基于海马CaMKⅡ信号紊乱的自闭症发病机制及药物调控研究
发布时间：2018-07-29 08:37
【摘要】：自闭症(Autism spectrum disorders)又称孤独症,属严重的神经发育障碍性疾病。自闭症患者大多见于儿童,主要表现为语言形成障碍、社交障碍,此外,另外的症状包括行为刻板与重复,动作僵硬、拒绝改变习惯以及兴趣狭窄等也非常常见。关于自闭症的发病机制与病因,围绕遗传因素、社会心理因素以及神经生物学因素等方面已进行了大量研究,随着对自闭症发病机制研究的深入,医学界已认识到自闭症是一种基于遗传因素作用,受多种环境因子刺激,而导致的弥漫性中枢神经系统发育障碍性疾病。基于遗传因素方面,研究者们开展了从分子遗传到神经免疫,脑功能影像到神经解剖和神经生物学等不同层面的研究。然而迄今为止,仍未能阐明其确切病因和发病机制。近两年在Nature、Science、Cell等刊物连续前瞻性地发表相关综述,建议将自闭症的认知特征与脑神经功能研究结合起来。最近的一些导向性研究均指向一个重要的神经细胞之间特异性通讯结构:突触(Synapse)。在神经生物化学层面,突触蛋白钙离子/甸调蛋白依赖性蛋白激酶 Ⅱ(calcium/calmodulin-dependent protein kinase,CaMKⅡ)成为研究突触功能所涉及到的重要信号分子的出发点。中枢神经系统CaMKⅡ通过参与突触可塑性调节、基因的转录调节、神经递质的合成与释放、细胞骨架蛋白磷酸化等发挥多种生理功能。其中,CaMKⅡ激酶活性在海马依存的学习和记忆功能方面的作用也得到公认。根据我们的文献调研,有关神经元CaMKⅡ信号转导网络紊乱与自闭症发病机制的内在关联性未见报道。所以,本研究以突触蛋白CaMKⅡ为切入点,探讨CaMKⅡ磷酸化/去磷酸化信号模块在介导自闭症模型海马突触关联调控网络紊乱的分子生物学机制及调控规律。目的:考察自闭症模型海马突触CaMKⅡ/PKA/PKC磷酸化/去磷酸化信号模块在自闭症发生病理过程中的变化规律,探索药物调控对自闭症大鼠行为学功能的影响。方法:采用丙戊酸(Valproate,VPA)建立自闭症动物模型,通过Western blotting技术和共聚焦免疫荧光技术考察自闭症模型病理过程中海马CaMKⅡ/PKA/PKC信号通路相关蛋白激酶磷酸化水平的改变。考察美拉托宁慢性给药后自闭症模型病理过程中海马CaMKⅡ磷酸化水平的变化情况和行为学功能的改善。结果:数据显示,自闭症模型海马突触CaMKⅡ、Synapsin Ⅰ、GluRl等CaMKⅡ/PKA/PKC通路相关蛋白的磷酸化水平下降,与自闭症模型鼠探索性差,趋避性严重、对新事物的学习和接受的能力下降,具有严重认知障碍的行为表现相一致,证实了海马突触的CaMKⅡ/PKA/PKC信号紊乱参与了自闭症的发病过程。自闭症模型组给予美拉托宁后,其降低的CaMKⅡ、SynapsinⅠ、GluRl等蛋白磷酸化水平显著上调,与电生理中LTP的改善及行为学中社交能力的提高相一致,提示美拉托宁慢性给药可以改善自闭症的症状,其机制可能是通过调控CaMKⅡ/PKA/PKC磷酸化/去磷酸化信号模块相关蛋白的表达。结论:海马突触的CaMKⅡ/PKA/PKC信号模块参与了自闭症的发病过程,美拉托宁可以通过上调降低的CaMKⅡ、Synapsin Ⅰ、GluRl等蛋白磷酸化水平来改善自闭症模型的行为学功能,为自闭症的防治药物的研发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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.94
本文编号：2152110
[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.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.94
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