体外诱导大鼠骨髓间充质干细胞向神经细胞分化的实验研究

Neuron-like cells differentiated from rat mesenchymal stem cells in vitro

关键词:  骨髓间充质干细胞  神经细胞  分化  诱导  培养

Key words:  [Key words]　mesenchymal stem cells  neuron  differentiate  induce  culture

摘要:

目的　对大鼠骨髓间充质干细胞（bone marrow mesenchymal stem cells，BMSCs）进行分离、培养与鉴定，并探讨全反式维甲酸(retinoic acid,RA)、碱性成纤维细胞生长因子（fibroblast growth factor,basic, bFGF）和表皮生长因子（epidermal growth factor, EGF）联合诱导BMSCs分化为神经细胞的可行性。 方法　全骨髓贴壁法分离培养BMSCs，观察细胞形态及生长增殖情况；流式鉴定细胞表面标志物CD29、CD34、CD90；选用第3代细胞，经RA、bFGF和EGF联合诱导后，细胞免疫化学染色检测神经细胞标志物神经元特异性烯醇化酶（neuron specific enolasen,NSE）的表达。 结果　体外培养的BMSCs呈成纤维细胞样，第3、4、5代BMSCs的生长曲线均呈S形，活性无明显差异。BMSCs的均一性较好，第3代细胞CD29、CD90阳性率均在90%以上，而CD34阳性率仅为0.58%；BMSCs经诱导后分化为神经细胞，并表达神经细胞标志NSE。 结论　成功建立BMSCs的体外培养体系，所得细胞纯度高、生物学特征稳定，并可诱导分化为神经细胞，为移植治疗神经系统损伤提供实验基础。

Abstract:

[Abstract] Objective　To isolate and culture rat bone marrow mesenchymal stem cells (BMSCs) and induce them differentiate to neural cells. Methods　BMSCs were isolated from rats by wall sticking method. Then the cells were identified with morphology, proliferation and the surface markers. Neuron-like cells from BMSCs were induced by the combination of retinoic acid(RA),fibroblast growth factor, basic (bFGF）and epidermal growth factor (EGF), and their neuron specific enolasen (NSE) were detected by NSE immunohistochemistry.Results　Cultured BMSCs like fibroblast-shaped in vitro. Growth curves of the 3rd, 4th and 5th-generation BMSCs and their activities were not significantly different. FCM detected that CD29 and CD90 were positively expressed, but CD34 was negatively expressed. The induced BMSCs showed neuron-like morphology and expressed NSE, the neuron marker.Conclusion　We have established an efficient and steady method to obtain BMSCs. BMSCs could differentiated into neurons which was an ideal source for clinical cell transplantation in treatment of nervous system diseases.