全骨髓贴壁法分离培养小鼠骨髓间充质干细胞

Isolation and Culture of Mouse Mesenchymal Stem Cells by Using the Whole bone Marrow Adherence Method

为体外建立一种高效、稳定的从小鼠骨髓中分离培养间充质干细胞的方法。采用全骨髓贴壁法分离C57BL/6小鼠骨髓间充质干细胞,传至第三代后运用流式细胞术检测第三代细胞的表面抗原;取第三代细胞,分别向成骨、成软骨诱导分化。结果显示:流式细胞术检测CD29、Scal-1、CD45的阳性率分别为97.1%、87.1%、0.7%;在成骨培养基的诱导下,碱性磷酸酶染色、茜素红染色均呈阳性;在成软骨培养基的诱导下,阿利辛蓝染色阳性。由此可知,通过此方法可以获得较高纯度的小鼠骨髓间充质干细胞,并且具有多向分化潜能。

To establish a stable and efficient method for isolation and culture of mouse bone marrow mesenchymal stem cells （MSCs）. MSCs were isolated by adherence to plastic plate from C57BL/6 mice bone marrow. The surface markers of the third generation were identified by flow cytometry. Osteogenic and chondrogenic inductions were performed in vitro to assess the differentiation capacity. As the result showed that the positive rates for CD29, Scal-1 and CD45 were 97.1%, 87.1%, 0.7%, respectively. The osteogenic differentiation of MSCs was verified by alkaline phosphatase staining and alizarin red staining; the chondrogenic differentiation of MSCs was identified by alcian blue staining. It is concluded that high-purity MSCs can be obtained by using the whole marrow adherence method and have muhipotential differentiation.