神经干细胞共移植体的体外构建  

Constructing the co-implant of neural stem cells in vitro

作　　者：朱晓峰[1] 齐志国[1] 张晓梅[1] 

机构地区：[1]佳木斯大学神经科学研究所,黑龙江省佳木斯市154007

出　　处：《中国组织工程研究与临床康复》2007年第24期4756-4759,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：国家自然科学基金项目(30450062);黑龙江省自然基金重点项目(ZJY0508)~~

摘　　要：目的:选择合适的细胞外基质,利用脑微血管内皮细胞、细胞外基质与神经干神胞构建神经干细胞共移植体,以提高移植神经干神胞向神经元的分化率。方法:实验于2005-11/2006-10在佳木斯大学神经病研究所免疫组织化学研究实验室完成。①实验材料:同一基因背景出生2h～7d清洁级Wistar大鼠由哈尔滨医科大学实验动物学部提供。取出生24h内Wistar大鼠大脑制备神经干细胞,采用免疫组织化学法和免疫荧光法做nestin染色鉴定;取出生1～7d Wistar大鼠大脑制备脑微血管内皮细胞,采用免疫细胞化学方法鉴定。②实验方法:不同细胞外基质的筛选:将多聚赖氨酸、层黏连蛋白和Matrigel分别与神经干细胞和脑微血管内皮细胞混合培养,接种于铺有盖玻片的6孔板中,并加入神经干细胞完全培养液,每3d换半量液,7d换全液。采用免疫组织化学观察抗微管相关蛋白阳性细胞。进行神经干细胞共移植体构建及检测。③实验评估:利用免疫荧光方法,以Ⅷ因子标记物标记脑微血管内皮细胞,用nestin标记神经干细胞,检测共移植体。结果:①神经干细胞和脑微血管内皮细胞形态学观察及鉴定:原代分离的单神经干细胞4d增生为神经细胞球,传代后,经nestin免疫荧光染色,细胞球呈阳性;原代培养脑微血管内皮细胞6～8d长满瓶壁,细胞呈多角形或扁平梭形,核卵圆形,Ⅷ因子相关抗原阳性。②3种细胞外基质对神经干细胞分化的影响:层黏连蛋白与两种细胞共培养,抗微管相关蛋白阳性细胞数明显高于Matrigel组和多聚赖氨酸组(P<0.01)。③共移植体活细胞镜下及免疫荧光结果所见:镜下可见共移植体细胞团不规则,神经干细胞被脑微血管内皮细胞包绕或相互连贴,脑微血管内皮细胞核大,胞体不规则,明显大于神经干细胞,神经干细胞折光性强。在200倍视野下,随机选取100个共移植体进行细胞计数,可见平均细胞AIM： To construct the combinative implant of neural stem cells which consist of neural stem cells （NSCs）, vascular endothelial cells （VECs） and appropriate extracellular matrix （ECM）, and improve the rate of differentiation from NSCs to neurons. METHODS： The experiment was carried out from November 2005 to October 2006 in the Immunohistochemistry Laboratory, Institute of Neuroscience in Jiamusi University.①Expedmental materials： Wistar rats, which were born from two hours to seven days, were supported by Board of Experimental Creature in Harbin Medical University; NSCs were made from Wistar rats which were born in 24 hours. Immunohistochemistry and immunofiuorescence label were used for identification of nestin pigmentation. VECs were made from brains of Wistar rats, which were born for 1-7 days, and were identified by immunohistochemistry. ②Filtration of the different ECMs： poly-I-lysine, Laminin and Matrigel were co-cultured with NSCs and VECs separately, and inoculated on coverslips in six poles plates added with NSC complete medium, and semi-serum was changed every 3 days and whole serum was changed every 7 days. Immunohistochemistry was used to observe microtubule-associated protein （MAP）-positive cells. The combinative implant of NSCs was constructed. ③Immunofluorescence label was to observe the structure of the combinative implant that VECs were labeled with Ⅶfactor and NSCs were labeled with nestin. RESULTS： ①Morphology and identification of NSCs and VECs： Primary isolated single NSC proliferated into neurosphere after 4 days. After passaging, neurosphere was positive when immunofiuorescence label was used for nestin. Isolated VECs grew to monolayer after 6-8 days of external cultivation. The cells were prese polygonal or fiat fusiform, and the nuclei shaped as orbicular-ovate. Correlation antigens of Ⅷ factor were positive by fluorescent stain.② Effect of three different ECMs on differentiation of NSCs： MAP-positive cells of co-culturing Laminin with NSCs and V

关 键 词：神经干细胞 移植体 构建 

分 类 号：R394.2[医药卫生—医学遗传学]