成人骨髓间充质干细胞的体外分离培养和生物学特性

Isolation, Cultivation and Biological Features of Adult Bone Marrow Mesenchymal Stem Cells in vitro

目的:建立一种成人骨髓间充质干细胞体外分离、培养和扩增的方法,并探讨其部分生物学活性及向脂肪细胞诱导分化的方法,并以此鉴定所获细胞。方法:采用1.073g/ml的Percoll分离液分离成人骨髓间充质干细胞。接种于纤维连接蛋白包被,含有表皮生长因子和血小板原性生长因子BB的2%胎牛血清培养液中,并利用其贴壁性进行纯化;观察其形态学变化及超微结构,流式细胞仪检测其表面标记物;采用10%胎牛血清和尼克酰胺诱导其向脂肪细胞的分化。结果:原代和传代培养均保持较强的增殖能力,超微结构显示了干细胞的幼稚特性,流式细胞仪检测示CD34、CD45、CD19、组织相容性抗原-DR阴性,CD44、CD10、CD29、CD13阳性。体外能够分化成为脂肪细胞,油红O染色阳性。结论:采用该方法骨髓间充质干细胞生长稳定,扩增较快;一定条件下能够定向分化成为脂肪细胞;我们所获得的细胞具有间充质干细胞的特性。

Objective:To establish a method for isolation, cultivation and expansion of adult bone marrow mesenchymal stem cells in vitro,and study their some biological features and a method for inducing their differentiation into adipocytes,which could be applied to identify what we cultured.Methods:MSCs were isolated from adult bone marrow by Percoll, cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% fetal bovine serum,and purified by their adhesiveness; the changes in cell morphology and the ultrastucture were observed,and cell surface markers were detected by Flow cytometry;MSCs were induced to differentiate into adipocyes by change to 10% fetal bovine serum and nicotinamide.Results:Both primary and passage MSCs have maintained highly proliferative capacity. The ultrastructure showed the young cell nature of stem cell.The cells were negative for CD34, CD45, CD19, HLA-DR,but positive for CD44,CD10, CD29,CD13.MSCs can be induced to differentiate into adipocytes that stained with Oil-red O.Conclusion:With our method, MSCs can be cultured stably,and expanded quickly; they can be induced to differentiate into adipocytes under given cultured conditions in vitro;and the cells we cultured have the same characters as MSCs.