小鼠小脑皮质发育与神经元凋亡关系的研究

发布时间：2018-02-25 09:24

本文关键词： 小脑皮质组织发生发育神经元凋亡关系　出处：《河南大学》2007年硕士论文　论文类型：学位论文

【摘要】： 小脑位于脑桥和延髓的背面及大脑枕叶的下方,是中枢神经系统中与执行运动机能有关的较高级调节中枢,并且还参与了学习记忆的神经活动过程。成熟小脑皮质有三层结构,包括五种神经元,在这些神经元中最具有特征性的是巨大的Purkinje细胞和较小的颗粒细胞,Purkinje细胞是小脑皮质的主要神经元,颗粒细胞是小脑数量最多的神经元。目前人们对小脑形态、细胞化学构筑、纤维联系、神经递质及不同神经元发生凋亡的可能机制等方面做了较为深入的研究。但有关小脑皮质的具体形态演变过程及发育中的神经元凋亡的变化规律及可能发生的机制的资料相对较少,所以此问题的阐清将更进一步加深对神经系统发育过程规律的认识,也为胚胎小脑移植和某些退化性神经系统疾病提供必要的帮助。为了探讨小鼠小脑皮质的组织发生过程和发育中神经元凋亡的规律和机制及两者之间的关系,我们应用光镜和电镜技术对胚胎和生后小脑皮质进行形态学观察,对生后第2天(P2)至成年鼠小脑皮质各层厚度和细胞密度行形态学测量;用激活型Caspase-3多抗免疫组化标记及Hoechst33258染色液染色检测从出生至成年小鼠小脑皮质中神经元的凋亡,用Western blotting方法对小脑组织中Caspase-3和Caspase-8的活化片段进行半定量测定。结果显示:①胚胎12d(E12)小脑原基有室管膜层、套层和边缘层构成;②约出生当日(P0)出现较典型外颗粒层、分子层、Purkinje细胞层和内颗粒层;③外颗粒层P6/7达最厚,至P20消失;④P0至P30,分子层原基呈CUB型曲线增厚,细胞密度呈S型曲线降低,内颗粒层厚度呈S型曲线增加,细胞逐步分化发育成熟,密度呈CUB型曲线先增加后降低,Purkinje细胞体积呈INV型曲线增大,单位长度细胞密度呈S型曲线降低,树突树逐渐形成,约P7时Purkinje细胞排列成单层;其发育过程中的差异有统计学意义,P0.001;⑤免疫组化:外颗粒层、Purkinje细胞层和内颗粒层凋亡细胞密度最高分别在出生后第8天(P8)、P5及P9左右,约P20各层细胞凋亡密度都很低;⑥Western blotting: Caspase-3活化片段的表达量在P5最高,以后渐降低至P14消失;Caspase-8活化片段的表达量从P0到P10都较高,以后略降低至P30基本消失;其发育过程中神经元凋亡的差异有统计学意义,P0.01。研究结果表明:E12～P0,片层化的小脑主要经历了细胞增殖、分化与迁移;P0～P30,片层化的小脑逐渐发育成熟,其中外颗粒层的消亡以细胞迁移和凋亡为主,而其他各层结构主要经历了细胞的分化、发育与凋亡。P0～P14是小脑皮质神经元迅速发育时期,同时也是其凋亡发生的高峰时期,通过启动Caspase-8的活化进而激活效应性Caspase-3的细胞凋亡途径存在于小脑皮质塑型成熟过程中。
[Abstract]:The cerebellum is located at the back of the pons and medulla oblongata and below the occipital lobe of the brain. It is a higher regulatory center in the central nervous system related to the execution of motor functions, and is also involved in the neural processes of learning and memory. The mature cerebellar cortex has a three-layer structure. Among the five kinds of neurons, the large Purkinje cells and the smaller granular cells are the main neurons in the cerebellar cortex, and the granulosa cells are the most numerous neurons in the cerebellum. At present, people are concerned about the cerebellar morphology, cytochemical architecture, fiber connection, Neurotransmitters and the possible mechanisms of apoptosis of different neurons have been studied in depth. However, the specific morphologic evolution of cerebellar cortex, the changes of neuronal apoptosis in development and the possible mechanism of apoptosis have been studied. There is relatively little data available. Therefore, the explanation of this problem will further deepen the understanding of the rules of nervous system development, and also provide necessary help for embryo cerebellum transplantation and some degenerative nervous system diseases. In order to investigate the histogenesis process of mouse cerebellar cortex, the regularity and mechanism of neuronal apoptosis during development and the relationship between them, we observed the morphology of embryonic and postnatal cerebellar cortex by light microscope and electron microscope. The thickness and cell density of cerebellar cortex from the second day after birth to adult rats were measured by morphology, and the apoptosis of neurons in cerebellar cortex of mice from birth to adult was detected by immunohistochemical staining of activated Caspase-3 and Hoechst33258 staining. The activation fragments of Caspase-3 and Caspase-8 in cerebellar tissue were determined by Western blotting method. The results showed that the primordium of cerebellum had ependymal layer, and the lamina and margin of cerebellum formed a typical outer granular layer (P0). The P6 / 7 of P6 / 7 was the thickest in the cell layer of Purkinje and the outer layer of P6 / 7 in the inner granular layer. By the time P20 disappeared from 4P0 to P30, the primordium of the molecular layer was thickened by CUB curve, the cell density was decreased by S-type curve, and the thickness of the inner granular layer was increased by S-type curve. The cell density increased firstly and then decreased. The cell volume increased as INV curve and the cell density per unit length decreased as S-shaped curve. Dendritic tree was gradually formed and Purkinje cells were arranged as monolayers at P7. The difference in development process was statistically significant (P 0.001): the density of apoptotic cells in Purkinje cell layer and inner granular layer was the highest at the 8th day after birth, and the density of apoptotic cells in the outer granular layer was about P8 + P5 and P9, respectively. The apoptotic density of P20 cells was very low. The expression of Caspase-3 activation fragment was the highest in P5, then decreased to P14 disappeared. The expression of Caspase-8 activated fragment was higher from P0 to P10, then decreased slightly to P30. The difference of neuronal apoptosis during its development was statistically significant (P 0.01). The results showed that the lamellar cerebellum mainly experienced cell proliferation, differentiation and migration, and the lamellar cerebellum matured gradually. The death of the outer granular layer was mainly cell migration and apoptosis. The other layers mainly undergo cell differentiation. Development and apoptosis. P0 P14 is the period of rapid development of cerebellar cortical neurons, and also the peak period of apoptosis. The apoptotic pathway of Caspase-3 by activating the activation of Caspase-8 exists in cerebellar cortical plastic maturation.
【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R33

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