蒙古羊脂肪来源间充质干细胞的分离培养及体外分化诱导

建立蒙古羊脂肪间充质干细胞（Adipose-derived mesenchymal stem ceils，ADSCs）体外分离培养方法，并对其生物学特性和多向分化潜能进行鉴定。利用I型胶原酶将蒙古羊脂肪组织消化后，离心得到单核细胞，并进行传代培养，测定其倍增时间。采用甲苯胺蓝染色和PAS染色法以及RT-PCR法，分别从组织学水平和基因水平对第3代蒙古羊ADSCs向成神经和成心肌的诱导分化情况进行鉴定。结果显示，分离得到的脂肪间充质干细胞大小较为均匀，呈梭形或星形的成纤维细胞样；传代接种后第4天细胞进入指数生长期，第8天进入平台期，前10代ADSCs的倍增时间平均为34．1h；经成神经诱导后，细胞呈胶质细胞状，RT-PCR检测EN02和GFAP基因表达呈阳性；心肌诱导后，细胞体积增大，多呈长梭形，平行排列，诱导15d后部分细胞可见类肌管样结构，PAS染色可见明显的糖原沉积，RT-PCR检测NKX2．5和GATA-4基因表达呈阳性。表明获得的蒙古羊ADSCs具有多向分化潜能。

Isolation,culture and multiple differentiation induction of mongolia sheep adipose tissue-derived mesenchymal stem cells

The aims of this paper are to explore the optimal method of isolating, purifying, and proliferating Mongolian sheep adipose-derived mesenchymal stern cells （ ADSCs）, and their multiple differentiation potentiality. ADSCs were harvested by centrifuging after collagenase I digestion and obtained mononuclear cells were cultured. The results showed that, the isolated adipose mesenchymal stem cells were uniform, a fibroblast like spindle or stellate. Analysis of the growth of the passage 1, 5, and 10 cultures revealed an S-shaped growth curve with the population doubling time of 34.1 h. The P3 ADSCs were cultured in vitro under inductive environments and induced into neurogenesis and cardio- myocytes. Their differentiation properties were confirmed by histological staining such as toluidine blue, and periodic acid schiff. RT-PCR showed that the specific genes to be induced were all expressed. This proves that the isolated cells are indeed the ADSCs, and also provides valuable materials for somatic cell cloning and transgenic research.