SD大鼠胚胎神经干细胞悬浮培养与鉴定  

Experimental Research of Suspension Culture and Identification of SD Rat Embryonic Neural Stem Cell

作　　者：贺菊芳[1] 余资江[1] 朱晓瓞 方丽丽[1] 王俊婕 HE Jufang YU Zijiang ZHU Xiaodie FANG Lili WANG Junjie(Department of Human Anatomy, Guizhou Medical University, Guiyang 550025, Guizhou, Chin)

机构地区：[1]贵州医科大学,人体解剖学教研室,贵州贵阳550025

出　　处：《贵州医科大学学报》2016年第10期1128-1132,共5页Journal of Guizhou Medical University

基　　金：国家自然科学基金(81060108);贵州省科技厅2012年社会发展攻关项目[黔科合SY字(2012)3153号]

摘　　要：目的:研究神经干细胞(NSCs)的分离、培养及鉴定方法。方法:以孕12.5 d SD胚胎鼠端脑为组织来源,应用无血清悬浮培养技术培养、扩增NSCs;NSCs传代2次后,应用Nestin免疫荧光检测NSCs的干细胞特性,Brd U标记法检测细胞增殖能力;诱导贴壁分化后,对分化细胞进行NSE、GFAP免疫荧光鉴定;扫描电镜观察神经球及单个细胞表型。结果:培养出大量悬浮生长的NSCs球,Nestin及Brd U表达阳性;神经球诱导分化后表达NSE、GFAP,表明NSCs可分化为神经元和星形胶质细胞;HE染色可见NSCs细胞核巨大,胞质较少;扫描电镜可观察到单个NSC表面较光滑,且神经球表面的NSCs连接较疏散。结论:应用无血清培养技术体外培养扩增出的胚胎神经干细胞具有自我更新和多向分化能力,经诱导后可分化为神经元和星形胶质细胞。Objective： To investigate isolation,culture and identification of Neural Stem Cells（ NSCs）. Methods： Pregnant 12. 5 d SD rats＇ embryonic brains tissue was collected and cultured by serum-free suspension for amplification of NSCs. 2 times after passage,Nestin immunofluorescence was adopted to detect NSCs characteristics of cells. Brd U labeling method was adopted to detect cells＇ proliferation ability. After induced adherence differentiation,the differentiated cells were identified by NSE,GFAP immunofluorescence. Scanning electron microscope was adopted to observe the phenotype of neural spheres and single cell. Results： A large number of NSCs ball of suspended growth were cultured with positive expression of Nestin and Brd U. The expression of NSE,GFAP after induced differentiation indicated that NSCs could be differentiated into neurons and astrocytes. HE staining showed that the NSCs nucleus was large and the cytoplasm was less. It was observed under scanning electron microscope that the surface of a single NSC was smooth,and the NSCs connection on the surface of the nerve ball was loose. Conclusion： The embryonic neural stem cells cultured and amplified in vitro with serum-free suspension have self-renewal and multi-directional differentiation ability,which can be differentiated into neurons and astrocytes after induction.

关 键 词：神经干细胞球 无血清 悬浮培养 多向分化 大鼠 Sprague Dawley 

分 类 号：R322.8[医药卫生—人体解剖和组织胚胎学]