【摘要】：自閉癥(Autism spectrum disorders)又稱孤獨(dú)癥,屬嚴(yán)重的神經(jīng)發(fā)育障礙性疾病。自閉癥患者大多見于兒童,主要表現(xiàn)為語言形成障礙、社交障礙,此外,另外的癥狀包括行為刻板與重復(fù),動(dòng)作僵硬、拒絕改變習(xí)慣以及興趣狹窄等也非常常見。關(guān)于自閉癥的發(fā)病機(jī)制與病因,圍繞遺傳因素、社會(huì)心理因素以及神經(jīng)生物學(xué)因素等方面已進(jìn)行了大量研究,隨著對自閉癥發(fā)病機(jī)制研究的深入,醫(yī)學(xué)界已認(rèn)識到自閉癥是一種基于遺傳因素作用,受多種環(huán)境因子刺激,而導(dǎo)致的彌漫性中樞神經(jīng)系統(tǒng)發(fā)育障礙性疾病�；谶z傳因素方面,研究者們開展了從分子遺傳到神經(jīng)免疫,腦功能影像到神經(jīng)解剖和神經(jīng)生物學(xué)等不同層面的研究。然而迄今為止,仍未能闡明其確切病因和發(fā)病機(jī)制。近兩年在Nature、Science、Cell等刊物連續(xù)前瞻性地發(fā)表相關(guān)綜述,建議將自閉癥的認(rèn)知特征與腦神經(jīng)功能研究結(jié)合起來。最近的一些導(dǎo)向性研究均指向一個(gè)重要的神經(jīng)細(xì)胞之間特異性通訊結(jié)構(gòu):突觸(Synapse)。在神經(jīng)生物化學(xué)層面,突觸蛋白鈣離子/甸調(diào)蛋白依賴性蛋白激酶 Ⅱ(calcium/calmodulin-dependent protein kinase,CaMKⅡ)成為研究突觸功能所涉及到的重要信號分子的出發(fā)點(diǎn)。中樞神經(jīng)系統(tǒng)CaMKⅡ通過參與突觸可塑性調(diào)節(jié)、基因的轉(zhuǎn)錄調(diào)節(jié)、神經(jīng)遞質(zhì)的合成與釋放、細(xì)胞骨架蛋白磷酸化等發(fā)揮多種生理功能。其中,CaMKⅡ激酶活性在海馬依存的學(xué)習(xí)和記憶功能方面的作用也得到公認(rèn)。根據(jù)我們的文獻(xiàn)調(diào)研,有關(guān)神經(jīng)元CaMKⅡ信號轉(zhuǎn)導(dǎo)網(wǎng)絡(luò)紊亂與自閉癥發(fā)病機(jī)制的內(nèi)在關(guān)聯(lián)性未見報(bào)道。所以,本研究以突觸蛋白CaMKⅡ?yàn)榍腥朦c(diǎn),探討CaMKⅡ磷酸化/去磷酸化信號模塊在介導(dǎo)自閉癥模型海馬突觸關(guān)聯(lián)調(diào)控網(wǎng)絡(luò)紊亂的分子生物學(xué)機(jī)制及調(diào)控規(guī)律。目的:考察自閉癥模型海馬突觸CaMKⅡ/PKA/PKC磷酸化/去磷酸化信號模塊在自閉癥發(fā)生病理過程中的變化規(guī)律,探索藥物調(diào)控對自閉癥大鼠行為學(xué)功能的影響。方法:采用丙戊酸(Valproate,VPA)建立自閉癥動(dòng)物模型,通過Western blotting技術(shù)和共聚焦免疫熒光技術(shù)考察自閉癥模型病理過程中海馬CaMKⅡ/PKA/PKC信號通路相關(guān)蛋白激酶磷酸化水平的改變�？疾烀览袑幝越o藥后自閉癥模型病理過程中海馬CaMKⅡ磷酸化水平的變化情況和行為學(xué)功能的改善。結(jié)果:數(shù)據(jù)顯示,自閉癥模型海馬突觸CaMKⅡ、Synapsin Ⅰ、GluRl等CaMKⅡ/PKA/PKC通路相關(guān)蛋白的磷酸化水平下降,與自閉癥模型鼠探索性差,趨避性嚴(yán)重、對新事物的學(xué)習(xí)和接受的能力下降,具有嚴(yán)重認(rèn)知障礙的行為表現(xiàn)相一致,證實(shí)了海馬突觸的CaMKⅡ/PKA/PKC信號紊亂參與了自閉癥的發(fā)病過程。自閉癥模型組給予美拉托寧后,其降低的CaMKⅡ、SynapsinⅠ、GluRl等蛋白磷酸化水平顯著上調(diào),與電生理中LTP的改善及行為學(xué)中社交能力的提高相一致,提示美拉托寧慢性給藥可以改善自閉癥的癥狀,其機(jī)制可能是通過調(diào)控CaMKⅡ/PKA/PKC磷酸化/去磷酸化信號模塊相關(guān)蛋白的表達(dá)。結(jié)論:海馬突觸的CaMKⅡ/PKA/PKC信號模塊參與了自閉癥的發(fā)病過程,美拉托寧可以通過上調(diào)降低的CaMKⅡ、Synapsin Ⅰ、GluRl等蛋白磷酸化水平來改善自閉癥模型的行為學(xué)功能,為自閉癥的防治藥物的研發(fā)提供了新思路和新策略。
[Abstract]:Autism spectrum disorders (autism), also known as autism, is a serious neurodevelopmental disorder. Most autistic patients are found in children, mainly manifested by language formation and social disorders. In addition, other symptoms include stereotyped and repetitive behavior, rigid movement, refusal to change habits, and narrow interest. The pathogenesis and etiology of autism, around genetic, psychosocial and neurobiological factors have been extensively studied. With the in-depth study of the mechanism of autism, the medical community has realized that autism is a diffuse center based on the role of genetic factors and stimulated by a variety of environmental factors. Neurodevelopmental disorders. Based on genetic factors, researchers have carried out studies from molecular to neurologic, brain function imaging to neuroanatomy and neurobiology. However, to date, the exact etiology and pathogenesis have not been clarified. In the last two years, the Nature, Science, Cell and other publications were in succession. A prospectively published review suggests that the cognitive characteristics of autism be combined with the brain function study. Recent guidance studies point to an important communication structure between neurons: synapses (Synapse). At the neurobiochemical level, synaptic egg white calcium / meadow protein dependent protein kinase II (CAL Cium/calmodulin-dependent protein kinase, CaMK II) is the starting point of the important signal molecules involved in the study of synaptic function. The central nervous system CaMK II plays a variety of physiological functions by participating in synaptic plasticity regulation, gene transcription regulation, neurotransmitter synthesis and release, cytoskeleton phosphorylation and so on. Among them, CaMK The role of II kinase activity in the learning and memory function of the hippocampus is also recognized. According to our literature research, there is no report on the intrinsic relationship between the neural CaMK II signal transduction network disorder and the mechanism of autism. Therefore, this study uses the synaptic protein CaMK II as an entry point to explore CaMK II phosphorylation / dephosphorylation. The molecular biological mechanism and regulation of the disturbance of the synaptic connections in the hippocampus of the autistic model are mediated by the chemical signal module. Objective: To investigate the changes of the CaMK II /PKA/PKC phosphorylation / dephosphorylation signal module in the autism model in the pathological process of autism, and to explore the behavior of drug control in autism rats. Methods: using Valproate (VPA) to establish an autistic animal model, the changes in the phosphorylation of CaMK II /PKA/PKC signaling pathway related protein kinase in the hippocampus of the hippocampus during the pathological process of autism were investigated by Western blotting technique and confocal immunofluorescence technique. Changes in the level of phosphorylation of hippocampal CaMK II in the pathological process and the improvement of behavioral function. Results: data show that the phosphorylation level of CaMK II, Synapsin I, GluRl and other CaMK II /PKA/PKC pathway related proteins in the hippocampus synapses of the autism model decreased, which was poor with the autism model rats, and the learning and connection of the new things. The decrease in ability and the consistent behavior of severe cognitive impairment confirmed the involvement of the CaMK II /PKA/PKC signal disorder in the hippocampal synapse in the process of autism. The level of CaMK II, Synapsin I, GluRl and other protein phosphorylation in the autistic group was significantly up-regulated after giving melatonin, and the improvement of LTP in electrophysiology and the improvement in electrophysiology. The improvement of social ability in behavioral studies suggests that chronic administration of melatonin can improve the symptoms of autism by regulating the expression of CaMK II /PKA/PKC phosphorylation / dephosphorylation signal module related proteins. Conclusion: the CaMK II /PKA/PKC signal module of the hippocampal synapse is involved in the process of autism, Melato It can improve the behavioral function of autism model by raising the level of reduced phosphorylation of CaMK II, Synapsin I, GluRl and other proteins, and provides new ideas and new strategies for the research and development of autism prevention and control drugs.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R749.94

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