骨髓间充质干细胞来源的神经干样细胞对急性重症一氧化碳中毒大鼠脑结构和功能的重塑

发布时间：2018-03-23 07:34

本文选题：骨髓间充质干细胞　切入点：神经干样细胞　出处：《浙江大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的探讨骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)来源的神经干样细胞(bone marrow mesenchymal stemcells-derived neural stem-like cells,MS-NSCs)对急性重症一氧化碳中毒大鼠脑结构和功能的影响,并对MS-NSCs和BMSCs进行移植后治疗效果的比较评价。方法体外分离90g大鼠长骨干内的BMSCs,运用全骨髓法贴壁培养,定时换液,祛除悬浮的血细胞和其它类干细胞如造血干细胞;运用生物因子的方法将BMSCs向MS-NSCs定向诱导,见克隆增生,并亦定时换液补充营养与能量;运用细胞免疫组化的方法监测两类干细胞在神经标志蛋白如神经特异性核蛋白(neuron-specific nuclearprotein,NeuN)、巢蛋白(nestin)、CD11b等表达与分泌情况;10μmol/l BrdU标记预移植的两类干细胞(BMSCs和MS-NSCs)。建立成年大鼠中毒模型并复苏24h后,将细胞经左颈内动脉移植入脑内,以神经功能严重度评分(neurological severity score,NSS)和Morris水迷宫试验(Morris Water Maze,MWM)评价脑功能的改善程度,5周后病理学上评价脑灰白质结构的重塑情况,包括HE染色,髓鞘染色,胶质纤维酸性蛋白(glial fibrillary acidic protein,GFAP)和2、3-环核苷酸磷酸二脂酶(2′3′-cyclic nucleotide 3′-phosphodiesterase,CNP)免疫组化染色,BrdU免疫组化染色,BrdU和神经标志蛋白如GFAP、CNP、CD11b及微管相关蛋白(microtubule-associatedprotein 2,MAP2)免疫荧光双染色等。结果重症CO中毒后5周,大鼠脑内白质存在局灶脱髓鞘现象,灰质如皮层、海马等处散在存在局灶坏死后腔隙;两类干细胞移植都能够改善白质结构的有序性和致密性及脱髓鞘情况,促进认知记忆功能的恢复,且这种功能恢复在MS-NSCs移植组中更明显(p＜0.05);同时在皮层、海马、基底节局部受损区域和疏松白质内检测到BrdU阳性细胞,且在MS-NSCs移植组中此阳性细胞数更多(p＜0.05)。同时MS-NSCs移植组中某些区域内干细胞向下游终末神经细胞分化的比例更高。结论MS-NSCs能重塑急性重症一氧化碳中毒大鼠的脑结构和功能,且这种作用较BMSCs更明显,提示前者可能是更佳的种子细胞。
[Abstract]:Objective to investigate the effects of bone marrow mesenchymal stemcells-derived neural stem-like cells MS-NSCs derived from bone marrow mesenchymal stem cells (BMSCs) on brain structure and function in acute severe carbon monoxide poisoning rats. Methods BMSCs were isolated from 90 g rat long diaphysis in vitro. The whole bone marrow adherent culture was used to remove suspended blood cells and other kinds of stem cells such as hematopoietic stem cells, and BMSCs was induced to MS-NSCs by biological factor method. Clone proliferation was also observed, and nutrition and energy were supplemented by regular fluid exchange. The expression and secretion of neural marker proteins such as neuron-specific nuclear protein (neuron-specific nuclear protein), nestin protein (nestin) and CD11b (CD11b) in neural marker proteins, including neuron-specific nuclear protein (neuron-specific nuclear protein) and CD11b, were detected by immunohistochemical method. Two types of stem cells, BMSCs and MS-NSCs, were labeled with 10 渭 mol/l BrdU to establish adult rats. After 24 hours of resuscitation, The cells were transplanted into the brain through the left internal carotid artery. Neurological severity score (NSS) and Morris water maze test (Morris water maze test) were used to evaluate the degree of improvement of brain function. Five weeks later, the remodeling of the gray matter structure, including HE staining, was evaluated histopathologically. Myelin staining, glial fibrillary acidic protein (GFAP) and 2- (3) -cyclic nucleotide 3- phosphoesterase (CNP) immunocytochemical staining, BrdU immunohistochemical staining and neural marker proteins such as GFAPCNPCD11b and microtubule-associated protein 2MAP2 immunofluorescent double staining, etc. Results at 5 weeks after severe CO poisoning, there was focal demyelination in the white matter of the rat brain, and the gray matter, such as cortex and hippocampus, were scattered in the space after focal necrosis. Both types of stem cell transplantation can improve the order and density of white matter structure and demyelination, promote the recovery of cognitive memory function, and the recovery of this function is more obvious in the MS-NSCs transplantation group than in the control group (p < 0.05), and also in the cortex and hippocampus. BrdU positive cells were detected in the damaged area of basal ganglia and loose white matter, and the number of BrdU positive cells in MS-NSCs transplantation group was more than 0.05, and the proportion of stem cells differentiated into downstream terminal nerve cells in some areas of MS-NSCs transplantation group was higher than that in MS-NSCs transplantation group. Conclusion MS-NSCs can reshape the brain structure and function of acute severe carbon monoxide poisoning rats, and this effect is more obvious than that of BMSCs, suggesting that the former may be a better seed cell.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

【共引文献】