成人骨髓间充质干细胞体外培养、鉴定与成骨诱导

摘要

背景：骨髓间充质干细胞具有自我增殖和多向分化潜能，然而间充质干细胞在骨髓中含量极少，体外纯化、扩增与成骨诱导是进行骨组织工程研究的关键。　　目的：拟建立一套简便可靠的成人骨髓间充质干细胞体外培养及鉴定体系，并定向诱导其向成骨细胞分化。方法：抽取髂前上棘处骨髓，采用密度梯度离心法与贴壁筛选法相结合，对骨髓间充质干细胞分离、纯化、培养并扩增。将地塞米松、β-甘油磷酸钠、维生素C配制成骨诱导液，将细胞分为成骨诱导组和空白对照组进行观察。结果与结论：成人骨髓间充质干细胞呈典型的长梭形、纺锤状。8-11 d细胞进入快速增殖期，生长曲线呈S型，CD44、CD90均呈阳性表达，而 CD34、CD45呈阴性表达。碱性磷酸酶活性随培养时间而增高，并在第12天达到高峰，成骨诱导组各时间点碱性磷酸酶活性均高于空白对照组。说明实验建立的成人骨髓间充质干细胞体外培养、鉴定及诱导成骨细胞分化体系可以获得纯度较高、成骨分化能力较好的间充质干细胞。%BACKGROUND:Bone marrow mesenchymal stem cells have the potential of self-proliferation and multi-directional differentiation, while mesenchymal stem cells are few in adult bone marrow. In vitro purification, amplification and osteoinduction are very important for the research of bone tissue engineering. OBJECTIVE:To establish a simple and reliable in vitro cultivation and identification system of adult bone marrow mesenchymal stem cells, and to induce the mesenchymal stem cells to differentiate into osteoblasts. METHODS:Bone marrow were extracted from adult anterior superior iliac, the density gradient centrifugation and adhesion method were used to isolate, purify, culture and amplify the bone marrow mesenchymal stem cells. Osteogenic medium was prepared by mixing appropriate amount of dexamethasone,β-glycerophosphate and ascorbic acid C. The cells were divided into osteoinduction group and blank control group for observation. RESULTS AND CONCLUSION:Adult bone marrow mesenchymal stem cells were in typical long spindle-shape. The cells grew into rapid proliferation phase at 8-11 days and the growth curve was S-shape. CD44 and CD90 were in positive expression, while CD34 and CD45 were negative. The alkaline phosphatase activity was increased with culturing time prolonging, and reached the summit at the 12th day. The alkaline phosphatase activities of osteoinduction group were higher than those in the blank control group at different time points. These results suggested that in vitro cultivation, identification and osteoinduction system could obtain mesenchymal stem cells with high purity and good osteogenic differentiation capacity.