表皮生长因子培养条件下脑源性神经生长因子诱导大鼠海马神经干细胞向神经元分化的最佳浓度(英文)  

Optimal concentration of brain-derived neurotrophic factors for rat hippocampus neural stem cell differentiation into neurons in the medium containing epidermal growth factors

作　　者：王振宇[1] 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

出　　处：《中国组织工程研究与临床康复》2008年第16期3171-3174,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助项目(30470963)~~

摘　　要：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�BACKGROUND： The differentiation of neural stem cells （NSCs） is a hot research. Whether brain-derived neurotrophic factors （BDNF） can induce the differentiation of NSCs into neurons has not been detailed studied. OBJECTIVE： To investigate the effect of different concentrations of BDNF on the differentiation of NSCs from adult rat hippocampus into neurons in the medium with epidermal growth factor. DESIGN： A controlled study. SETTING： Department of Human Anatomy, China Medical University. MATERIALS： The experiment was performed at the Neurotomia Laboratory of China Medical University in August 2007. NSCs isolated from hippocampus of adult SD rats were inoculated in a serum-free medium. Three clean adult SD rats （200-250 g each） were provided by Laboratory Animal Department of China Medical University. Dispositions to the rats were consistent with ethical standards of animals. Epidermal growth factor （EGF） and BDNF were bought from R＆D Company. METHODS： （1）NSCs obtained from rat hippocampus were cultured in the serum-free medium containing basic fibroblast growth factor （bFGF）, EGF and B27. （2）Single cell clone was obtained via limited dilution for the fourth passage of cells, Identification of NSCs for the passage of ceils from monoclonal spheres was performed by Nestin immunocytochemistry. Neurons and astroglial cells were identified by immunocytochemistry for neuron specific enolase （NSE） and glial fibrillary acidic protein （GFAP） a week after differentiation. （3）Differentiation for the monoclonal spheres was done to appraisal the proportion of neurons. NSCs were divided for several groups containing 0 μg/L, 50 μg/L, 100 μg/L, 150 μg/L, and 200 μg/L groups according to the doses of BDNF. EGF was added in the media of each group. Immunocytochemistry for NSE was done a week later to count the positive cells. MAIN OUTCOME MEASURES： Proportion of neurons differentiated from NSCs. RESULTS： （1）NSCs immunocytochemical staining showed that Nes

关 键 词：神经干细胞 大鼠海马 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]