表皮生长因子对脑源性神经生长因子诱导体外培养大鼠脑海马神经干细胞向神经元分化的影响  

Effect of epidermal growth factor on brain-derived neurotrophic growth factor-induced differentiation of rat hippocampus neural stem cells into neurons in vitro

作　　者：王振宇[1] 朱志[2] 佟雷[1] 季丽莉[1] 赵久红[1] 唐源远[1] 

机构地区：[1]中国医科大学基础医学院人体解剖学教研室,辽宁省沈阳市110001 [2]中国医科大学第一临床学院肿瘤外科,辽宁省沈阳市110001

出　　处：《中国组织工程研究与临床康复》2008年第12期2317-2320,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金(30470963)~~

摘　　要：目的：获得大量神经干细胞并且能够控制其向神经元定向分化可为进一步修复损伤大脑和治疗退行性神经系统疾病奠定种子细胞基础,实验观察了表皮生长因子对脑源性神经生长因子诱导神经干细胞向神经元分化过程中的影响。方法：实验于2007-08在中国医科大学神经解剖研究室完成。①材料：清洁级雄性成年SD大鼠3只,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。②实验方法：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＋条件培养液（细胞原代培养第7天的上清液的过滤液）,进行单细胞克隆培养,传代的神经干细胞分成空白对照组、表皮生长因子组、脑源性神经生长因子组、表皮生长因子＋脑源性神经生长因子组。空白对照组培养液为含体积分数0.1胎牛血清的DMEM/F12,其余3组培养液在此基础上分别加入各自对应的细胞因子培养1周,表皮生长因子浓度为20μg/L,脑源性神经生长因子浓度为50μg/L。③实验评估：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：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,表皮生长因子组有所降低;脑源性神经生长因子组、表皮生长因子＋脑源性神经生长因子组均明显提高（t=2.309～2.779,P〈0.05）,且后者提高幅度尤为显著（t=2.309,P〈0.05）。结论：表皮生长因子可以提高脑源性神经生长因子诱导成年大鼠脑海马神经干�AIM： Neural stem cells （NSCs） and neurons differentiated from neural stem cells play an important role in repairing damaged brains and treating degenerative nervous system disease. This study aimed to study the effects of epidermal growth factor （EGF） on brain-derived neurotrophic growth factor （BDNF） induced differentiation of NSCs into neurons. METHODS： The experiment was performed at the Neurotomia Laboratory of China Medical University in August 2007. （1）Three adult male SD rats were provided by Experimental Animal Center of China Medical University. Dispositions to the rats were consistent with ethical standards of animals. （2）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 μ m by mixture of DMEM/F12 containing 2% B27, 20 μ g/L EGF, 20 μ g/L basic fibroblast growth factor （BFGF） and conditional media containing filtration of the seventh day media. Monoclonal cells were passaged. NSCs were divided into blank control, EGF, BDNF and EGF＋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 EGF, BDNF and EGF＋BDNF respectively for 1 week. The concentration of EGF was 20 μ g/L and the concentration of BDNF was 50 μ g/L. （3） Immunocytochemistry for identification of NSCs were done to detect positive rate of neuron specific enolase and the differentiation of NSCs into neurons. RESULTS： （1）The monoclonal cells expressed nestin and the differentiated cell expressed neuron specific enolase and glial fibrillary acidic protein. （2）The proportion in the blank control group was higher than in the EGF group. BDNF group and EGF＋BDNF group were much higher than in blank control and EGF groups （t =2.309-2.779, P 〈 0.05） with the highest in EGF＋BDNF group （t =2.309, P

关 键 词：神经干细胞 神经元 表皮生长因子 脑源性神经生长因子 分化 

分 类 号：R394.2[医药卫生—医学遗传学]