不同胎龄大鼠神经干细胞体外诱导向多巴胺能神经元分化的比较(英文)  

Dopaminergic neuronal differentiation of neural stem cells in rats at different gestational ages

作　　者：柯春龙[1] 陈白莉[2] 金华伟[1] 杨超[1] 

机构地区：[1]中山大学附属第一医院神经外科,广东省广州市510080 [2]中山大学附属第一医院消化内科,广东省广州市510080

出　　处：《中国组织工程研究与临床康复》2008年第25期4991-4995,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金资助(30300115)~~

摘　　要：背景：胚胎来源干细胞的分化潜能不仅受到诱导条件影响，也受来源胚胎胚龄的影响。目的：实验拟观察不同胎龄大鼠中脑神经干细胞体外诱导向多巴胺能神经元分化能力。设计：随机对照观察。单位：中山大学附属第一医院神经外科。材料：实验于2007—03／2007—09在中山大学附属第一医院实验室完成。成年孕SD大鼠30只，体质量350～400g，由中山大学动物实验中心提供。许可证号码为SCXK（粤）2007—0034。实验过程中对动物处置符合动物伦理学标准。DMEM／F12无血清培养液、B27添加剂、表皮生长因子、碱性成纤维细胞生长因子、体积分数为0．1的胎牛血清为英国Gibco公司产品；白细胞介素1α，白细胞介素11、胶质细胞源性神经营养因子购自美国R＆D公司；白血病抑制因子购自英国PEPROTECH公司；酪氨酸羟化酶抗体购自美国Santa Cruz公司；巢蛋白抗体、微管相关蛋白2抗体及胶质纤维酸性蛋白抗体为美国CHEMICON公司产品。方法：①分别于大鼠孕10，12，14，16，18天摸球法随机选取6只，麻醉后处死孕鼠，无菌条件下剖腹取胚胎，分离中脑腹侧脑组织进行神经干细胞培养。在含表皮生长因子及碱性成纤维细胞生长因子的无血清培养液中分别培养不同胎龄大鼠脑神经干细胞，经传代扩增后，在含白细胞介素1α、白细胞介素11、白血病抑制因子、胶质细胞源性神经营养因子的诱导分化液中向多巴胺能神经元分化。②诱导分化6d后观察分化细胞生长情况并进行免疫细胞化学鉴定；酪氨酸羟化酶染色标记后通过流式细胞仪检测分化的多巴胺能神经元比率。主要观察指标：①不同胎龄大鼠神经干细胞诱导分化后生长情况及免疫细胞化学鉴定结果。②神经干细胞诱导分化后酪氨酸羟化酶染色阳性细胞的比率。结果：①孕10，12，14，16，18d胚胎大鼠中脑神经干细BACKGROUND： Differentiation inducing factors and gestational age influence the differentiation potential of embryonic neural stem cells. OBJECTIVE： This study was designed to observe the differentiation potential of rat mesencephalic neural stem cells at different gestational ages towards dopaminergic neurons. DESIGN： A randomized controlled observation. SETTING： Department of Neurosurgery, First Affiliated Hospital of Sun Yat-sen University, Guangzhou City, Guangdong Province, China. MATERIALS： This study was performed at the Laboratory of the First Affiliated Hospital of Sun Yat-sen University between March and September 2007. Thirty adult gestational SD rats, weighing 350-400 g, were provided by the Laboratory Animal Center of Sun Yat-sen University （Permission No. 2007-0034）. The protocol was performed in accordance with ethical guidelines stated in Guide for the use and care of laboratory animals, approved by the Committee on the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources Commission on Life Sciences, National Research Council, China （1985）. DMEM/F 12 serum-free medium, B27 additives, epidermal growth factor, basic fibroblast growth factor, and feral bovine serum （volume fraction：0.1） were purchased from Gibco Company, British; Interleukin la, interleukin 11, and glial cell-derived neurotrophic factors were purchased from R＆D Company, USA; In addition, leukaemia inhibitory factor （Perpotech, British）, tyrosine hydroxylase（Santa Cruz, USA）, nidogen antibody, microtubule-associated protein 2 antibody, and glial fibrillary acidic protein antibody（Chemicon, USA） were also used. METHODS： Six rats were randomly selected at each time point （on days 10,12,14,16, and 18 after gestation）. After anesthesia, the rats were sacrificed. Under the aseptic condition, fetal rat was harvested. Rat mesencephalic ventral brain tissue was isolated for culture of neural stem cells. Different gestational ages of rat brain-derived neural ste

关 键 词：神经干细胞 诱导分化 多巴胺能 神经元 

分 类 号：R394.2[医药卫生—医学遗传学]