TAG1-APP信号通路在成体神经发生中的作用
发布时间：2018-08-13 08:47
【摘要】：[目的]阿尔茨海默病(AD,俗称老年痴呆)由德国外科医生Alzheimer首次发现,是一种进行性发展、致死性中枢神经系统(central nervesystem,CNS)退退行性疾病。自1906年发现该病至今,其发病机制仍未完全明确,也无精准的早期诊断手段及有效的治疗方法。近年来大量研究证实成年哺乳动物的CNS终身存在着神经发生,这为脑损伤和神经系统变性疾病(如AD)的治疗带来了希望。此外,越来越多的有关衰老和诸如AD等神经系统退行性疾病的研究发现神经发生减少,且与AD发生发展相关的关键分子和信号通路同时影响了神经发生。可见,神经发生能力的减退可能与AD的发病密切相关。本课题组前期研究已经证实TAG1-APP信号通路通过Fe65负性调控胚胎神经源性的脑室区的神经发生,但是在成体神经发生中的作用及其在神经系统变性疾病(如AD)中的作用尚不清楚。据此,本研究将进一步探讨TAG1-APP信号通路对成体神经发生的影响,以期为AD病理机制的理解及探索AD治疗的新靶点提供重要依据。大量文献报道,在成年哺乳类动物脑内,主要在SVZ-RMS-OB系统和海马DG系统存在持续的神经发生。神经干细胞(Neural Stem Cells,NSCs)在这两个系统则主要表达于脑室室管膜下区(subventricularzone,SVZ)和海马齿状回颗粒下区(subgranularzone,SGZ)。这两个脑区的NSCs通过不对称分裂成为定向祖细胞或神经前体细胞(Neural Precusor Cells,NPCs),并逐渐向功能区域迁移,在那里分化为成熟的神经元,并与其它神经元建立突触联系,近而整合入现有的神经网络发挥作用,这一系列的过程称之为神经发生。由于解剖结构的差异,位于SVZ区的NSCs需长距离迁移至功能区域——OB颗粒细胞层,而位于SGZ区的NSCs则短距离即可迁移至功能区域——海马颗粒细胞层,并在它们各自的功能区分化为颗粒细胞,即颗粒神经元。近而分别整合到嗅球(olfactory bulb,OB)及海马的神经元通路中去,可能对脑损伤及AD等神经变性疾病的神经功能的恢复有潜在的治疗作用。据文献报道,SVZ区相较SGZ区能更快速的产生较多的神经元,SVZ区和SGZ区产生的新生神经元对嗅球依赖和海马依赖的学习、记忆有重要作用。神经发生的程度与这两个成体神经发生的关键脑区NSCs和NPCs的增殖、NPCs的迁移和分化及新生神经元的存活密切相关。为延续本课题组前期研究工作,本课题先重点研究SVZ区的成体神经发生,来初步探究TAG1-APP信号通路对成体神经源性的SVZ区新生细胞向神经元分化的作用。另一重要系统的神经发生,将在后续研究中进行。在此,本研究分两部分拟解决如下问题:第一部分 TAG1和APP在成体神经发生的两个关键脑区表达的研究1.用Western Blot方法,检测TAG1和APP在成年小鼠SVZ区及DG区是否有表达?2.用免疫荧光双标的方法,检测TAG1和APP是否在成年小鼠SVZ区及DG区共表达及能否与参与神经发生过程的NSCs和NPCs的标记物共定位?第二部分TAG1-APP信号通路对成体SVZ源性的NSCs和NPCs向神经元分化的研究1.TAG1对成年小鼠SVZ区神经发生是否有影响?2.APP对成年小鼠SVZ区神经发生是否有影响?3.TAG1和APP相互作用对成年小鼠SVZ区神经发生是否有影响?[方法](一)选取8-10周龄的成年C57BL/6J雄性小鼠作为研究对象:1.快速断头取脑,将脑组织放入4℃ HBSS溶液中。用振动切片机冠状切小鼠脑袋,并将脑片收集在4℃ HBSS溶液中。用解剖显微镊于体视显微镜下分离SVZ区和海马齿状回(DG)区的脑组织。采用Western Blot的方法,检测TAG1和APP在成体神经发生的这两个脑区表达情况;2.麻醉灌注后断头取脑,OCT包埋,制作SVZ区和海马DG区冠状连续冰冻切片。每组选取3张非连续切片(按解剖标志保证组间位于相同平面)。采用免疫荧光双标的方法检测TAG1和APP分别在SVZ区和海马DG区的共定位情况,以及TAG1和APP分别与SVZ区和海马DG区的神经干细胞和神经前体细胞标记物(Nestin/GFAP/SOX2/EGFR/PSA-NCAM/DCX)的共定位情况。(二)选取8-10周龄的成年APP基因敲除(APP-KO)小鼠和TAG1基因敲除(TAG1-KO)小鼠及APP和TAG1双敲除(DKO)小鼠与他们同窝的野生型(WT)雄性小鼠作为研究对象:1.采用PCR法进行基因型鉴定,获得APP-KO、TAG1-KO、DKO及同窝WT的雄性实验小鼠;2.按100mg/kg注射剂量,腹腔注射Brdu,每天三次,持续注射两天,并于4周后取材;3.麻醉灌注后断头取脑,OCT包埋,制作嗅球区(OB)区、SVZ区和海马DG区冠状非连续冰冻切片,每张切片放3个非连续脑片,间隔240um。每只老鼠选取5张非连续切片(按解剖标志保证组间位于同一平面)。通过Brdu标记增殖的细胞,NeuN标记成熟神经元。采用免疫荧光双标的方法检测OB区新生神经元(Brdu+ NeuN+)的数量。通过细胞计数,比较基因敲除组与同窝野生型对照组的Brdu+ NeuN+细胞数,分析TAG1-APP信号通路对SVZ区成体神经发生的影响。[结果]1.Western Blot结果显示,TAG1和APP在成体神经发生的两个关键脑区均有表达;2.免疫荧光双标的结果显示,TAG1和APP能共表达于成体神经发生的这两个脑区,且TAG1和APP可分别与SVZ区和海马DG区的NSCs和NPCs的标记物共定位;3.成体SVZ-RMS-OB系统神经发生的结果显示:在OB区,大多数的Brdu+细胞分布在颗粒细胞层(GCL),且大多数能与NeuN+细胞共定位。Brdu+/NeuN+细胞即为从SVZ区迁移到OB的新生神经元。统计结果显示,OB区的新生神经元的数量:TAG1-KO组(152.17±23.48)比同窝 TAG1-WT 组(109.50±23.46)明显增多(P=0.003);APP-KO 组(208.50±57.98)比同窝 APP-WT 组(175.42±31.45)多(P=0.011);TAG1/APP-DKO组(210.14±37.45)比 WT 组(100.59±24.95)多(P=0.028)。[结论]1.TAG1和APP可在成体神经发生的两个关键脑区(SVZ区和DG区)表达并能共表达,且能表达于这两个关键脑区的NSCs和NPCs上;2.TAG1和APP有负性调控成体SVZ区的成体神经发生的趋势;TAG1-APP信号通路主要使SVZ区的新生细胞分化成神经元的数量减少,而抑制神经发生。
[Abstract]:[Objective] Alzheimer's disease (AD), first discovered by German surgeon Alzheimer, is a progressive, fatal degenerative disease of the central nervous system (CNS). Since its discovery in 1906, the pathogenesis of AD has not been fully clarified, and there is no precise early diagnosis and effective treatment. In recent years, a large number of studies have confirmed the existence of neurogenesis in adult mammals'CNS, which brings hope to the treatment of brain injury and neurodegenerative diseases (such as AD). In addition, more and more studies on aging and neurodegenerative diseases such as AD have found that neurogenesis is reduced and is related to the occurrence and development of AD. The key molecules and signaling pathways affect neurogenesis at the same time. It can be seen that the decline of neurogenesis may be closely related to the pathogenesis of AD. Previous studies have confirmed that TAG1-APP signaling pathway negatively regulates embryonic neurogenic ventricular neurogenesis via Fe65, but its role in adult neurogenesis and its role in adult neurogenesis have been demonstrated. The role of TAG1-APP signaling pathway in adult neurogenesis is still unclear in neurodegenerative diseases such as AD. This study will further explore the effect of TAG1-APP signaling pathway on adult neurogenesis in order to provide important evidence for understanding the pathological mechanism of AD and exploring new targets for AD treatment. Neural Stem Cells (NSCs) are mainly expressed in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. (Neural Precusor Cells, NPCs) migrate gradually to functional areas, where they differentiate into mature neurons, establish synaptic connections with other neurons, and integrate into existing neural networks to function. This series of processes is called neurogenesis. The functional area, the OB granular cell layer, whereas the NSCs located in the SGZ area, migrate to the hippocampal granular cell layer in a short distance and differentiate into granular cells, i.e. granular neurons, in their respective functional areas. It is reported that SVZ can produce more neurons faster than SGZ, and the new neurons produced by SVZ and SGZ play an important role in olfactory bulb dependence and hippocampal learning and memory. The proliferation of NSCs and NPCs, the migration and differentiation of NPCs, and the survival of new neurons are closely related. In order to continue our previous work, we first focused on the adult neurogenesis of SVZ region to explore the role of TAG1-APP signaling pathway in the differentiation of adult neurogenic SVZ cells into neurons. Nervogenesis of important systems will be carried out in the follow-up study. Here, this study is divided into two parts to solve the following problems: Part I: TAG1 and APP in adult neurogenesis in the two key areas of brain research 1. Western Blot method, detection of TAG1 and APP in adult mice SVZ and DG region whether there is expression? Methods: TAG1 and APP were co-expressed in the SVZ and DG regions of adult mice and co-localized with the markers of NSCs and NPCs involved in neurogenesis. Part 2 TAG1-APP signaling pathway on the differentiation of adult SVZ-derived NSCs and NPCs into neurons. 1. Does TAG1 affect neurogenesis in the SVZ region of adult mice. 2. Does APP affect neurogenesis in adult mice? Does the interaction between TAG1 and APP affect the neurogenesis of SVZ region in mice? 3. Does the interaction between TAG1 and APP affect the neurogenesis of SVZ region in adult mice? [Methods] (1) Male C57BL/6J mice aged 8-10 weeks were selected as the subjects of study. 1. Brain tissue was quickly cut off and put into 4 C HBSS solution. The brain tissue of SVZ and DG regions were separated by anatomical microforceps under stereomicroscope. The expression of TAG1 and APP in the two brain regions of adult neurogenesis was detected by Western Blot method. 2. After anesthesia and perfusion, the brain was cut off and OCT was embedded to make coronary continuous ice in SVZ and DG regions. Frozen section. Three discontinuous sections were selected from each group (at the same level as the anatomical markers). The co-localization of TAG1 and APP in SVZ and DG regions of hippocampus was detected by immunofluorescence double labeling method, and the co-localization of TAG1 and APP with neural stem cells and neural precursor cell markers (Nestin/GFAP/SOX2/EG) in SVZ and DG regions of hippocampus, respectively. Co-localization of FR/PSA-NCAM/DCX. (2) Adult APP knockout (APP-KO) mice aged 8-10 weeks, TAG1 knockout (TAG1-KO) mice and wild type (WT) male mice with their siblings (APP and TAG1 double knockout (DKO) mice were selected as the research objects. (1) The genotypes of APP-KO, TAG1-KO, DKO and WT were identified by PCR. Male experimental mice; 2. Brdu was injected intraperitoneally three times a day for two days at a dose of 100mg/kg, and the samples were taken after 4 weeks; 3. After anesthesia and perfusion, the brain was cut off and encapsulated with OCT to make coronal non-continuous frozen sections of olfactory bulb area (OB), SVZ area and DG area of hippocampus. Each section contained three non-continuous brain slices with an interval of 240um. The number of Brdu + NeuN + cells in the OB region was detected by immunofluorescence double labeling method. The number of Brdu + NeuN + cells in the knockout group was compared with that in the wild type control group of the same nest, and TA was analyzed. [Results] Western Blot analysis showed that TAG1 and APP were expressed in the two key brain regions of adult neurogenesis; 2. Immunofluorescence double labeling showed that TAG1 and APP could co-express in the two brain regions of adult neurogenesis, and TAG1 and APP could be associated with SVZ and hippocampal DG respectively. The results of neurogenesis in adult SVZ-RMS-OB system showed that most of Brdu+ cells were distributed in the granulosa cell layer (GCL) and most of them could co-localize with NeuN+ cells. Brdu+/NeuN+ cells were new neurons migrating from SVZ to OB. Dosage: TAG1-KO group (152.17 (+ 23.48) significantly increased (P = 0.003) in 152.17 (+ 23.48) compared with homomomomomomomomomogroupsTAG1-WT group (109.50 (+ 23.46) (P = 0.003) significantly increased (P = 0.003); APP-KO group (208.50 (+ 57.98) more than homomomomomomomomomomomomomomomomomohomomomomomomomomomomomomomomomomomomomomomomomomomomomohomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomoBrain area (SVZ) TAG1 and APP negatively regulate the adult neurogenesis in the adult SVZ region. TAG1-APP signaling pathway mainly reduces the number of neurons differentiated into new cells in the SVZ region and inhibits neurogenesis.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
本文编号：2180466
[Abstract]:[Objective] Alzheimer's disease (AD), first discovered by German surgeon Alzheimer, is a progressive, fatal degenerative disease of the central nervous system (CNS). Since its discovery in 1906, the pathogenesis of AD has not been fully clarified, and there is no precise early diagnosis and effective treatment. In recent years, a large number of studies have confirmed the existence of neurogenesis in adult mammals'CNS, which brings hope to the treatment of brain injury and neurodegenerative diseases (such as AD). In addition, more and more studies on aging and neurodegenerative diseases such as AD have found that neurogenesis is reduced and is related to the occurrence and development of AD. The key molecules and signaling pathways affect neurogenesis at the same time. It can be seen that the decline of neurogenesis may be closely related to the pathogenesis of AD. Previous studies have confirmed that TAG1-APP signaling pathway negatively regulates embryonic neurogenic ventricular neurogenesis via Fe65, but its role in adult neurogenesis and its role in adult neurogenesis have been demonstrated. The role of TAG1-APP signaling pathway in adult neurogenesis is still unclear in neurodegenerative diseases such as AD. This study will further explore the effect of TAG1-APP signaling pathway on adult neurogenesis in order to provide important evidence for understanding the pathological mechanism of AD and exploring new targets for AD treatment. Neural Stem Cells (NSCs) are mainly expressed in the subventricular zone (SVZ) and the subgranular zone (SGZ) of the hippocampal dentate gyrus. (Neural Precusor Cells, NPCs) migrate gradually to functional areas, where they differentiate into mature neurons, establish synaptic connections with other neurons, and integrate into existing neural networks to function. This series of processes is called neurogenesis. The functional area, the OB granular cell layer, whereas the NSCs located in the SGZ area, migrate to the hippocampal granular cell layer in a short distance and differentiate into granular cells, i.e. granular neurons, in their respective functional areas. It is reported that SVZ can produce more neurons faster than SGZ, and the new neurons produced by SVZ and SGZ play an important role in olfactory bulb dependence and hippocampal learning and memory. The proliferation of NSCs and NPCs, the migration and differentiation of NPCs, and the survival of new neurons are closely related. In order to continue our previous work, we first focused on the adult neurogenesis of SVZ region to explore the role of TAG1-APP signaling pathway in the differentiation of adult neurogenic SVZ cells into neurons. Nervogenesis of important systems will be carried out in the follow-up study. Here, this study is divided into two parts to solve the following problems: Part I: TAG1 and APP in adult neurogenesis in the two key areas of brain research 1. Western Blot method, detection of TAG1 and APP in adult mice SVZ and DG region whether there is expression? Methods: TAG1 and APP were co-expressed in the SVZ and DG regions of adult mice and co-localized with the markers of NSCs and NPCs involved in neurogenesis. Part 2 TAG1-APP signaling pathway on the differentiation of adult SVZ-derived NSCs and NPCs into neurons. 1. Does TAG1 affect neurogenesis in the SVZ region of adult mice. 2. Does APP affect neurogenesis in adult mice? Does the interaction between TAG1 and APP affect the neurogenesis of SVZ region in mice? 3. Does the interaction between TAG1 and APP affect the neurogenesis of SVZ region in adult mice? [Methods] (1) Male C57BL/6J mice aged 8-10 weeks were selected as the subjects of study. 1. Brain tissue was quickly cut off and put into 4 C HBSS solution. The brain tissue of SVZ and DG regions were separated by anatomical microforceps under stereomicroscope. The expression of TAG1 and APP in the two brain regions of adult neurogenesis was detected by Western Blot method. 2. After anesthesia and perfusion, the brain was cut off and OCT was embedded to make coronary continuous ice in SVZ and DG regions. Frozen section. Three discontinuous sections were selected from each group (at the same level as the anatomical markers). The co-localization of TAG1 and APP in SVZ and DG regions of hippocampus was detected by immunofluorescence double labeling method, and the co-localization of TAG1 and APP with neural stem cells and neural precursor cell markers (Nestin/GFAP/SOX2/EG) in SVZ and DG regions of hippocampus, respectively. Co-localization of FR/PSA-NCAM/DCX. (2) Adult APP knockout (APP-KO) mice aged 8-10 weeks, TAG1 knockout (TAG1-KO) mice and wild type (WT) male mice with their siblings (APP and TAG1 double knockout (DKO) mice were selected as the research objects. (1) The genotypes of APP-KO, TAG1-KO, DKO and WT were identified by PCR. Male experimental mice; 2. Brdu was injected intraperitoneally three times a day for two days at a dose of 100mg/kg, and the samples were taken after 4 weeks; 3. After anesthesia and perfusion, the brain was cut off and encapsulated with OCT to make coronal non-continuous frozen sections of olfactory bulb area (OB), SVZ area and DG area of hippocampus. Each section contained three non-continuous brain slices with an interval of 240um. The number of Brdu + NeuN + cells in the OB region was detected by immunofluorescence double labeling method. The number of Brdu + NeuN + cells in the knockout group was compared with that in the wild type control group of the same nest, and TA was analyzed. [Results] Western Blot analysis showed that TAG1 and APP were expressed in the two key brain regions of adult neurogenesis; 2. Immunofluorescence double labeling showed that TAG1 and APP could co-express in the two brain regions of adult neurogenesis, and TAG1 and APP could be associated with SVZ and hippocampal DG respectively. The results of neurogenesis in adult SVZ-RMS-OB system showed that most of Brdu+ cells were distributed in the granulosa cell layer (GCL) and most of them could co-localize with NeuN+ cells. Brdu+/NeuN+ cells were new neurons migrating from SVZ to OB. Dosage: TAG1-KO group (152.17 (+ 23.48) significantly increased (P = 0.003) in 152.17 (+ 23.48) compared with homomomomomomomomomogroupsTAG1-WT group (109.50 (+ 23.46) (P = 0.003) significantly increased (P = 0.003); APP-KO group (208.50 (+ 57.98) more than homomomomomomomomomomomomomomomomomohomomomomomomomomomomomomomomomomomomomomomomomomomomomohomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomomoBrain area (SVZ) TAG1 and APP negatively regulate the adult neurogenesis in the adult SVZ region. TAG1-APP signaling pathway mainly reduces the number of neurons differentiated into new cells in the SVZ region and inhibits neurogenesis.
【学位授予单位】：昆明医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16
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1 Lei Zhang;Xiao Han;Xiang Cheng;Xue-feng Tan;He-yan Zhao;Xin-hua Zhang;;Denervated hippocampus provides a favorable microenvironment for neuronal differentiation of endogenous neural stem cells[J];Neural Regeneration Research;2016年04期
相关硕士学位论文 前1条
1 朱茜;20S蛋白酶体参与调控神经干细胞衰老进程[D];山西医科大学;2012年
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