TRIM32的缺失导致小鼠大脑皮层神经元发生障碍的研究

发布时间：2018-05-18 04:45

本文选题：TRIM32 + 放射胶质细胞　；参考：《南方医科大学》2017年硕士论文

【摘要】：研究背景大脑皮层的发育(cortex development)是一个复杂而又精细的过程。在啮齿类动物中,神经元主要在胚胎发育的E12到E18之间产生,E15时达到高峰,出生后停止;神经胶质细胞在E18天左右开始产生,在新生期达到高峰。在脑室区域(ventricular zone,VZ)的背侧,分布着一群放射状胶质细胞(Radial glial cells,RGCs)的细胞,这群细胞通过增殖分化产生了皮层的兴奋性椎体神经元。RGCs在自我更新的同时,迁移分化为椎体神经元,组成分为六层的脑皮层。TRIM32(Tripartite motif 32)是一种具有E3泛素链接酶活性的蛋白,其蛋白结构中具有TRIM家族特征性的锌指结构域、B-boxes结构域和卷曲结构域,以及特征性的6个重复性的NHL结构域。TRIM32参与了某些中枢神经系统的疾病发生,例如:阿尔茨海默病(Alzhermer' s Disease,AD)、注意力缺陷多动症(Attention Deficit Hyperacitivity Disorder,ADHD),我们前期研究发现,TRIM32高表达于小鼠成熟的大脑皮层神经元以及胎脑的MGE区中间神经前体细胞中,TRIM32的缺失能够导致中间神经节隆起(Medial ganglionic eminence,MGE)区域的中间神经前体细胞增殖分裂减少,进而导致了 γ-氨基丁酸能(GABAergic)抑制性神经元的减少,从而引起了小鼠自闭症样的行为学表现。本研究主要探讨的是TRIM32在调控皮层兴奋性神经元的产生过程中的作用。研究目的1.TRIM32基因在小鼠大脑皮层细胞表达情况的研究;2.TRIM32的遗传缺失对小鼠胚胎时期大脑皮层神经元产生的影响研究;3.TRIM32的遗传缺失对小鼠胚胎时期皮层VZ/SVZ区神经前体细胞产生的影响;4.TRIM32的遗传缺失影响小鼠胚胎时期皮层VZ/SVZ区神经前体细胞产生的机制研究。研究方法1.TRIM32基因在小鼠大脑皮层细胞表达情况野生型小鼠交配后母鼠孕第E13.5、E16.5、E18.5时取大脑标本,切片后进行TRIM32荧光染色。2.TRIM32的遗传缺失对小鼠皮层成熟神经元产生的影响DAPI染色比较皮层CNx和CP的厚度,然后通过大脑各皮层神经元的特异性marker来标记相应神经元,计数统计后比较差异。3.TRIM32的遗传缺失对小鼠胚胎时期皮层VZ/SVZ区神经前体细胞产生的影响PAX6、TBR2荧光染色标记皮层VZ/SVZ区神经前体细胞,计数统计后比较差异。4.TRIM32的缺失对神经前体细胞分裂、增殖的影响Edu和PH3荧光分别标记增殖和分裂的细胞,计数统计后比较差异。5.TRIM32的缺失对皮层神经细胞凋亡的影响Caspase3荧光染色标记凋亡状态细胞,计数统计后比较差异。6.统计学分析统计结果采用均数±标准差(x± s)表示,采用SPSS20.0软件结果,以P0.05认为有统计学差异。研究结果1.TRIM32基因在小鼠大脑皮层细胞表达情况E13.5时,TRIM32主要表达在VZ与SVZ区细胞的胞质中,E16.5时TRIM32高表达于IZ区细胞,而到E18.5时,TRIM32高表达于TBR1阳性成熟神经元胞核。2.TRIM32的遗传缺失导致了皮层厚度缩小以及小鼠皮层成熟神经元数量减少E18.5 时,TRIM32-/-胎鼠皮层 NCx 和 CP 的均缩小(NCx:t=-4.709,P=0.026;CP:t=-7.634,P=0.011);E14.5,E16.5 和 E18.5 三个时期,TRIM32-/-胎鼠 TBR1阳性神经元数量均减少,有统计学差异(E14.5,t=-4.433,P=0.032;E16.5,t=-5.549,P=0.022;E18.5,t=-7.793,P=0.021);E16.5和 E18.5 两个时期,TRIM32-/-胎鼠BCL11b阳性神经元数量均减少,有统计学差异(E16.5,t=-6.691,P=0.037;E18.5,t=-12.047,P=0.036);E18.5 和 P30 两个时期,TRIM32-/-胎鼠 CUX1 阳性神经元数量明显减少,有统计学差异(E18.5,t=-7.622,P=0.047;P30,t=33.956,P=0.014)。3.TRIM32的遗传缺失导致神经前体细胞的数量减少E14.5和E16.5两个时期,TRIM32-/-胎鼠VZ/SVZ区PAX6和TBR2阳性的神经前体细胞数量减少,有统计学差异(PAX6,E14.5,t=9.125,P=0.018;E16.5,t=13.230,P=0.018;TBR2,E14.5,t=7.143,P=0.028,E16.5,t=12.548,P=0.024)。4.TRIM32的遗传缺失导致神经前体细胞增殖、分裂能力减弱E14.5,E16.5时,TRIM32-/-胎鼠Edu阳性细胞数目减少,有统计学差异(E14.5,t=14.061,P=0.000;E16.5,t=8.715,P=0.038)。E14.5时,TRIM32-/-胎鼠basal区和apical区PH3阳性细胞数减少,有统计学差异(basal 区:t=16.234,P=0.000;apical 区:t=26.415,P=0.000)。E16.5时,TRIM32-/-胎鼠basal区PH3阳性细胞数减少,但差异不显著(t=1.784,P=0.231),apical区细胞减少差异仍然有显著性(t=4.051,P=0.022)。5.TRIM32的遗传缺失对大脑皮层神经细胞的凋亡无影响E14.5,E18.5时,TRIM32-/-与TRIM32+/+胎鼠之间Caspase3阳性细胞数目无学统计差异(E14.5,t=0.083,P=0.938;E18.5,t=-1.350,P=0.283)。
[Abstract]:Cortex development is a complex and fine process. In rodents, neurons are produced mainly between E12 and E18 from embryonic development. E15 reaches the peak and stops after birth; glial cells begin to produce at about E18 days and peak in the newborn period. In the ventricle region (ventricula) The back side of R zone, VZ, distributed in a group of cells of Radial glial cells, RGCs, which proliferate and differentiate to produce cortical excitatory vertebral neurons.RGCs, while self renewal, migration and differentiation into vertebral neurons, and the composition of the six layers of cerebral cortex.TRIM32 (Tripartite motif 32) is a kind of E3. Ubiquitin linked enzyme activity protein with TRIM family characteristic domain of zinc finger, B-boxes domain and curl domain, and 6 repetitive NHL domain.TRIM32, characterized by the disease in some central nervous systems, such as Alzheimer's disease (Alzhermer's Disease, AD), and attention deficit Attention Deficit Hyperacitivity Disorder (ADHD), our previous study found that TRIM32 was highly expressed in the mature cerebral cortex neurons in mice and the intermediate nerve precursor cells in the MGE region of the fetal brain. The deletion of TRIM32 could lead to the increase of the intermediate ganglion (Medial ganglionic eminence, MGE) region of the intermediate nerve precursor cells. The decrease of the colonization resulted in the reduction of the inhibitory neurons of gamma aminobutyric acid (GABAergic), which resulted in the behavioral performance of the autistic mice. This study mainly discussed the role of TRIM32 in the regulation of the production of cortical excitatory neurons. The purpose of the study was to express the expression of 1.TRIM32 gene in the cerebral cortex cells of mice. A study of the effects of the genetic deletion of 2.TRIM32 on the production of cortical neurons in the embryonic stage of the mouse; the effect of the genetic deletion of 3.TRIM32 on the production of neural precursor cells in the VZ/SVZ region of the mouse embryo; the genetic deletion of 4.TRIM32 affects the mechanism of the production of neural precursor cells in the VZ/ SVZ region of the mouse embryo. The expression of 1.TRIM32 gene in the mouse cerebral cortex cells was expressed in the cerebral cortex cells of mice. The brain specimens were taken at E13.5, E16.5, and E18.5 after mating in the wild type mice. The effects of the genetic deletion of TRIM32 fluorescent staining.2.TRIM32 on the production of mature neurons in the cortex of mice were compared with the thickness of CNx and CP compared with the cortical CNx and CP, and then through the brain. The specific marker of cortical neurons was used to mark the corresponding neurons. After counting the statistical difference, the effects of the genetic deletion of.3.TRIM32 on the production of neural precursor cells in the VZ/SVZ region of the mouse embryonic period were PAX6, and the TBR2 fluorescence staining was used to mark the neuronal precursor cells in the cortical VZ/SVZ region, and the difference of.4.TRIM32 loss was compared to the neural precursor after the counting system. Cell division and proliferation effects of Edu and PH3 fluorescence labeling of cells with proliferation and division respectively. After counting statistics, the effects of.5.TRIM32 loss on apoptosis of cortical neurons were compared and Caspase3 fluorescent staining was used to mark apoptotic cells. After counting the statistical difference, the statistical results of.6. statistical difference (x + s) were indicated by the statistical difference (x + s). The results of SPSS20.0 software showed that there were statistical differences between P0.05 and 1.TRIM32. When the expression of 1.TRIM32 gene in the cerebral cortex cells of mice was E13.5, TRIM32 was mainly expressed in the cytoplasm of VZ and SVZ cells, and TRIM32 was highly expressed in IZ region cells when E16.5, and TRIM32 was highly expressed in the positive mature neuron nucleus when E18.5. When the cortical thickness was reduced and the number of mature neurons in the cortex was reduced by E18.5, the NCx and CP in the cortex of TRIM32-/- mice were reduced (NCx:t=-4.709, P=0.026; CP:t=-7.634, P=0.011), and the number of positive neurons in E14.5, E16.5 and E18.5 decreased in the three periods. 4.433, P=0.032, E16.5, t=-5.549, P=0.022; E18.5, t=-7.793, P=0.021); E16.5 and E18.5 two periods, the number of BCL11b positive neurons in TRIM32-/- fetal mice decreased. The genetic deletion (E18.5, t=-7.622, P=0.047; P30, t=33.956, P=0.014).3.TRIM32 causes the decrease of the number of neural precursor cells in the two periods of E14.5 and E16.5. The genetic deletion of =7.143, P=0.028, E16.5, t=12.548, P=0.024).4.TRIM32 leads to the proliferation of neural precursor cells, and the division ability weakens the E14.5, E16.5, the number of Edu positive cells in TRIM32-/- fetal mice decreases. Fewer, there were statistical differences (basal region: t=16.234, P=0.000; apical region: t=26.415, P=0.000).E16.5, the number of PH3 positive cells in basal region of TRIM32-/- fetal mice decreased, but the difference was not significant (t=1.784, P=0.231). When E14.5 and E18.5 were affected, there was no statistical difference in the number of Caspase3 positive cells between TRIM32-/- and TRIM32+/+ fetal rats (E14.5, t=0.083, P=0.938, E18.5, t=-1.350, P=0.283).
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R741

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