摘要
目的:将由神经干细胞、层黏连蛋白、脑微血管内皮细胞构建的共移植体植入缺血模型鼠大脑内,观察共移植体中神经干细胞的增殖和凋亡方法:实验于1996-01/1996-12在佳木斯大学神经科学所完成实验材料:同一基因背景Wistar大鼠,体质量300～350g,雄性,6月龄,购自哈尔滨医科大学实验动物学部实验方法:①取出生24h内Wistar新生鼠,培养神经干细胞②将传代脑微血管内皮细胞消化后,以3×107L-1密度接种于培养瓶中,12h贴壁后吸出脑微血管内皮细胞培养液,涂以一层细胞外基质后,接种经0.125%胰酶消化20min后吹打成单细胞的神经干细胞悬液,密度为6×108L-1,加入神经干细胞完全培养液培养3～6h,镜下观察神经干细胞完全贴壁后,吸出培养液,再涂以一薄层细胞外基质,接种以上相同密度脑微血管内皮细胞,加入神经干细胞完全培养液共培养12～24h,镜下观察脑微血管内皮细胞利用免疫荧光方法检测共移植体③制备大鼠脑缺血模型模型制备完成后大鼠虽然有脑缺血症状,但伴下列情形之一的不纳入:神经学症状评分低于2分;;取脑时发现蛛网膜下腔出血;;脑组织石蜡切片苏木精-伊红染色无缺血病理改变;;未到观察时相点便死亡的共48只大鼠模型制作成功将Wistar大鼠随机分为神经干细胞组、共移植体组,每组24只2组分4个时间点3,7,14,60d,每个时间点6只模型建立后3d,神经干细胞组移植神经干细胞,共移植体组移植共移植体,各5μL移植前神经干细胞经Brdu5mmol/L标记3d,再将其溶解于PBS中,细胞密度调整为2×1011L-1,移植部位为右侧纹状体区通过免疫组织化学和免疫荧光方法观察神经干细胞增殖和凋亡情况结果:48只大鼠均进入结果分析①神经干细胞增殖情况:细胞数3d开始增加,14d达高峰,60d减少两组3,7,14,60d细胞数相比,差异显著(神经干细胞组:19.57±4.27,22.25±4.53,39.13±4.52,7.13±2.75;共移植体组:39.88�
AIM： Transplants constructed by neural stem cells, laminin and brain vascular endothelial cells （VECs） were implanted into cerebrums of ischemic model rats. This study was to observe proliferation and apoptosis of the neural stem cells of co-cultured body. METHODS： The experiment was conducted at the Institute of Neuroscience, Jiamusi University from January to December 1996. Experimental materials： The identical gene background male Wistar rats with the body mass of 300- 350 g, 6 months of age, was purchased from Experimental Animal Education Ministry of Harbin Medical College. Experimental technique： （1）The Wistar rats newborn within 24 hours were obtained and neural stem cells were cultured. （2）After brain VECs were digested, VECs were seeded at a density of 3×10^7 L^-1 in the culture flask. After 12 hours, culture medium was sucked, and brain VECs were smeared with laminin, then neural stem cell suspension at a density of 6×10^8 L^-1 was put into the same culture flask, and then complete culture medium of neural stem cells was added. Three to six hours after culture, neural stem cell adhered to the wall was observed under microscope, and then smeared with a thin layer of laminin after sucking culture medium. Brain VECs of the same density were vaccinated and complete culture medium for neural stem cells was added. 12-24 hours after co-culture, VECs were observed under microscope and co-cultured transplants were tested by immunofluorescence method. （3）Cerebral ischemia rat models were prepared. Exclusive criteria were as follows： rat models of cerebral ischemia with less than 2 points of neurological symptom, subarachnoid hemorrhage, no ischemic pathological change were found in paraffin section of brain tissues by hematoxylin-eosin （HE） and rats died before observation. Totally 48 rat models were successfully established. The Wistar rats were randomly divided into neural stem cell group and co-cultured transplant group with 24 in each group. There were 4 time points, namely 3,
出处
《中国组织工程研究与临床康复》
CAS
CSCD
北大核心
2007年第20期3960-3963,共4页
Journal of Clinical Rehabilitative Tissue Engineering Research
基金
国家自然基金项目(批准号30450062)
黑龙江省自然基金重点项目(批准号ZJY0508)~~
