绵羊脐带间充质干细胞的分离、体外培养及诱导分化

Isolation,Cultivation in Vitro and Inductive Differentiation of Sheep Umbilical Mesenchymal Stem Cells

通过多重酶混合消化法和胰酶消化筛选法体外分离、纯化及培养绵羊脐带间充质干细胞(MSCs),并观察其生物学特性,以此建立绵羊脐带间充质干细胞的分离培养流程。试验采集健康的哈萨克羔羊脐带组织,剔除血管后剪碎,添加复合消化酶消化24h得到细胞,胰酶消化法筛选除杂并观察细胞形态,绘制第1代、第5代及第10代细胞生长曲线;取第10代细胞检测其体外诱导成骨细胞能力和诱导成脂细胞能力,cAKP染色检测其分化状态。结果表明,获得的细胞经传代培养达10代后无明显的形态和增殖能力改变;体外诱导成骨细胞和成脂细胞试验证明其具有被诱导分化成骨细胞和脂肪的能力,cAKP染色证明其处于未分化状态。试验获得的绵羊脐带间充质干细胞能在体外培养、扩增并且具有和骨髓间充质干细胞类似的生物学特征。

The objective of this paper was to isolate in vitro,purify and cultivate sheep umbilical mesen chymal stem cells （MSCs） and observe their biological characteristics through multiple enzyme mix-diges tion and pancreatic enzyme screening digestion, by which the process of isolation and cultivation of the sheep umbilical mesenchymal stem cell was established. The test was carried out to collect umbilical organs of the healthy Kazakhstan lamb,which were cut into pieces before artery and vein were got rid of. The cells were obtained when mix-digestion enzyme was added for 24 h. The cell morphology were observed after wi- ping the impurity off by pancreatic enzyme screening digestion method,then the cell growth curve of the first, the fifth and tenth generations was drawed from observing the cell morphology. The induced in vitro osteogenic cell capacity and induced adipogenous cell capacity were replaced by P10 generations;The differ entiation state was detected by cAKP stain. The results showed that there was no obvious changes in mor- phology and proliferation capacity in the cells after 10 generations being continuously cultivated. The test for the induced adipogenic and osteogenic differentiation induced in vitro proved that they both had induced differentiation osteogenic cell capacity and adipogenous cell capacity, cAKP stain proved that it stayed in the undifferentiation state. The above results showed that sheep umbilical mesenchymal stem cell can be cultivated and amplified in vitro, which were similar to biological characteristics of sheep umbilical mesenchymal stem cell.