碱性成纤维生长因子和神经生长因子联合应用培养成年大鼠海马神经干细胞向神经元样细胞的分化

背景：影响神经干细胞向神经元分化的因素很多,各种营养因子可以不同程度地刺激神经干细胞向神经元分化,如何使神经干细胞大量分化为神经元是研究的热点问题。目的：观察联合应用碱性成纤维生长因子和神经生长因子对成年大鼠海马神经干细胞为神经元的影响。方法：无菌条件下分离大鼠脑海马组织,传至第4代克隆球直径约为200μm时,滴加DMEM/F12＋2%B27＋20μg/L表皮生长因子＋20μg/L碱性成纤维细胞生长因子,进行单细胞克隆培养,传代的神经干细胞分成空白对照组、碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子＋神经生长因子组。观察传代后的克隆球进行神经干细胞免疫细胞化学染色鉴定,计数神经元特异性烯醇化酶阳性细胞率,检测神经干细胞向神经元的分化情况。结果与结论：①单细胞克隆培养后,克隆球细胞表达巢蛋白,诱导分化后神经元特异性烯醇化酶、胶质纤维酸性蛋白均呈阳性表达。②与空白对照组神经干细胞分化为神经元的比例比较,碱性成纤维细胞生长因子组、神经生长因子组、碱性成纤维细胞生长因子组＋神经生长因子组均明显提高（P〈0.05）,且碱性成纤维细胞生长因子组＋神经生长因子组神经元的比例最高（P〈0.05）。提示,碱性成纤维细胞生长因子可以提高神经生长因子诱和神经生长因子均可促进神经干细胞向神经元分化,且二者联合应用效果更佳。

Combination of basic fibroblast growth factor and nerve growth factor affects the differentiation of adult rat hippocampal neural stem cells into neuron-like cells

BACKGROUND：Differentiation of neural stem cells （NSCs） was mediated by many factors. Several factors could induce NSCs to differentiate into neurons in varying degrees and it is now a focus on the control of NSCs differentiation. OBJECTIVE：To study the effects of combination of basic fibroblast growth factor （bFGF） and nerve growth factor （NGF） on the differentiation of adult rat hippocampal NSCs into neurons. METHODS：The rat brain hippocampus was removed sterilely. Cell suspension was prepared and diluted when the diameter of the fourth passage of clone sphere was 200 μm by mixture of DMEM/F12 containing 2% B27,20 μg/L epidermal growth factor （EGF） and 20 μg/L bFGF. Monoclonal cells were passaged. NSCs were divided into blank control,bFGF,NGF and bFGF＋NGF groups. Immunocytochemistry for identification of NSCs was done to detect positive rate of neuron specific enolase （NSE） and the differentiation of NSCs into neurons. RESULTS AND CONCLUSION：①The monoclonal cells expressed nestin and the differentiated cells expressed NSE and glial fibrillary acidic protein. ②The proportions of NSE-positive cells in bFGF group,NGF group and bFGF＋NGF group were much higher than in blank control group （P 0.05）,with the highest in bFGF＋NGF group （P 0.05）. These indicate that bFGF and NGF can elevate the differentiation of NSCs into neurons,and their combination obtains better outcomes.