犬骨髓间充质干细胞体外诱导分化为胰岛样细胞的研究

Study of the differentiation of canine bone marrow mesenchymal stem cells into pancreatic islet-like cells in vitro

目的研究犬骨髓间充质干细胞(MSC)体外诱导分化为胰岛样细胞的可能性。方法分离纯化犬骨髓MSC,予EGF、bFGF和β细胞调节素、尼克酰胺、B27添加剂诱导。双硫腙染色、Westernblot、免疫荧光染色、胰岛素分泌量测定和葡萄糖刺激胰岛素释放试验鉴定诱导前后的细胞功能。结果未诱导细胞呈长梭形贴壁生长,诱导第2周细胞逐渐变圆,聚集成团,双硫腙染色呈棕红色;Westernblot检测诱导后细胞表达PDX-1;免疫荧光染色显示诱导后细胞表达胰岛素、胰升血糖素、生长抑素;ELISA结果表明诱导后细胞可分泌胰岛素,体外葡萄糖刺激胰岛素释放试验阳性。结论犬骨髓MSC在体外能被诱导分化为胰岛样细胞。

Objective To explore the possibility of canine bone marrow mesenehymal stem cells （MSCs） differentiating into pancreatic islet- like cells（ILCs） in vitro. Methods Mesenchymal stem cells were isolated from canine bone marrow. The pure MSCs were induced into ILCs under a two-stage protocol. The MSCs were cultured and induced in HG-DMEM （ FBS free） supplemented with epidermal growth factor （EGF）, basic fibroblast growth factor （bFGF）, Beta-cellulin, nieotinamide, B27 supplement, and β-mercaptoethanol. Morphological changes of the induced cells were observed. The identification of the function of the induced cells were performed by dithizone （DTZ） staining, Western blot, immunofluorescence staining, insulin quantitative secretion and glucose-simulated insulin secretion. Results MSCs were spindle-shaped adherent cells. After induction, cells were getting round and beginning to gather as clusters similar to pancreatic islet, and to secrete insulin, and could be stained with DTZ on the 2nd week. Western blot showed the pancreatic and duodenal homeobox 1 （PDX-1） expression, the expression reinforced gradually from 7th to 21st day. Immunofluorescenee assay showed positive insulin, glucagon and somatostaitin in the induced cells. The clusters were positive for the test of glucose-stimulated insulin secretion in vitro by ELISA. Conclusions MSCs from canine bone marrow can he induced into pancreatic islet- like cells in vitro.