骨髓间充质干细胞体外分离培养及其生物学特性的初步研究  

Primary study of isolation and culture in vitro of adult human bone marrow-derived mesenchymal stem cells and their biological properties.

作　　者：吴涛[1] 白海[1] 王存邦[1] 路继红[1] 欧剑锋[1] 王茜[1] 

机构地区：[1]兰州军区兰州总医院全军血液病中心,兰州730050

出　　处：《中国综合临床》2007年第8期705-707,共3页Clinical Medicine of China

基　　金：兰州军区医学科研基金资助项目(LXH-2005013)

摘　　要：目的建立成人骨髓间充质干细胞(MSCs)体外培养和扩增的方法,探讨其生物学特性,建立稳定的MSCs体外培养扩增体系。方法取正常成人骨髓5 ml,用Percoll分离液(密度1.073 g/ml)经密度梯度离心法分离得到单个核细胞,以2×10~5个细胞/cm^2的密度接种于含10%新生牛血清的LG-DMEM培养液。经培养、扩增后,应用倒置相差显微镜观察其形态,MTT法测定生长曲线,流式细胞仪行细胞表面抗原及细胞周期的检测,并在透射电镜下观察其超微结构。结果培养扩增获取的成人骨髓MSCs形态均一,为梭形或纺锤形的成纤维细胞样外观,生长曲线示其增殖能力强。流式细胞仪检测显示有90%以上的细胞处于G0/G1期,表面标记物中CD44表达阳性,而造血细胞表面标志CD3、CD4、CD7、CD13、CD14、CD15、CD19、CD22、CD33、CD34、CD45和与移植排斥发生密切相关的HLA-DR表达阴性。超微结构显示细胞内有丰富的粗面内质网,细胞器发达。结论所建立的分离和培养方法可获取骨髓粘附细胞中一组独特的细胞群,具有MSCs的生物学特性,可作为组织工程中的种子细胞。Objective To establish a method of culture and amplification of adult human bone marrow-derived mesenchymal stem cells （ MSCs ） in vitro and to explore their biological properties. Methods Mononuclear cells were obtained from 5 ml adult human bone marrow by density gradient centrifugation with Percoll solution. Adult human MSCs were cultured in Dulbecco＇s Modified Eagle＇s Medium with low glucose （ LG-DMEM） containing 10% fetal calf serum at a density of 2 × 10^5 cell/cm^2. The morphocytology was observed under phase-contrast microscope. The cell growth was measured by MTT method. The flow cytometer was performed to examine the expression of cell surface molecules and cell cycle. The ultrastructure of MSCs was observed under transmission electron microscope. Results The MSCs harvested were homogenous population and exhibited a spindle-shaped fibroblastic morphology. The cell growth curve showed that MSCs had a strong ability of proliferation. The cells were positive for CD44,while negative for hematopeietic cell surface marker such as CD3,CD4,CD7,CD13,CD14,CD15,CD19,CD22,CD33, CD34,CD45 and HLA-DR,which was closely related to graft versus host disease. More than 90% cells of MSCs were found at G0/G1 phase. The ultrastructure of MSCs indicated that there were plenty of rough endoplasmic reticula. Condusion The method for culture and amplification of adult human bone marrow-derived MSCs in vitro is successfully established. MSCs is a homogenous population that has unique growth phenotype and multilineage potential which may act as seed cell of tissue engineering.

关 键 词：骨髓间充质干细胞 细胞培养 流式细胞术 组织工程 

分 类 号：R551.3[医药卫生—血液循环系统疾病]