments of adipogenic and osteogenic differentiation were carried out and flow cytometry was employed to detect surface antigens.The cells were purified by planting in lower density（3×10^3/cm^2）and by erasing the peripheral large-and-flattened cells that surrounded the ＂colony＂.The biological behaviors of these cells such as colony-forming-unit test,growth curve,and cell cycle analysis were further compared to those planted in a higher density（3×10^4/cm^2）.ResultsBMSCs were successfully cultured by whole bone marrow method,and they could be induced into adipocytes and osteocytes in vitro.Flow cytometry demonstrated that the expressions of CD34 and CD45 were lower than 2%,and those of CD73 and CD90 were more than 95%.Furthermore,the purified cells showed stronger stemness features during their growth.The efficacy of CFU test was（28.80±5.00）%（P〈0.01）.The cell amount of those cultured for 12 d was increased by 26.18±4.80 times（P〈0.05）.The percentage of the cells at S＋G2/M phase was（45.98±1.60）%（P〈0.01）.ConclusionMore purified BMSCs are obtained by culturing cells in lower density combining with erasing peripheral large-and-flattened cells,and the cells have better biological properties.

关 键 词：骨髓间充质干细胞 细胞生物学行为 细胞培养 条件优化 

分 类 号：R329.33[医药卫生—人体解剖和组织胚胎学] R329.28[医药卫生—基础医学]