联合应用碱性成纤维细胞生长因子和脑源性神经营养因子对成年大鼠脑海马神经干细胞分化为神经元的影响

背景:调控神经干细胞分化的微环境因素很多,数种因素可以不同程度地刺激神经干细胞向神经元分化,如何使神经干细胞更有效地分化为神经元尤为关键.目的:观察碱性成纤维细胞生长因子与脑源性神经营养因子联合应用对成年大鼠脑海马神经干细胞分化为神经元的影响.设计、时间及地点;细胞学体外观察,于2008-05在中国医科大学神经解剖研究室完成.材料:清洁级雄性SD大鼠3只,由中国医科大学实验动物部提供.方法:无菌条件下分离大鼠脑海马组织,胰蛋白酶消化后采用无血清培养技术体外培养神经干细胞,传至第4代克隆球直径约为200 pm时,滴加含B27、表皮生长因子及碱性成纤维细胞生长因子的DMEM/F12进行单细胞克隆培养.传代的神经干细胞分成4组:空白对照组加入含体积分数为10%胎生血清的DMEM/F12培养液,碱性成纤维细胞生长因子组、脑源性神经营养因子组、联合组分别在空白对照组培养液的基础上加入10 pg/L碱性成纤维细胞生长因子、200 μg/L脑源性神经营养因子、10 μg/L碱性成纤维细胞生长因子+200 μg/L脑源性神经营养因子,培养1周.主要观察指标:免疫细胞化学染色鉴定神经干细胞,检测神经干细胞分化为神经元的比例.结果: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.与空白对照组比较.碱性成纤维细胞生长因子组、脑源性神经营养因子组、联合组神经干细胞分化为神经元的比例均明显提高(t=3.409～7.558,P<0.05),且联合组升高幅度尤为显著.结论:适宜浓度的碱性成纤维细胞生长因子和脑源性神经营养因子联合应用,比其单独应用具有更强的促神经干细胞向神经元分化的作用.

Combination of basic fibroblast growth factor and brain-derived neurotrophic growth factor influences the differentiation of adult rat hippocampus neural stem cells into neurons

BACKGROUND: Differentiation of neural stem calls (NSCs) was mediated by many environmental factors. Several factors can 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 brain-derived neurotrophic growth factor (BDNF) on the differentiation of NSCs into neurons.DESIGN, TIME AND SETTING: The in vitro cytology observation was performed at the Neurotomia Laboratory of China Medical University in May 2008.MATERIALS: Three adult male SD rats were provided by Experimental Animal Center of China Medical University.METHODS: Dispositions to the rats were consistent with ethical standards of animals. The rat brain hippocampus was removed sterilely. After trypsin digestion, NSCs were cultured in serum-free medium. 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 of epidermal growth factor (EGF), and 20 μg/L bFGF. Monoclonal calls were passagad. NSCs were divided into blank control, bFGF, BDNF and bFGF+BDNF groups by different growth factors added into the media. Fetal bovine serum of 0.1 volume fraction was added in blank control group. The media in the other three groups were added bFGF, BDNF and bFGF+BDNF respectively for 1 week.The concentration of bFGF was 10 μg/L and the concentration of BDNF was 200 μg/L.MAIN OUTCOME MEASURES: Immunocytochemistry staining was used to identify NSCs as well as to detect the differentiation of NSCs into neurons.RESULTS: The monoclonal calls expressed nestin and the differentiated call expressed neuron specific enolase and glial fibrillary acidic protein. Compared to blank control group, the proportion NSCs into neurons in the bFGF group, BDNF group and bFGF+BDNF group were much higher (t=3.409-7.558, P < 0.05), with the highest in bFGF+BDNF group (t =7.558, P < 0.05).CONCLUSION: Combination of bFGF and BDNF can promote the differentiation of adult hippocampus NSCs into neurons.