人胚神经干细胞植入新生鼠侧脑室后迁移与分化的研究

发布时间：2018-06-24 16:51

本文选题：人胎脑神经干细胞 + 新生鼠　；参考：《安徽医科大学》2008年硕士论文

【摘要】： 目的建立人胎脑神经干细胞(hNSC)在新生鼠脑内迁移与分化的模型,采用免疫组化的方法比较外源性神经干细胞球和单细胞悬液在植入宿主体内迁移与分化规律,为选择合适的hNSC移植方法治疗各种新生儿脑病提供实验基础。材料与方法 1.细胞培养用添加表皮生长因子(EGF)+碱性成纤维细胞生长因子(bFGF)+白血病抑制因子(LIF)的N2培养基从流产的人胚胎前脑组织培养出神经干细胞球;取部分神经干细胞球通过免疫荧光技术检测神经干细胞巢蛋白(Nestin)鉴定其干细胞属性,诱导分化神经干细胞,并以抗-人神经丝蛋白(NF)和抗-人酸性胶质纤维蛋白(GFAP)分别鉴定神经干细胞(NSC)分化成的神经元及星形胶质细胞,证实神经干细胞具有分化能力。 2.将神经干细胞分为细胞球和单细胞悬液(采用Dispase酶消化)两种,调整浓度为5.0×10~4cells/ul待移植时用。 3.动物分组36只9日龄、体重20-25g的清洁级雌雄不限的SD大鼠,随机分为3组,分别植入细胞球(细胞球组)、单细胞悬液(单细胞悬液组)和F12培养液(对照组)。 4.于移植24、48、72 h通过取脑,制作石蜡切片,免疫组织化学观察植入细胞在受体鼠内的迁移分化情况,并比较两组实验结果的差异。结果 1.在添加EGF等的培养基上培养出来的是典型的神经细胞球,培养6 d,观察到大量的形态并不规整的小细胞集落,12 d后形成典型的神经球。免疫荧光鉴定细胞网络分化的细胞中有NF(+)、GFAP(+),GalC(+),证实细胞球能分化成神经元、星形胶质细胞和少突胶质细胞。 2.移植入脑室内的两种类型细胞,其在脑内存活迁移的结果明显不同。①细胞球移植组迁移情况:移植后24 h可在脑室内见到大量Anti-hNuc(+)细胞:48 h后可看到侧脑室、脑室壁上、脉络膜及嗅球附近见到大量Anti-hNuc(+)细胞;72 h大量Anti-hNuc(+)细胞在鼠脑额叶、枕叶和海马均有分布。72h后分化情况:大脑皮层的颗粒层和锥体细胞层及嗅球处有分化成熟的神经元,胞体中央有不着色的圆形细胞核,胞浆和突起均发出红色荧光,并可观察到阳性细胞间有突起-突起间和突起-胞体间的连接;②单细胞悬液组:移植24 h可在脑室见到大量Anti-hNuc(+)细胞,72 h在脑室和脑室内壁上仅有少量Anti-hNuc(+)细胞分布;72h后分化情况;未见明显分化。结论 1.体外培养获得的人神经干细胞具有分化为神经元及胶质细胞的潜能。 2.人胎脑神经干细胞(hNSC)移植入鼠侧脑室后不仅能够存活、迁移和分化为神经元和胶质细胞。而且细胞之间突起-突起间和突起-胞体间的连接。 3.细胞球移植法迁移分化能力显著高于单细胞悬液移植法。
[Abstract]:Objective to establish a model of migration and differentiation of human fetal neural stem cells (hNSC) in the brain of newborn rats, and to compare the migration and differentiation of exogenous neural stem cell balls and single cell suspensions in the implanted host by immunohistochemical method. To provide experimental basis for the selection of appropriate transplantation of hNSC for the treatment of various neonatal encephalopathy. Materials and methods 1. Neural stem cells were cultured in N2 medium supplemented with epidermal growth factor (EGF) basic fibroblast growth factor (bFGF) leukemia inhibitory factor (LIF) from aborted human embryonic forebrain tissue. Some neural stem cell spheres were used to detect the nestin of neural stem cells (NSCs) by immunofluorescence to identify their stem cell properties and to induce the differentiation of neural stem cells (NSCs). Neurons and astrocytes differentiated from neural stem cells (NSC) were identified by anti-human neurofilament protein (NF) and anti-human acidic glial fibrin (GFAP), respectively. Neural stem cells (NSCs) were divided into cell spheres and single cell suspensions (digested by Dispase) with an adjusted concentration of 5.0 脳 10~4cells/ul. Thirty-six 9-day-old SD rats weighing 20-25 g were randomly divided into three groups: cell ball group (cell ball group), single cell suspension group (single cell suspension group) and F12 culture medium (control group). After 24 minutes and 48 minutes 72 hours of transplantation, paraffin sections were made and the migration and differentiation of implanted cells in recipient mice were observed by immunohistochemistry, and the differences between the two groups were compared. Result 1. Typical neuronal spheres were cultured on the medium supplemented with EGF. After 6 days of culture, a large number of irregular small cell colonies were observed to form typical neuronal spheres after 12 days. NF-GFAP () GFAP () GalC (), showed that the cells could differentiate into neurons, astrocytes and oligodendrocytes by immunofluorescence. 2. The survival and migration of the two types of cells transplanted into the ventricle were significantly different from those in the 1-cell ball transplantation group: a large number of Anti-hNuc () cells could be seen in the ventricle at 24 h after transplantation, and the lateral ventricle and the wall of the ventricle could be seen at 48 h after transplantation. A large number of Anti-hNuc () cells were observed in the vicinity of choroid and olfactory bulb for 72 h. A large number of Anti-hNuc () cells were distributed in frontal lobe, occipital lobe and hippocampus. 72 h later, there were differentiated neurons in granular layer, pyramidal cell layer and olfactory bulb of cerebral cortex. There are round nuclei in the center of the cell body, and the cytoplasm and process emit red fluorescence, and the connections between the positive cells and the processes between the positive cells can be observed. 2 single cell suspension group: after 24 h transplantation, a large number of Anti-hNuc () cells could be seen in the ventricle. After 72 h, there were only a few Anti-hNuc () cells distributed in the ventricle and the inner wall of the ventricle. Conclusion 1. In vitro cultured human neural stem cells have the potential to differentiate into neurons and glial cells. 2. Human fetal neural stem cells (hNSC) can not only survive, migrate and differentiate into neurons and glial cells, but also be transplanted into the lateral ventricle of rats. And the connections between processes-processes and processes-bodies. 3. The migration and differentiation ability of cell ball transplantation was significantly higher than that of single cell suspension transplantation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329

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