密度梯度离心和贴壁法分离大鼠骨髓间充质干细胞增殖活性和成骨功能比较

目的　通过比较密度梯度离心和贴壁筛选两种方法所获MSCs的增殖活性和成骨功能,探索骨髓MSCs体外分离和纯化较好的方法,为MSCs进一步的试验研究提供依据。方法　应用密度梯度离心和贴壁筛选两种方法分离纯化骨髓MSCs ,并通过MTT法检测其增殖活性。用第三代MSCs进行骨向诱导,以观察两种方法所获MSCs是否有相似的骨向分化能力。结果　密度梯度离心法所获MSCs纯度高,杂质细胞少,增殖活性强,常表现为梭形和小多角形,7到1 0天内便可达到融合。而贴壁法所获MSCs纯度低,红细胞、破骨样细胞等杂质细胞较多,增殖速度相对较慢,且经数次传代仍有较多杂质细胞存在。经骨向诱导后,两种方法所获MSCs表现出相似的ALP活性和矿化结节形成能力。矿化结节数目在两组比较无显著性差异(P >0 .0 5 )。结论　密度梯度离心法是体外分离和纯化骨髓MSCs较好的方法,所获细胞增殖活性高。两种方法所获MSCs具有相似的骨向分化能力。

Comparison of proliferation ability and osteogenic potential of rat bone marrow mesenchymal stem cells isolated by two different methods

Objective To explore a practical method of isolation and purification,in vitro,of bone marrow MSCs through comparison of proliferation ability and osteogenic differentiation potential of the cells obtained by two different methods.Methods Density gradient centrifugation and plastic adherence methods were used for isolation of bone marrow MSCs from adult SD rats and the proliferation ability was analyzed by MTT examination. The MSCs were also undergone osteogenic differentiation by addition of osteogenic supplements to verify whether the MSCs obtained by the two methods had similar osteogenic differentiation potential or not.Results High purity of MSCs was obtained by gradient centrifugation and they always showed spindle and small polygonal shape. These cells proliferated fast and got confluence in 7 to 10 days. Little other cells like red blood cells and osteoclast-like cells were observed.While MSCs obtained by plastic adherent method showed irregular shape in primary culture and proliferated a little slow when compared with MSCs obtained by gradient centrifugation.Red blood cells and other cells could be observed even after several passages.When MSCs were undergone osteogenic differentiation, they showed similar ALP activity and mineralization ability.There was no significant difference between the numbers of mineralization node in the MSCs obtained by above two methods.Conclusion Gradient centrifugation is a better method for isolation and purification of MSCs from bone marrow and the cells showed perfect proliferation ability.Both methods can get MSCs with similar osteogenic differentiation potential.