TAG1-APP信號通路在成體神經(jīng)發(fā)生中的作用
發(fā)布時間：2018-08-13 08:47
【摘要】：[目的]阿爾茨海默病(AD,俗稱老年癡呆)由德國外科醫(yī)生Alzheimer首次發(fā)現(xiàn),是一種進行性發(fā)展、致死性中樞神經(jīng)系統(tǒng)(central nervesystem,CNS)退退行性疾病。自1906年發(fā)現(xiàn)該病至今,其發(fā)病機制仍未完全明確,也無精準(zhǔn)的早期診斷手段及有效的治療方法。近年來大量研究證實成年哺乳動物的CNS終身存在著神經(jīng)發(fā)生,這為腦損傷和神經(jīng)系統(tǒng)變性疾病(如AD)的治療帶來了希望。此外,越來越多的有關(guān)衰老和諸如AD等神經(jīng)系統(tǒng)退行性疾病的研究發(fā)現(xiàn)神經(jīng)發(fā)生減少,且與AD發(fā)生發(fā)展相關(guān)的關(guān)鍵分子和信號通路同時影響了神經(jīng)發(fā)生�？梢�,神經(jīng)發(fā)生能力的減退可能與AD的發(fā)病密切相關(guān)。本課題組前期研究已經(jīng)證實TAG1-APP信號通路通過Fe65負(fù)性調(diào)控胚胎神經(jīng)源性的腦室區(qū)的神經(jīng)發(fā)生,但是在成體神經(jīng)發(fā)生中的作用及其在神經(jīng)系統(tǒng)變性疾病(如AD)中的作用尚不清楚。據(jù)此,本研究將進一步探討TAG1-APP信號通路對成體神經(jīng)發(fā)生的影響,以期為AD病理機制的理解及探索AD治療的新靶點提供重要依據(jù)。大量文獻報道,在成年哺乳類動物腦內(nèi),主要在SVZ-RMS-OB系統(tǒng)和海馬DG系統(tǒng)存在持續(xù)的神經(jīng)發(fā)生。神經(jīng)干細胞(Neural Stem Cells,NSCs)在這兩個系統(tǒng)則主要表達于腦室室管膜下區(qū)(subventricularzone,SVZ)和海馬齒狀回顆粒下區(qū)(subgranularzone,SGZ)。這兩個腦區(qū)的NSCs通過不對稱分裂成為定向祖細胞或神經(jīng)前體細胞(Neural Precusor Cells,NPCs),并逐漸向功能區(qū)域遷移,在那里分化為成熟的神經(jīng)元,并與其它神經(jīng)元建立突觸聯(lián)系,近而整合入現(xiàn)有的神經(jīng)網(wǎng)絡(luò)發(fā)揮作用,這一系列的過程稱之為神經(jīng)發(fā)生。由于解剖結(jié)構(gòu)的差異,位于SVZ區(qū)的NSCs需長距離遷移至功能區(qū)域——OB顆粒細胞層,而位于SGZ區(qū)的NSCs則短距離即可遷移至功能區(qū)域——海馬顆粒細胞層,并在它們各自的功能區(qū)分化為顆粒細胞,即顆粒神經(jīng)元。近而分別整合到嗅球(olfactory bulb,OB)及海馬的神經(jīng)元通路中去,可能對腦損傷及AD等神經(jīng)變性疾病的神經(jīng)功能的恢復(fù)有潛在的治療作用。據(jù)文獻報道,SVZ區(qū)相較SGZ區(qū)能更快速的產(chǎn)生較多的神經(jīng)元,SVZ區(qū)和SGZ區(qū)產(chǎn)生的新生神經(jīng)元對嗅球依賴和海馬依賴的學(xué)習(xí)、記憶有重要作用。神經(jīng)發(fā)生的程度與這兩個成體神經(jīng)發(fā)生的關(guān)鍵腦區(qū)NSCs和NPCs的增殖、NPCs的遷移和分化及新生神經(jīng)元的存活密切相關(guān)。為延續(xù)本課題組前期研究工作,本課題先重點研究SVZ區(qū)的成體神經(jīng)發(fā)生,來初步探究TAG1-APP信號通路對成體神經(jīng)源性的SVZ區(qū)新生細胞向神經(jīng)元分化的作用。另一重要系統(tǒng)的神經(jīng)發(fā)生,將在后續(xù)研究中進行。在此,本研究分兩部分?jǐn)M解決如下問題:第一部分 TAG1和APP在成體神經(jīng)發(fā)生的兩個關(guān)鍵腦區(qū)表達的研究1.用Western Blot方法,檢測TAG1和APP在成年小鼠SVZ區(qū)及DG區(qū)是否有表達?2.用免疫熒光雙標(biāo)的方法,檢測TAG1和APP是否在成年小鼠SVZ區(qū)及DG區(qū)共表達及能否與參與神經(jīng)發(fā)生過程的NSCs和NPCs的標(biāo)記物共定位?第二部分TAG1-APP信號通路對成體SVZ源性的NSCs和NPCs向神經(jīng)元分化的研究1.TAG1對成年小鼠SVZ區(qū)神經(jīng)發(fā)生是否有影響?2.APP對成年小鼠SVZ區(qū)神經(jīng)發(fā)生是否有影響?3.TAG1和APP相互作用對成年小鼠SVZ區(qū)神經(jīng)發(fā)生是否有影響?[方法](一)選取8-10周齡的成年C57BL/6J雄性小鼠作為研究對象:1.快速斷頭取腦,將腦組織放入4℃ HBSS溶液中。用振動切片機冠狀切小鼠腦袋,并將腦片收集在4℃ HBSS溶液中。用解剖顯微鑷于體視顯微鏡下分離SVZ區(qū)和海馬齒狀回(DG)區(qū)的腦組織。采用Western Blot的方法,檢測TAG1和APP在成體神經(jīng)發(fā)生的這兩個腦區(qū)表達情況;2.麻醉灌注后斷頭取腦,OCT包埋,制作SVZ區(qū)和海馬DG區(qū)冠狀連續(xù)冰凍切片。每組選取3張非連續(xù)切片(按解剖標(biāo)志保證組間位于相同平面)。采用免疫熒光雙標(biāo)的方法檢測TAG1和APP分別在SVZ區(qū)和海馬DG區(qū)的共定位情況,以及TAG1和APP分別與SVZ區(qū)和海馬DG區(qū)的神經(jīng)干細胞和神經(jīng)前體細胞標(biāo)記物(Nestin/GFAP/SOX2/EGFR/PSA-NCAM/DCX)的共定位情況。(二)選取8-10周齡的成年APP基因敲除(APP-KO)小鼠和TAG1基因敲除(TAG1-KO)小鼠及APP和TAG1雙敲除(DKO)小鼠與他們同窩的野生型(WT)雄性小鼠作為研究對象:1.采用PCR法進行基因型鑒定,獲得APP-KO、TAG1-KO、DKO及同窩WT的雄性實驗小鼠;2.按100mg/kg注射劑量,腹腔注射Brdu,每天三次,持續(xù)注射兩天,并于4周后取材;3.麻醉灌注后斷頭取腦,OCT包埋,制作嗅球區(qū)(OB)區(qū)、SVZ區(qū)和海馬DG區(qū)冠狀非連續(xù)冰凍切片,每張切片放3個非連續(xù)腦片,間隔240um。每只老鼠選取5張非連續(xù)切片(按解剖標(biāo)志保證組間位于同一平面)。通過Brdu標(biāo)記增殖的細胞,NeuN標(biāo)記成熟神經(jīng)元。采用免疫熒光雙標(biāo)的方法檢測OB區(qū)新生神經(jīng)元(Brdu+ NeuN+)的數(shù)量。通過細胞計數(shù),比較基因敲除組與同窩野生型對照組的Brdu+ NeuN+細胞數(shù),分析TAG1-APP信號通路對SVZ區(qū)成體神經(jīng)發(fā)生的影響。[結(jié)果]1.Western Blot結(jié)果顯示,TAG1和APP在成體神經(jīng)發(fā)生的兩個關(guān)鍵腦區(qū)均有表達;2.免疫熒光雙標(biāo)的結(jié)果顯示,TAG1和APP能共表達于成體神經(jīng)發(fā)生的這兩個腦區(qū),且TAG1和APP可分別與SVZ區(qū)和海馬DG區(qū)的NSCs和NPCs的標(biāo)記物共定位;3.成體SVZ-RMS-OB系統(tǒng)神經(jīng)發(fā)生的結(jié)果顯示:在OB區(qū),大多數(shù)的Brdu+細胞分布在顆粒細胞層(GCL),且大多數(shù)能與NeuN+細胞共定位。Brdu+/NeuN+細胞即為從SVZ區(qū)遷移到OB的新生神經(jīng)元。統(tǒng)計結(jié)果顯示,OB區(qū)的新生神經(jīng)元的數(shù)量:TAG1-KO組(152.17±23.48)比同窩 TAG1-WT 組(109.50±23.46)明顯增多(P=0.003);APP-KO 組(208.50±57.98)比同窩 APP-WT 組(175.42±31.45)多(P=0.011);TAG1/APP-DKO組(210.14±37.45)比 WT 組(100.59±24.95)多(P=0.028)。[結(jié)論]1.TAG1和APP可在成體神經(jīng)發(fā)生的兩個關(guān)鍵腦區(qū)(SVZ區(qū)和DG區(qū))表達并能共表達,且能表達于這兩個關(guān)鍵腦區(qū)的NSCs和NPCs上;2.TAG1和APP有負(fù)性調(diào)控成體SVZ區(qū)的成體神經(jīng)發(fā)生的趨勢;TAG1-APP信號通路主要使SVZ區(qū)的新生細胞分化成神經(jīng)元的數(shù)量減少,而抑制神經(jīng)發(fā)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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16
本文編號：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.
【學(xué)位授予單位】：昆明醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.16
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