新生小鼠海马、嗅球及皮质神经干细胞的分离培养及鉴定  

Isolation and identification of neural stem cells from newborn mouse hippocampus, olfactory bulb and cortex

作　　者：马浚宁 高俊玮[1] 侯博儒[1] 任海军[1,2] 陈四化[1] 刘吉星[1] 严贵忠 

机构地区：[1]兰州大学第二临床医院神经外科,甘肃省兰州市730000 [2]兰州大学生命科学学院,甘肃省兰州市730000

出　　处：《中国组织工程研究》2014年第45期7266-7272,共7页Chinese Journal of Tissue Engineering Research

基　　金：甘肃省自然科学基金(1208RJZA184)~~

摘　　要：背景:从体外分离培养出高纯度、生物学性能均一的神经干细胞,建立起一套完整的神经干细胞培养体系,是进行神经干细胞研究的基础。目的:建立新生小鼠海马、嗅球、皮质组织神经干细胞的分离培养体系,并对其生物学特性进行分析。方法:分离新生昆明小鼠海马、嗅球、皮质组织,采用机械分离和胰酶消化法提取原代神经干细胞。采用无血清培养技术、机械吹打和酶消化法进行传代培养神经干细胞。以体积分数为10%的胎牛血清诱导分化神经干细胞。对神经干细胞及其分化产物行CD133、巢蛋白、β-微管蛋白Ⅲ、胶质纤维酸性蛋白免疫荧光染色鉴定。结果与结论:从新生小鼠海马、嗅球、皮质可提取出具有自我更新和多向分化能力的神经干细胞,经巢蛋白、CD133免疫荧光染色检测呈阳性;神经干细胞经胎牛血清诱导后可分化为β-微管蛋白Ⅲ、胶质纤维酸性蛋白阳性细胞,并证实染色阳性细胞为神经元和星形胶质细胞。该实验建立了一套神经干细胞体外分离培养、纯化、鉴定、诱导分化方案,为后续神经干细胞研究的顺利进行奠定了实验基础。BACKGROUND：To in vitro isolate neural stem cel s with high purity and uniform biological properties and to establish a complete set of neural stem cel culture system is the basis for neural stem cel research. OBJECTIVE：To establish an isolation and culture system for neural stem cel s from newborn mouse hippocampus, olfactory bulb and cortex and to analyze the biological properties of cel s. METHODS：Neural stem cel s were isolated from the hippocampus, olfactory bulb and cortex tissue of newborn Kunming mice by mechanical separation and trypsin digestion. Serum-free culture technology, mechanical pipetting and trypsin digestion were used for subculture of neural stem cel s. 10%fetal bovine serum was used to induce differentiation of neural stem cel s. Neural stem cel s and their differentiated products were identified by 〈br〉 immunofluorescent staining of Nestin, CD133,β-TubulinIII, glial fibril ary acidic protein. RESULTS AND CONCLUSION：The neural stem cel obtained from newborn mouse hippocampus, olfactory bulb and cortex had the capacity of self-renewal and differentiation which were positive for Nestin and CD133. After induction with fetal bovine serum, neural stem cel could differentiation toβ-tubulinIII or glial fibril ary acidic protein positive cel s that were neurons and astrocytes. This experiment has successful y established the neural stem cel isolation, culture, identification and induction system, providing experimental basis for subsequent studies of neural stem cel s.

关 键 词：干细胞 培养 神经干细胞 神经球 海马 嗅球 皮质 CD133 巢蛋白 神经干细胞库 甘肃省自然科学基金 

分 类 号：R394.2[医药卫生—医学遗传学]