rhEGF和rhbFGF诱导胎鼠脑组织神经干细胞向神经元样细胞分化的研究

Study on the rhEGF and rhbFGF induced neurotropic-like cell differentiation of neural stem cell in fetal rat brain

目的观察在重组人表皮生长因子和重组人碱性纤维母细胞生长因子诱导下胎鼠脑组织神经干细胞向神经元样细胞分化的超微结构及细胞标志物表型特征。方法采用孕16d大鼠胚胎脑组织进行神经干细胞体外分离、培养传代及鉴定,分别于倒置相差显微镜及电子显微镜下观察经体外诱导培养后神经干细胞的组织形态及超微结构变化;免疫细胞化学染色检测神经微丝、微管相关蛋白-2、去甲肾上腺素、乙酰胆碱、γ-氨基丁酸以及多巴胺等蛋白质标志物的表达。结果倒置相差显微镜观察显示,经体外培养的神经干细胞随着培养时间的延长逐渐形成神经干细胞球体,经体外诱导培养至第7天时即开始出现明显分化趋向,第14天时部分细胞分化发育完全;且蛋白质标志物神经微丝、微管相关蛋白-2、去甲肾上腺素、乙酰胆碱、γ-氨基丁酸及多巴胺均表达阳性。电子显微镜观察经体外诱导培养至第14天时,细胞出现明显的成熟分化趋向,细胞质中含有大量神经微丝和神经内分泌颗粒,呈典型的神经元分化特征。结论神经干细胞在体外诱导培养条件下短期即可向神经元方向成熟分化。

Objective To investigate the ultrastructure and cell marker phenotype characteristics of the differentiated neurotropic-like cell of fetal rat brain neural stem cells （NSCs） in the induction of recombinant human epidermal growth factor （rhEGF） and recombinant human basic fibroblast growth factor （rhbFGF）. Methods The NSCs in embryonic 16 d rat brain were isolated, cultured, passaged and identified in vitro. After induction of rhEGF and rhbFGF, inverted phase contrast microscope and electron microscope were used to examine the histological and ultrastructural changes of the cultured tissues, respectively. The expressions of protein markers including neurofilament （NF）, microtubule-associated protein-2 （MAP-2）, noradrenaline （NA）, acetycholine （ACh）, γ-aminobutyric acid （GABA） and dopamine were detected by immunohistochemical staining. Results Under the observation of inverted phase contrast microscope, the NSCs were gradually trans- formed into neurospheres with time in culture. On the 7th day of induction for the-cultured cells, differential trend presented obviously. On the 14th day part of differentiated cells developed completely, and protein markers （NF, MAP-2, NA, ACh, GABA and dopamine） expressed positively; and at the same day, electron microscopic observation showed apparent trend of mature cell differentiation. In the cytoplasm, there were amount of NF and neuroendocrine granules which presented typical characteristics of neuron differentiation. Conclusion Neural stem cells can be differentiated into neurotropic-like cells in short time by induction and culture in vitro.