成人间充质干细胞体外成骨的初步研究

目的 建立一种分离和培养成人骨髓来源的间充质干细胞（MSCs）的方法，观察成人MSCs体外成骨潜能。方法 用Percoll分离液分离出骨髓中的单个核细胞，并在含10％胎牛血清的低糖DMEM培养液中培养。通过传代培养扩增MSCs。为促进成人MSCs体外成骨性分化，第5传代培养时加入成骨性添加剂，培养第4、12天分别用流式细胞仪分析成人MSCs表面分子的表达，并用碱性磷酸酶组化染色和Von Kossa染色。结果 成人MSCs是骨髓黏附细胞中有相应细胞表面蛋白表达和形态均的一细胞群。成骨性添加剂可作用于传代培养的成人MSCs，表现为培养皿表面有相互连接的结节状聚合体、碱酶染色阳性细胞数量增多、Von Kossa染色可见钙化的基质沉积。结论 所建立的成人MSCs的分离和培养条件可分选出骨髓黏附细胞中一组独特的细胞群，成人MSCs具有体外成骨潜能。

A preliminary study of in vitro osteogenesis of culture expanded,bone marrow derived adult human mesenchymal stem cells

Objective　To establish a method of isolating and culturing adult human bone marrow-derived mesenchymal stem cells(MSCs) and to investigate their ex vivo osteogenic potential. Methods　Adult human MSCs derived from marrow were isolated from the Percoll fraction of mononuclear cells, cultured in Dulbecco's Modified Eagle's Medium with low glucose containing 10% fetal bovine serum, and proliferated through a process of subculturing. For in vitro osteogenic differentiation,adult hMSCs from the fifth passage were grown in the presence of osteogenic supplements (100 nmol/L dexamethasone, 10 mmol/L glycerophosphate,and 50 μmol/L vitamin C). On days 4 and 12 of culture, flow cytometric analysis was performed to examine the expression of cell surface molecules on adult hMSCs and samples were assayed for alkaline phosphatase histochemistry and for mineral deposition by Von Kossa staining respectively. Results　Adult human MSCs were a homogenous population of adherent bone marrow cells with a distinct cell surface protein expression and morphology. Passaged adult hMSCs responded to the osteogenic supplements by forming interconnected nodular aggregates on the surface of the dish, increasing A Pase-postive cells, and depositing a calcified matrix. Conclusion　The isolation and culture conditions established for adult hMSCs may select a distinct population of bone marrow-derived adherent cells. Adult human MSCs possess osteogenic potential in vitro.