人骨髓间充质干细胞分离培养及体外诱导分化为成骨细胞

Isolation and culture of human bone marrow mesenchymal stem cells and induction of its differentiating into osteoblasts in vitro

[目的]建立人骨髓来源的间充质干细胞(hBMSCs)分离、培养及传代的方法,观察成hBMSCs体外成骨潜能。[方法]采用全骨髓贴壁筛选法分离培养hBMSCs,流式细胞仪检测细胞表型;所得细胞第3代用含100 nmol/L地塞米松、5mmol/Lβ-甘油磷酸钠,50μg/ml抗坏血酸的条件培养基进行骨诱导,茜素红染色鉴定。[结果]分离培养的细胞流式细胞术检测显示CD44、CD105阳性,而CD34、CD45阴性,符合MSCs特征;hBMSCs经骨诱导后可形成钙结节,茜素红染色显示阳性。[结论]全骨髓贴壁筛选法可分离获得高纯度的hBMSCs,其在体外具有成骨潜能。

[ Objective] To establish the experimental method for isolating and culturing human bone -derived mesenchymal stem cells （hBMSCs） and to investigate their osteogenic potential in vitro. [ Methods] hBMSCs were isolated and expanded in culture by bone marrow direct plating method and identified by flow cytometry method to detect cell phenotype. For osteogenic differentiation, the third passage hBMSCs were grown in the presence of osteogenic supplements （100 nmol/L dexamethasone, 5 mmol/Lβ- sodium glycerophosphate, 50 μg/ml ascorbic acid） . Calcium deposition was detected using alizarin red stain. [Resets] The signs of hBMSCs with positive CD44, CD105 and negative CD34, CD45 were detected by the flow cytometzy, and that was consistent with the characteristics of MSCs. hBMSCs were cuhured in medium and supplemented with components for osteogenic differentiation. Red - stained calcium deposits were detected in differentiated cultured hBMSCs. [ Condusion] Highly purified hBMSCs can be obtained by bone marrow direct plating method, hBMSCs possess osteogenic potential in vitro. Key words： mesenchymal stem cell, osteoinduction, osteoblasts