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背景:目前神经干细胞的定向分化是神经干细胞研究的热点。脑源性神经生长因子作为诱导剂可否诱导神经干细胞分化为神经元。目的:观察在表皮生长因子培养条件下,不同质量浓度脑源性神经生长因子对成年大鼠海马神经干细胞向神经元分化的影响。设计:对比实验。单位:中国医科大学人体解剖学教研室。材料:实验于2007-08在中国医科大学神经解剖研究室完成。提取成年SD大鼠海马神经干细胞,无血清技术培养。清洁级成年SD大鼠3只,体质量200~250g,由中国医科大学实验动物部提供,实验过程中对动物的处置符合动物伦理学标准。实验过程中应用的表皮生长因子、脑源性神经生长因子均由R&D公司提供。方法:①无菌条件下分离大鼠海马组织,用含碱性成纤维生长因子、表皮生长因子、B27的无血清细胞培养技术体外培养神经干细胞。②取第4代神经干细胞,利用有限稀释法进行单细胞克隆培养。将单细胞克隆传代后的克隆球细胞进行神经干细胞的鉴定。克隆球细胞行巢蛋白免疫细胞化学染色,诱导分化1周后进行神经元特异性烯醇化酶、胶质纤维酸性蛋白免疫细胞化学染色。③单细胞克隆的神经干细胞脑源性神经生长因子诱导分化实验,根据脑源性神经生长因子质量浓度不同分成0μg/L浓度的对照组和50、100、150、200μg/L浓度的4个实验组,各组的培养液中均加入表皮生长因子,诱导分化1周后进行神经元特异性烯醇化酶免疫细胞化学染色,检测神经干细胞向神经元分化情况。主要观察指标:神经干细胞分化为神经元的比例。结果:①神经干细胞免疫细胞化学染色结果显示,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。②50,100μg/L组脑源性神经生长因子组神经干细胞分化为神经元比例明显高于对照组、150,200μg/L脑源性神经生长因子组组(t=2.502~5.025,P<0.05);50,100μg/L脑源性神经生长因子组之间神经干细胞分化为神经元的比例差异不显著(P>0.05);对照组、150,200μg/L组3组间神经干细胞分化为神经元比例差异不显著(P>0.05)结论:在20μg/L表皮生长因子培养条件下,脑源性神经生长因子促使神经干细胞向神经元分化的较佳质量浓度为50μg/L。
Background: Currently, the directional differentiation of neural stem cells is a hotspot in neural stem cell research. Brain-derived nerve growth factor as an inducer can induce neural stem cells to differentiate into neurons. OBJECTIVE: To observe the effect of different concentrations of brain derived growth factor on differentiation of neural stem cells into neurons in adult rat hippocampus under epidermal growth factor (EGF) culture. Design: Contrast experiment. Unit: Department of Human Anatomy, China Medical University. Materials: The experiment was performed at the Department of Neuroanatomy, China Medical University from 2007-08. Adult SD rat hippocampal neural stem cells were extracted and cultured without serum. Three adult SD rats of clean grade with a body weight of 200-250g were provided by Laboratory Animal Department of China Medical University. The handling of animals during the experiment accorded with the standards of animal ethics. Epidermal growth factor and brain derived nerve growth factor used in the experiment were provided by R & D company. Methods: ① Separation of rat hippocampus under aseptic conditions, the use of basic fibroblast growth factor, epidermal growth factor, B27 serum-free cell culture technology in vitro culture of neural stem cells. ② take the 4th generation of neural stem cells, using limited dilution method for single cell cloning. Identification of neural stem cells by cloned single-cell clone cloned spherocysts. Nestin immunocytochemical staining of cloned spherocysts showed that neuron-specific enolase and glial fibrillary acidic protein were immunocytochemically stained one week after differentiation. ③ Single cell cloned neural stem cells induced differentiation of brain-derived neurotrophic factor, divided into 0μg / L concentration of control group and 50,100,150,200μg / L concentration of 4 experiments according to the concentration of brain-derived neurotrophic factor The epidermal growth factor (EGF) was added into the culture medium of each group. Neuronal specific enolase immunocytochemical staining was performed 1 week after induction of differentiation, and the differentiation of neural stem cells into neurons was detected. MAIN OUTCOME MEASURES: The proportion of neural stem cells differentiated into neurons. Results: (1) Immunocytochemical staining of neural stem cells showed that the nestin was expressed in the spheroplasts cultured in a single cell clone, and the expression of neuron specific enolase and glial fibrillary acidic protein was positive after differentiation. ② The percentage of neural stem cells differentiated into neurons in brain derived nerve growth factor group of 50 and 100μg / L group was significantly higher than that of the control group, 150,200μg / L brain derived neurotrophic factor group (t = 2.502 ~ 5.025, P <0.05); There was no significant difference in the percentage of NSCs differentiated into neurons between 50,100μg / L brain-derived NGF group (P> 0.05). There was no significant difference in proportion of neural stem cells differentiated into neurons in 150,200μg / L group (P> 0.05) .Conclusion: Under the condition of 20μg / L epidermal growth factor, the best concentration of brain derived neurotrophic factor to differentiate into neural stem cells is 50μg / L.