脑神经元Actin骨架蛋白重构在阿片成瘾记忆中的作用及机制研究

Drug abuse and addiction is a kind of chronic, relapsed brain disease and is coincident with abnormal learning behavior and memory disorder, which often cause drug-related personal injury and death. These cases are the important content and research in the field of Forensic Medicine. Though the study on the mechanism of opioid addiction has gained great progress, however, is still unclear. The difficult withdrawal and high relapse is an important cause keeping the opioid addiction in large population. Recently, more and more researches have indicated that the abnormal learning behavior and memory disorder are consistent with the neural plasticity of synaptic. Moreover, cytoskeletal proteins, especially synthesis and extension of F-actin, are playing a significant role respectively in formation, consolidation and extraction of the memory process. However, if during different periods of drug addiction, abnormal process of memory is related to the functions and mechanisms of F-actin and other associated protein, such as Arc, Cofilin and Akt, it is still mysterious. In this study, when drug is administrated to the animals, we choose to observe whether the process of turning to addiction and the parallel different stages of memory associated with it are companied by the changes of F-actin, Arc Cofilin, and Akt and want to find out this very mechanisms of these proteins in the Prefrontal Cortex (PFC) and dorsal Hippocampal (dHip) Circuit which is key neuron substrates and responsible for the brain diseases and learning and memory. By using sensitization, CPP and MWM behavioral experiment, and molecular, biochemistry and histomorphology methods, including RT-PCR, Western Blot, co-IP Immunoflorescence, electronic microscope ( EM )， microinjection in ，et al, the biological effect of actin cytoskeleton in the process of morphine addiction memory formation to be investigated from multi- aspects. The aims are to clarify the function and the regulation mechanism of F-actin and G-actin，actin cytoskeleton rearrangement, Arc, Cofilin and Akt on cytoskeleton rearrangement and synaptic structure. As the cytoskeletal elements in neurons give the cell dynamic structure and are involved in a myriad of processes, including developmental ones like axon pathfinding and synapse formation, this study will provide new ideas on the molecular mechanisms of opioid addiction memory formation , and will give effective target for the opioid addiction treatment.

毒品滥用和成瘾是一种慢性复发性脑疾病，易引起人身损害、死亡和事故等，是法医鉴定的重要内容及研究领域。阿片毒品成瘾机制研究虽然取得长足进展，目前仍然不十分清楚；毒品滥用导致的成瘾记忆持久、顽固，是戒断难、复吸快、吸毒人数居高不下的重要原因。近来研究显示细胞骨架是成瘾记忆形成的重要分子；本课题选择与成瘾记忆密切相关的PFC和dHip两个重要脑功能区域，采用行为学、RT-PCR、Western Blot、Co-IP、免疫荧光、电镜、核团注射阻断等技术，从多个方面研究吗啡成瘾记忆形成过程中神经元Actin骨架蛋白的生物学效应，明确F-actin和G-actin在吗啡成瘾记忆中作用和调控机制，探索Actin骨架蛋白重排对Arc转运的作用及Arc、Cofilin和Akt对骨架蛋白重排和突触结构的调控；该研究对阐明阿片成瘾记忆形成的分子机制具有重要的意义，将为阿片毒品治疗提供新的思路和有效靶点

毒品成瘾是一种慢性复发性脑疾病。目前全球毒品持续泛滥，我国毒品问题突出，毒品成瘾和复吸机制仍然不十分清楚，是目前脑神经科学领域研究热点之一；在法医学领域，毒品滥用引起躁狂、冲动行为及情感等障碍，导致他杀、自杀、伤害事故等恶性案件，毒品相关死亡、损伤是法医病理、毒理等司法鉴定实践中常见问题。因此，对阿片毒品成瘾神经生物学机制的深入研究，将有助于发现新的治疗靶点，减少毒品社会和公共安全危害，具有重要科学意义和社会影响。本课题选择与吗啡成瘾记忆密切的前额叶皮层（PFC）、伏隔核（NAc）和海马（Hip）脑区，采用成瘾相关经典行为学实验（如CPP, BS, FST, SNI等），通过WB、免疫荧光、RT-PCR、脑核团定位、脑片膜片钳等技术，探讨阿片成瘾记忆中神经元骨架蛋白系统在毒品成瘾记忆及疼痛等诱发疾病中作用和机制；结果显示阿片类等毒品可引起PFC内锥体神经元突触可塑性变化，且与成瘾记忆强度相关；刺激PFC内投射至伏隔核的谷氨酸能神经元，诱导小鼠产生复吸行为。双侧VLO内注射组蛋白酶乙酰化酶抑制剂对大鼠慢性吗啡给药所致行为敏化表达具有抑制作用，对大鼠急性吗啡给药行为敏化表达具有增强作用；在大鼠行为敏化过程中，骨架蛋白可通过ERK/CREB分子信号通路激活，参与VLO内组蛋白乙酰化对吗啡成瘾行为调控；表观修饰KDM6B/ H3K27me3/ NR2A 和 lncRNA MALAT1通路也参与骨架蛋白及成瘾调控。PFC中DRD3可能通过骨架蛋白系统及相关BDNF等参与吗啡成瘾调控。慢性给予吗啡，NAc Shell内骨架蛋白发生重排，神经元棘突密度增加；5HT-6R介导骨架蛋白信号通路可能参与该过程调节海马中骨架蛋白参与吗啡成瘾环境线索记忆的巩固过程。本研究为毒品成瘾治疗提供新的靶点，为毒品相关死亡案件法医鉴定生物标记筛选提供新的视野。