大鼠骨髓间质干细胞体外分离培养及生物学特性研究

Isolation,Culture and Basic Biological Properties of Rat Bone Marrow Mesenchymal Stem Cells in Vitro

目的 :建立大鼠骨髓间质干细胞 (mesenchymalstemcells ,MSCs)体外分离培养方法 ,并对大鼠MSCs的生物学特性进行研究。方法 :分离 5～ 8周龄的大鼠股骨骨髓进行体外培养 ,取 5代以后的MSCs观察形态特征 ,绘制生长曲线 ,进行细胞化学染色 ,并用流式细胞仪检测其表面标志和进行细胞周期分析。结果 :大鼠MSCs细胞形态呈长梭形或多边形 ,细胞生长曲线呈S形 ,群体倍增时间为 30h ,细胞化学染色显示碱性磷酸酶 (ALP)、过氧化物酶 (POX)、苏丹黑B(SB)阴性 ,而α 醋酸萘酚酯酶 (α NAE)与糖原 (PAS)阳性 ,流式细胞仪检测结果显示CD2 9、CD4 4、CD90表达阳性 ,而CD34、CD38、CD4 5表达阴性 ;细胞周期分析显示 ,G0 /G1期 :79.4 8% ,G2 /M期 :6 .10 % ,S期 :14 .4 2 %。结论 :本方法分离纯化出的是MSCs,其在体外具有生长稳定 ,增殖较快的特点 ,是组织工程研究中良好的细胞来源 ,也是转基因研究优良的载体细胞。

Objective:To seek the way of isolating and culturing rat bone marrow mesenchymal stem cells(MSCs), and to investigate the basic biological characteristics of rat bone marrow MSCs. Methods: Bone marrow was collected from femur of 5～8 weeks old female rats and seeded in cell culture flask. the morphology of MSCs after passage 5 were analyzed. The growth curve and cytochemistry were observed. The surface antigens and cell cycle were analysed by flow cytometer(FCM). Results:MSCs had spindle and polygon shape . The growth curve likes 'S' shape. The double time of MSCs was about 30 hours. Cytochemistry evaluation showed that MSCs were homogeneously negative for ALP, POX, SB and positive for α-NAE, PAS. MSCs expressed CD29,CD44,CD90 and didn't express CD34,CD38,CD45.Cell cycle included G 0/G 1 phase:79.48%,G 2/M phase: 6.10%,S phase 14.42%. Conclusion: The MSCs from rat bone marrow showed stable, long-term growth and proliferated quickly. It can be served as optimal cell source for tissue engineering and for transgenic research.