摘要
目的:观察不同比例的脑微血管内皮细胞对体外培养的神经干细胞向神经元分化的影响。方法:实验于2005-12/2006-10在佳木斯大学神经病学研究所完成。①实验动物:取新生6h内Wistar小鼠4只用于神经干细胞的培养。另取新生1～7d Wistar大鼠4只用于脑微血管内皮细胞的培养。②实验方法:取新生神经球按5×108L-1接种于预先置有盖玻片的6孔板中,2mL/孔,并加入含表皮生长因子和碱性成纤维细胞生长因子的DMEM/F12条件培养基,于24h后行巢蛋白免疫组化鉴定。取脑微血管内皮细胞按3×107L-1密度接种于铺有盖玻片的6孔板中,2mL/孔,采用Ⅷ因子相关抗原进行鉴定。神经干细胞与脑微血管内皮细胞共同接种于6孔板,接种密度分别为100∶1,10∶1,1∶1,1∶10,1∶100,每种密度均设6孔,以单纯神经干细胞作为对照。培养液为不含胎牛血清的神经干细胞培养液,2mL/孔,每2～3d全量换液,共培养14d,密切观察细胞的生长情况。③实验评估:利用免疫组化法计数神经微管相关蛋白2阳性细胞数,观察神经干细胞定向分化为神经元的情况。结果:①细胞形态观察:神经干细胞聚集成球状,细胞团呈棕黄色,神经球呈悬浮生长,细胞排列紧密;脑微血管内皮细胞呈“鹅卵石样”或“铺路石样”排列生长,细胞呈多角形或扁平梭形,核卵圆形,可见核仁。②神经干细胞与脑微血管内皮细胞鉴定结果:新生神经球巢蛋白免疫组化染色阳性,细胞胞浆深染呈棕黄色,胞核不着色。脑微血管内皮细胞Ⅷ因子相关抗原免疫组化染色胞浆显示棕黄色或褐色颗粒。③神经微管相关蛋白2免疫化学检测:培养14d与单纯神经干细胞对照组比较,神经干细胞与脑微血管内皮细胞100∶1,10∶1,1∶1,1∶10,1∶100接种密度共培养组神经微管相关蛋白2阳性细胞数均明显升高(19.00±5.12),(34.46±11.57),(72.83±7.54),(60.71±10.45),(43.08±7.46),(31.08±4.60)个,F=35
AIM： To observe the effect of brain microvascular endothelial cell （VEC） at various proportions on the differentiation of neural stem cells （NSCs） into neurons in vitro. METHODS： The experiment was carded out in the Institute of Neuroscience of Jiamusi University from December 2005 to October 2006. ①Four Wistar rats of 6 hours old were selected for the culture of NSCs, meanwhile, four Wistar rats of 1-7 days old were selected for the culture of VECs. ②5×10^8L^-1 neural spheres were seeded in 6-well culture plate with coverslips, 2 mL per well, in which DMEM/F12 culture medium with epidermal growth factor （EGF） and basic fibroblast growth factor （bFGF） were added. Twenty-four hours later, Nestin immunohistochemistry identification was done. Meanwhile, 3×10^7 L^-1 VECs were seeded in the 6-well culture plate with coverslip, 2 mL per well, then identified with factor Ⅷ related antigen. Additionally, NSCs and VECs were co-cultured in a 6-well culture plate at proportions of 100：1, 10：1, 1：1, 1：10, and 1：100, 6 wells for every concentration in the NSC culture fluid without fetal bovine serum, 2 mL per well, changed every 2-3 days for 14 days, and the simple NSCs served as control. The growth of cells was observed carefully. ③Immunohistochemistry technique was employed to count the number of neurotubule-associated protein-2 positive cells and the differentiation of neurons from NSCs was examined. RESULTS： ①Cell morphous： NSCs integrated globulady, and the cell group was brownish yellow; neural sphere was floating, and cell arrayed tightly; VECs grew in cobble-stone or paved-stone, and the cells were polygon or fiat fusiform in shape with orbicular-ovate nucleus. ② Identification of NSCs and VECs： Neural sphere was positive in nestin immunohistochemistry and the cytoplasm was deeply stained in buffy, but the nucleus was not stained. Factor Ⅷrelated antigen immunohistochemistry staining showed that the cytoplasm of VECs was buff5, or brown. ③The number of positive neu
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第24期4670-4673,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然科学基金项目(30450062)
黑龙江省自然科学基金重点项目(ZJY0508)~~
