NeuroD在大鼠脑发育过程中的差异性表达

发布时间：2018-03-20 07:19

本文选题：碱性螺旋-环-螺旋　切入点：神经源性分化因子　出处：《福建医科大学》2008年硕士论文　论文类型：学位论文

【摘要】： 目的探讨神经源性分化因子(Neurogenic Differentiation,NeuroD)在大鼠神经系统发育过程中,表达量及部位的变化情况,进而研究其对神经分化的影响。方法利用神经细胞标志分子NSE和星形胶质细胞标记分子GFAP,对发育不同时期的大鼠脑组织进行NeuroD与NSE、GFAP的共标记免疫荧光染色,激光共聚焦显微镜检测蛋白在鼠脑中的分布情况;利用蛋白质免疫印迹(Western Blot)对NeuroD蛋白在各脑组分和不同发育阶段的表达进行半定量分析,利用RT-PCR方法检测NeuroD mRNA在大鼠脑不同发育时段的表达变化,进而确定NeuroD在鼠脑发育过程中表达的时空特异性。结果免疫荧光染色结果显示:NeuroD蛋白(绿)分布区域与神经元(NSE)(红)相重合,与星形胶质细胞(GFAP)(红)部分相符。NeuroD主要分布于大脑皮质,其次在小脑也有部分表达,在间脑内,NeuroD在侧脑室附近见有多量表达。蛋白质免疫印迹结果显示,NeuroD在脑组织各组分中均有表达,其中以大脑皮质表达最为丰富,其次为间脑和小脑。NeuroD蛋白在大鼠脑发育的不同时期都有表达,从神经管发生的E9.5天到脑形态初步建立的E12.5天,NeuroD的表达呈现明显升高,E12.5天达到最高峰,在胚胎发育的晚期,脑组织形态得到进一步的完善,在E21.5天脑组织各部分进一步成熟,此时NeuroD达到第二次高峰,而出生后NeuroD表达无明显的差异。NeuroD mRNA在E7.5天时即开始有表达,目的基因和β-actin在E10.5天至E12.5天之间先后达到高峰,出生前(E21.5天)形成第二次表达高峰,RT-PCR结果与Western-blotting结果基本一致。结论在不同发育时期的大鼠脑组织中,NeuroD的表达在时间和空间上呈现出明显的差异性:在神经系统发育早、中期,即从E9.5天神经管闭合至E12.5天形成前脑、中脑和后脑的发育原基,和胚胎发育的后期,即E21.5天脑组织各部分进一步成熟,脑组织形态得到进一步的完善,NeuroD的表达均有明显升高,而出生后NeuroD表达无明显的差异,推测NeuroD的主要作用有:1、对神经细胞的早期形成、分化起作用;2、在神经系统成熟阶段对神经细胞间的突触联系的进一步完善也有一定作用;3、NeuroD在侧脑室附近也见有明显表达,推测其可能与室管膜上皮下的神经干细胞迁移分化有关,还有待进一步实验证实。
[Abstract]:Purpose. To investigate the changes of neurogenic differentiation factor NeuroDduring the development of rat nervous system, and to study the effect of neurogenic differentiation on neurogenic differentiation. Method. NSE and GFAP were used to detect the distribution of NeuroD and NSE GFAP in the brain of rats at different stages of development. The distribution of protein in the brain was detected by confocal laser microscopy. The expression of NeuroD protein in different brain components and different developmental stages was semi-quantitatively analyzed by Western blotting. The expression of NeuroD mRNA in different developmental stages of rat brain was detected by RT-PCR method. Furthermore, the spatiotemporal specificity of NeuroD expression during brain development was determined. Results. The results of immunofluorescence staining showed that the distribution of neuroD protein (green) coincided with that of NSEP (red), and that the neuroD was mainly distributed in the cerebral cortex, and was also partially expressed in the cerebellum. In diencephalon, neuroD was expressed in the vicinity of lateral ventricle. Western blot showed that NeuroD was expressed in all components of brain, especially in cerebral cortex. Secondly, diencephalon and cerebellar neuroD protein were expressed at different stages of brain development in rats. The expression of NeuroD increased significantly from the day of neurotubule development to the day 12. 5, and reached the peak at day 12. 5, and reached the peak at the late stage of embryonic development. The morphology of brain tissue was further improved, and the parts of brain tissue matured further on the day of E21.5, at which NeuroD reached the second peak, but there was no significant difference in the expression of NeuroD after birth. NeuroD mRNA began to express at E7.5. Objective the gene and 尾 -actin reached the peak between E10.5 and E12.5 days, and the second peak expression of 尾 -actin was observed on E21.5 days before birth. The results of RT-PCR were consistent with the results of Western-blotting. Conclusion. The expression of NeuroD in brain tissues of rats at different developmental stages showed significant difference in time and space: the developmental primordia of forebrain, midbrain and hindbrain were formed in the early stage of nervous system development, in middle stage, i.e. from day 9.5 to day 12.5. The expression of neuroD was significantly increased in the later stage of embryonic development, i.e. the maturation of all parts of brain tissue at E21.5 day, but there was no significant difference in the expression of NeuroD after birth. It is speculated that NeuroD plays a major role in the early formation and differentiation of nerve cells, and in the further improvement of synaptic connections between nerve cells in the mature stage of the nervous system. It may be related to the migration and differentiation of neural stem cells under ependymal subcutaneous.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R329.21

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