巴马香猪骨髓间充质干细胞的分离扩增及初步鉴定

目的建立巴马香猪骨髓问充质干细胞（mesenchymal stem cells，MSCs）分离、培养、扩增的方法，并进行鉴定。方法采用梯度离心法分离并结合贴壁筛选法传代纯化培养猪的MSCs；采用倒置相差显微镜观察MSCs的形态学特征；流式细胞仪检测第3代细胞表面标志抗原CD71、CD34的表达率。结果原代培养的MSCs于6h后贴壁，贴壁细胞呈集群生长趋势。培养7～9d后可见细胞融合，14～21d达到95％融合。第3代细胞表面标记物CD71阳性率为95％，CD34阳性率为O％。结论采用全骨髓梯度离心法分离并结合贴壁筛选法能够成功分离和培养巴马香猪的MSCs，在第3代可获得高纯度MSCs。

Isolation,cultivation and identification of mesenchymal stem cells in Bama minipigs

Objective To establish a method for isolation and culture of bone marrow mesenchymal stem cells （MSCs） from Bama minipigs in vitro and to identify characteristic of the ceils. Methods MSCs were isolated and cultured from the bone marrow of Bama minipigs by gradient centrifugation followed by adherent method. The morphology of MSCs was observed under phase contrast microscope. The expression of CD71 and CD34 of the third generation cells were analyzed by flow cytometry. Results After 6 hours primary culture, MSCs adhered to plastic surface of the culture dish. About 7 - 9 days later, the cells proliferated in colonies. Primary MSCs reached 95% of confluence between 14 and 21 days approximately. The positive rates of CD71 and CD34 in MSCs at third generation were 95%, 0%, respectively. Conclusion MSCs can be successfully isolated and cultivated by gradient ceutrifugation followed by adherent method. And higher purity MSCs can be picked up after culture expansion at passage 3.