脑源性神经营养因子促进神经干细胞在体内外的定向分化  

Brain derived neurotrophic factor promotes the oriental differentiation of neural stem cells in vitro and in vivo

作　　者：黄保胜[1] 李立新[1] 谢青松[1] 田和平[1] 胡卫星[1] 傅震[1] 

机构地区：[1]南京医科大学第一附属医院神经外科,江苏省南京市210029

出　　处：《中国组织工程研究与临床康复》2007年第37期7353-7357,共5页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：江苏省"一三五工程"医学重点人才基金资助项目(RC2003086)~~

摘　　要：目的:观察脑源性神经营养因子促进神经干细胞在体内外条件下定向分化的作用,探索神经干细胞在体内外定向分化的规律。方法:实验于2006-09/2007-04在南京医科大学分子生物学实验室和动物实验中心完成。①体外研究:取孕14～16d的胚胎大鼠脑组织,以无血清培养基培养获得神经干细胞,随即分成两组进行体外研究。实验组(n=10),加入5%胎牛血清和20μg/L脑源性神经营养因子诱导其体外条件下定向分化,对照组(n=10),仅加入5%胎牛血清,用免疫荧光及流式细胞仪的方法来检测定向分化得到的神经元及其比例。②体内研究:参照Bavetta等的方法制作大鼠脊髓(T10)右半切4mm长块状缺损模型。实验组(n=10):注射移植溴脱氧尿苷标记的细胞密度为1×109L-1的神经干细胞悬液20μL。对照组(n=10):注射移植20μLPBS于模型动物中。12周后,行辣根过氧化物酶神经逆行示踪评价脊髓感觉和传导功能的恢复程度,并取损伤处脊髓组织行免疫组织化学染色,观察缺损处打入的神经干细胞的存活,分化和迁移情况。结果:参加体内实验的20只大鼠均进入结果分析。①体外条件下,两组神经干细胞定向分化后均得到微管相关蛋白2阳性神经元,并且实验组微管相关蛋白2阳性神经元的比例在分化后第3天达最高峰,约为60%,而同期对照组仅约为30%,差异有统计学意义(P<0.05)。②体内条件下,移植12周后,免疫组织化学染色显示:体内实验组损伤脊髓处可见溴脱氧尿苷标记的阳性细胞,且缺损处可见大量神经微丝相关蛋白和神经生长相关蛋白染色阳性的神经元,体内对照组则未见以上两种蛋白染色阳性的神经元。辣根过氧化物酶神经逆行示踪显示:体内实验组脑组织可见到部分辣根过氧化物酶标记阳性神经元,而体内对照组未见辣根过氧化物酶阳性神经元。结论:脑源性神经营养因子促进神经干细胞体内外条件下AIM： To observe the role of brain derived neurotrophic factor （BDNF） on the oriental differentiation of neural stem cells （NSCs） both in vitro and in vivo, and investigate the differentiation law. METHODS： The experiments were conducted at the Molecular Biology Laboratory and the Animal Experiment Center of Nanjing Medical University from September 2006 to April 2007. ①In vitro study： NSCs were isolated from 14-16 days embryonic rat cerebral tissue and cultured in serum-free medium. After NSCs were harvested, they were randomly divided into two groups： experimental group （n =10） was added into 5% fetal bovine serum and 20 μg/L BDNF for in vitro differentiation; control group （n =10） was added into 5% fetal bovine serum only. Immunofluorescence and flow cytometer were applied to detect the neurons and their percentage after differentiation. ② In vivo study： According to Bavetta＇s method, animal model was made in right side half-cut lump of the spinal cord in 20 rats by cutting at T10 and then were randomly divided into two groups. The experimental group （n =10） was transplanted with 20 μL suspension of 1×10^9L^-1 NSCs marked with 5-bromodeoxyuridine （BrDU）. The control group （n =10） was transplanted with 20 μL phosphate buffered solution only. The recovery of sensory and conduction functions of spinal cord was evaluated with horseradish peroxidase （HRP,） tracing technique after 12 weeks. The survival, differentiation and migration of NSCs in the injured spinal cord were detected with immunohistochemistry. RFSULTS： Twenty rats entering in vivo study were all involved in the result analysis.①Two groups of NSCs differentiated into microtubule-associated protein （MAP）-2 positive neurons in vitro. The proportion of MAP-2-positive neuron in the experimental group reached its peak on the 3^rd day, which was approximately 60%, but only about 30% was found in the control group. Statistical analysis showed the significant differences between the two groups （P 〈

关 键 词：神经干细胞 脑源性神经营养因子 神经元 定向分化 

分 类 号：R394.2[医药卫生—医学遗传学]