小鼠胚胎间充质干细胞系C3H／10T1／2的多向分化潜能研究

Multilineage differentiation of murine embryonic mesenchymal stem cell line C3H/10T1/2

目的建立小鼠胚胎间充质干细胞系C3H/10T1/2细胞成肌、成脂、成内皮、成神经元分化的细胞实验模型。方法用化学诱导剂5-氮杂胞苷诱导C3H/10T1/2细胞成肌、成脂,血管内皮生长因子(vascular endothelial growth factor,VEGF)和碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)联合诱导C3H/10T1/2细胞成内皮,bFGF、β-巯基乙醇(β-mercaptoethanol,β-ME)、DMSO诱导C3H/10T1/2细胞成神经元细胞分化。诱导期间应用细胞形态学观察、油红"O"染色、免疫细胞化学染色检测内皮细胞表面标志CD31和神经元特异性烯醇化酶(neuron-specific enolase,NSE)对分化细胞进行鉴定。结果5-氮杂胞苷诱导C3H/10T1/210d后细胞变长,17d后可见明显肌管形成,15d少量细胞出现脂滴,25d油红"O"染色见细胞质大量红色脂滴;VEGF和bFGF诱导5d后细胞呈现"鹅卵石"样形态,8d后阳性表达CD31;bFGF、β-巯基乙醇和DMSO联合诱导3d后细胞胞体收缩,突起变长,15dNSE染色阳性。结论C3H/10T1/2细胞具有多向分化的潜能,可用作研究间充质干细胞生物学特性的模型。

Objective To establish experimental cell models of myogenic cells, adipocytes, endothelial cells and neurocytes differentiated from murine embryonic mesenchymal stem cell line C3H/10T1/2. Methods Myogenic and adipogenic differentiation of C3H/10T1/2 cells were induced by 5-azacytidine, angiogenic differentiation by vascular endothelial growth factor （VEGF） and basic fibroblast growth factor （bFGF）, and neurogenic differentiation by bFGF, β-mercaptoethanol （β-ME） and DMSO. The differentiated cells were identified by observing cell morphology, Oil Red O staining and immunohistochemical staining to detect endothelial cell surface marker CD31 and neuron-specific enolase （NSE）. Results C3H/IOT1/2 cells were morphologically elongated at the 10th day, and myotube-like cells appeared at the 17th day after 5-azacytidine induction. Meanwhile, adipose globelets were observed in a small proportion of cells at the 17th day, and Oil Red O staining confirmed adipose accumulation in the cytoplasm at the 25th day after 5-azacytidine induction. Cobble stonelike structure was observed since 5th day after inducement of VEGF and bFGF, and at the 8th day the induced cells exhibited typical CD31 positive staining. After treated with bFGF, β-ME in combination with DMSO, cytoplasm retracted towards the nucleus and the cell processes prolonged at the 3th day. Immunohistochemical staining showed that differentiated cells expressed NSE at the 15th day. Conclusion C3H/10T1/2 cells have the potential of muhilineage differentiation and can be used as an alternative source of mesenchymal stem cells. Growth factor-induced endothelial differentiation of C3H/10T1/2 cells was established .