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建立蒙古羊脂肪间充质干细胞(Adipose-derived mesenchymal stem cells,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34.1 h;经成神经诱导后,细胞呈胶质细胞状,RT-PCR检测ENO2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15 d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。
To establish a method for the isolation and culture of Adipose-derived mesenchymal stem cells (ADSCs) in vitro and to identify its biological characteristics and multi-directional differentiation potential. The type I collagenase was used to digest Mongolia sheep adipose tissue and then mononuclear cells were obtained by centrifugation and subcultured to determine the doubling time. Toluidine blue staining, PAS staining and RT-PCR were used to identify the differentiation of ADSCs from 3rd generation Mongolian sheep into adult and isolated myocardium from histological level and gene level respectively. The results showed that the isolated adipose-derived mesenchymal stem cells were relatively uniform in size and spindle-shaped or star-shaped fibroblast-like cells. On the 4th day after inoculation, the cells entered the exponential growth phase and reached the plateau on the 8th day. The ADSCs The average doubling time was 34.1 h. After induced by the axons, the cells were glial cells, and the positive expression of ENO2 and GFAP gene was detected by RT-PCR. After myocardial induction, the cells increased in size, After 15 days of induction, the myotube-like structures were observed in some cells. PAS staining showed obvious glycogen deposition. The expression of NKX2.5 and GATA-4 gene was positive by RT-PCR. The results showed that ADSCs obtained from Mongolian sheep had multidirectional differentiation potential.