脑源性神经生长因子促进成年大鼠脑海马神经干细胞定向分化的浓度选择  

Concentrations of brain-derived neurotrophic factors influence the directional differentiation of neural stem cells from the hippocampus of adult rats

作　　者：季丽莉[1] 佟雷[1] 唐源远[1] 赵久红[1] 王振宇[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》2007年第46期9255-9258,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:探讨培养基中表皮生长因子与血清两者浓度一定的条件下,不同浓度的脑源性神经生长因子对成年大鼠脑海马神经干细胞向神经元分化的影响。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1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。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。②实验方法:无菌条件下分离大鼠脑海马组织,剪碎后胰蛋白酶消化,过滤、离心、弃上清,加入含2%B27、20μg/L表皮生长因子、20μg/L碱性成纤维生长因子的DMEM/F12无血清条件培养基体外培养神经干细胞,传至第4代时利用有限稀释法进行单克隆培养,100倍镜下克隆球直径约为200μm时制备单细胞悬液,稀释后滴加于96孔板内,设立两组,全量新鲜培养基组加入刚配置未使用过的DMEM/F12无血清培养基100μL,半量条件培养基组加入上述曾用于神经干细胞培养且含有其代谢产物的1/2DMEM/F12无血清培养基100μL。③实验评估:观察神经干细胞的单克隆培养增殖情况。对克隆球行巢蛋白、神经元特异性烯醇化酶、胶质原纤维酸性蛋白免疫细胞化学染色。按培养基中脑源性神经生长因子终浓度的不同将所培养细胞设立0,50,100,150,200μg/L组,各组均加入20μg/L表皮生长因子和体积分数为0.1的胎牛血清,1周后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。结果:①神经干细胞单克隆培养结果:单克隆培养开始时神经干细胞增殖缓慢,半量条件培养基组细胞增殖速度快于全量新鲜培养基组,随着细胞数的增多,两组细胞增殖速度也相应加快,分别在培养后第12天、第15天形成直径为200μm的克隆球。②神经干细胞免疫细胞化学染色结果:单克隆培养后克隆球表达巢蛋白,诱导�AIM： To research the influence of brain-derived neurotrophic factor on the proliferation and differentiation of neural stem cells from hippocampus of adult rats when the concentrations of epidermal growth factor and serum are invariable. METHODS： The experiment was finished in Neurotomia Laboratory of China Medical University in August 2007. ①A clean adult male SD rat was provided by Laboratory Animal Department of China Medical University. Dispositions to the rat were consistent with ethical standard of animals. Epidermal growth factor and brain-derived neurotrophic factor were bought from R＆D company. ②The hippocampus was removed sterilely and cut into cubes. The tissues were placed in digestion media consisting of trypsin dissolved in PBS, and then filtered the cells, and the supernatant was removed. The serum-free medium containing 20 μg/L basic fibroblast growth factor, 20 μg/L epidermal growth factor and 2% B27 could culture neural stem cells. Monoclone that utilized limiting dilution assay was done when neural stem cells was passaged for three times. Cell suspension was prepared and diluted when diameters of most spheres were 200 μm at 100 times microscope. Cells were dropped into 96-well plate. Monoclone spheres were divided into two parts： full dose of fresh media group and half dose of fresh media group. Neural stem cells in the full dose of fresh media group were incubated in 100 μL fresh DMEM/F12. Neural stem cells in the half dose of fresh media group were incubated in 100 μL fresh 1/2 DMEM/F12. ③The condition of monoclone was observed. Immunocytochemical stain for Nestin, neurone specific enolase and glial fibrillary acidic protein were done to monoclone neural stem cells. Neural stem cells were divided into several groups that included 0,50,100,150,200 ③g/L groups according to the doses of brain-derived neurotrophic factor. 20 ③g/L epidermal growth factor and fetal bovine serum of 0.1 volume fraction were added in each group. Immunocytochemical stain of neurone specific eno

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]