克隆筛选法体外纯化骨髓间充质干细胞

目的尝试使用简单的克隆筛选法来纯化骨髓间充质干细胞（Bone marrow mesenchymal stem cells，BMSCs）并进行鉴定。方法对早期分离培养的BMSCs，采取低密度接种（10／cm^2），待有单克隆形成，用细胞刮除去其他细胞，将克隆的细胞以低密度接种，待再次有单克隆形成时可重复以上操作，反复进行筛选。对筛选的BMSCs生物学特性进行观察，FACs鉴定BMSCs的标记物的表达，并进行成脂、成骨诱导试验。结果采用克隆筛选法培养BMSCs在传代筛选2—3代后即可获得形态均一的细胞群，FACs鉴定细胞高表达BMSCs的阳性标记物（CD2998．8％，CD9098．4％），低表达BMSCs的阴性标记物（CD312．6％，CIM53％）。BMSCs能被高效地诱导向脂肪和骨分化。结论本实验采用的克隆筛选法可在短期内获得高纯度的BMSCs，方法简单易行而效率高。

Purification of bone marrow mesenchymal stem cells by a Colony-Forming system

Objective To purify BMSCs by a simple Colony-Forming system and identify BMSCs in vitro. Methods BMSCs were planted at low-density （ 10/cm^2 ） in the early stage of isolation, additional cells were scraped until a colony formed, secondly seeded those Colony-Forming cells at low-density. This process was repeat again when passaged ceils formed another colony. This so called Colony-Forming selection was repeated several times until highly purified cells were obtained. Biological characters of BMSCs were observed. The surface antigens of BMSCs were identified by flow cytometry. The multipotentiality of BMSCs was assayed for differentiation into either osteoblasts or adipocytes. Results Homogeneous cells were obtained after BMSCs were passaged twice to thrice by Colony-Forming system. These BMSCs highly expressed positive surface antigens of BMSCs （ CD29 in 98. 8% , CD90 in 98.4% ） meanwhile seldom expressed negative surface antigens of BMSCs （CD31 in 2. 6%, CD45 in 3% ）. BMSCs efficiently differentiated into osteoblasts and adipocytes. Conclusions Colony-Forming system is a simple and effective way to get highly purified BMSCs in a short time.