中文摘要：

背景：神经干细胞的临床应用前景广阔，寻找多种方法体外促进神经干细胞的增殖和分化为今后的发展方向。目的：介绍一种省时且经济的神经干细胞的培养方法。设计：观察对比实验。单位：北京市神经外科研究所。材料：选择4只孕14天Wistar大鼠，常温常湿环境饲养，体质量（180±20）g，均购自中国医学科学院动物所（实验动物许可证号码：SCXK1100—0006）。DMEM／F12（1：1）以及B27购自Gibco公司；碱性成纤维细胞生长因子和表皮生长因子购自PeproTech公司；巢蛋白单克隆抗体、胶质纤维酸性蛋白多克隆抗体、半乳酸脑苷多克隆抗体和微管相关蛋白2单克隆抗体购自Chemicon公司。胎牛血清购自Hyclone公司。方法：实验于2004—05／2004—10在北京市神经外科研究所损伤修复室完成。将Wistar大鼠脱臼处死，每次取4只胎鼠，将其脑组织置于Hank’s液中，解剖显微镜下剥离软脑膜及血管，眼科剪剪碎脑组织并收集细胞于2个离心管中离心，弃上清液。①根据给予血清预培养与否将细胞分成血清预培养组及对照组。血清预培养组细胞加入含100g／L胎牛血清DMEM培养液，培养48h后换含表皮生长因子、碱性成纤维细胞生长因子、B27的DMEM／F12培养液；对照组细胞中直接加含表皮生长因子、碱性成纤维细胞生长因子、B27的DMEM／F12培养液，37℃．体积分数为0．05的CO2培养箱培养1周。通过相差显微镜观察血清预培养组对照组培养48h后神经干细胞的生长情况。②用100g／L胎牛血清诱导分化后的第5天和第10天行nestin、胶质纤维酸性蛋白多克隆抗体、半乳酸脑苷多克隆抗体和微管相关蛋白2单克隆抗体免疫荧光染色，采用荧光显微镜观察检测两组神经干细胞的表达；对照组采用PBS缓冲液替代一抗，其它步骤同血清预培养组。主要观察指标：①相差显微镜下观察血清预培养

英文摘要：

BACKGROUND： The neural stem cells （NSCs） will be clinically applied extensively in the future, and seeking of more promoting methods of the proliferation and differentiation of NSCs in vitro will be the study direction of NSCs. OBJECTIVE： To introduce an economic and time-saving method for NSCs proliferation. DESIGN： Observation and control experiment. SETTING： Beijing Neurosurgical Institute. MATERIALS： Four Wistar rats pregnant for 14 days with the body mass of （180±20） g （bought from Animal Department of Chinese Academy＇of Medical Sciences with the batch number of SCXK1100-0006 for experiment animals） were selected and fed at common temperature and humidity. The DMEM/F12 and B27 were bought from Gibco Corporation. The basic fibroblast growth factor （bFGF） and epidermal growth factor （EGF） were bought from PeproTech Company. The nidogen monoclonal antibody （MA）, glial fibrillary acidic protein （GFAP） polyclonal antibody, galactocerebroside polyclonal antibody and microtubule-associated protein 2 （MAP2） were obtained from Chemicon Company. The fetal bovine serum （FCS） was provided by Hyclone company. METHODS： The experiment was conducted in the Department of Injury and Repair of Beijing Neurosurgical Institute from May to October 2004. Wistar rats were executed by dislocation and sterilized by putting into 75% alcohol. Four rats were used each time, the brain tissues of which were put into Hank＇s fluid. The cerebral pia mater and vessels were isolated under anatomical microscope, and the brain tissues were sheared off with eye scissors, which were filtered by 200 mesh copper grid and collected into 2 centrifuge tubes, and the supernatant was gotten rid off after that.① Cells were divided into serum pre-culture group and control group according to whether there were serum pre-culture. The DMEM nutrient fluid of FCS （100 g/L） was added to serum pre-culture group, which was replaced by DMEM/F12 nutrient fluid of EGF, bFGF and B27 at 48 hours after culture.