【摘要】 [目的]探讨成人骨髓间充质干细胞分离、纯化、培养及鉴定的方法,观察其成骨分化过程中Runx2基因的动态表达以及生物学特性。[方法]取自人股骨近端骨髓标本,利用联合密度梯度离心和差异贴壁法分离骨髓间充质干细胞,体外扩增和传代培养,流式细胞仪检测细胞表面标记,诱导向成骨细胞分化,并采用RT-PCR和Western blot方法检测Runx2的动态表达。[结果]原代和传代细胞呈纺锤状外观,生长增殖能力良好,骨髓间充质干细胞的生长曲呈成"S"形,细胞表面标记物CD90阳性表达,CD34和CD45阴性表达。经定向诱导分化后,细胞分别呈现成骨细胞的表型特征,随着诱导时间的增加,Runx2的表达也明显增加,与对照组相比有统计学差异(P<0.05)。[结论]该方法能从人骨髓中高效分离和扩增MSCs,生物学性状稳定,具有成骨分化潜能,为骨组织工程提供理想的种子细胞,同时证实Runx2在成骨分化中起到重要的调控作用。更多还原

【Abstract】 [Objective] The purposes of this study were to establish a method for effective isolation of human bone marrow- derived mesenchymal stem cells( MSCs),to verify the morphology,cell surface markers,and osteogenic differentiation potential of these cells,as well as to analyze the dynamic expression of Runx2 in the process of osteogenic differentiation. [Methods]MSCs were isolated from human bone marrow using a combination of density gradient centrifugation and different adherent time method. The morphology and growth characteristics of the MSCs were examined using phase contrast microscopy. The presence of cell surface markers CD90,CD34,and CD45 on the MSCs was analyzed using flow cytometry. Glycerophosphate,L- ascorbic acid,and dexamethasone were used to induce the in vitro differentiation of MSCs into osteoblasts. Real time RT- PCR and western blot were performed to detect the expression of the Runx2 gene in the process of osteogenic differentiation. [Results] The MSCs were spindle- shaped and showed active proliferation in primary and passage cultures. Among the cell surface markers,the MSCs were positive for CD90 and negative for CD34 and CD45. The cells were successfully induced to differentiate into osteoblasts,and the expression of Runx2 during the osteogenic differentiation process gradually increased. [Conclusion] This method is successful for the isolation and multiplication of MSCs as seed cells from human bone marrow,which can be verified by the cell morphology and osteogenic differentiation potential. Runx2 plays a key role in regulating the process of osteogenic differentiation of MSCs.更多还原