Reelin基因在恐惧记忆表达及其易感性中的表观遗传调控机制研究

Post-traumatic stress disorder (PTSD), as the main representative of the mental injury, is both involving the traumatic event type, intensity and duration, but also related to individual susceptibility. The inconsistant results of correlation analysis have been shown in PTSD population genetics study, due to inability to strictly controlling environmental factors, Cumulative effect related to the occurrence of PTSD development and its susceptibility mechanism, perhaps is related to epigenetic gene. Reelin is an important protein regulating neuronal plasticity and learning and memory. Adverse early environmental factors may act though epigenetic mechanism by Reelin, become a risk factor for development of PTSD characteristic symptoms, such as fear conditioning, and increase disease susceptibility. In order to verify the scientific hypothesis, this present projecr plan to use contextual fear conditioning, behavioral pharmacology, electrophysiology and molecular biology, to analyze the consolidation and reconsolidation of fear memory and hippocampal Schaffer-CA1 synaptic pathway plasticity, and explore epigennetic regulation of Reelin gene transcription in fear memory and vulnerability.

创伤后应激障碍（PTSD）为主要代表的精神损伤案件，既涉及创伤性事件的类型、强度及持续时间，也涉及个体的发病易感性差异。PTSD群体遗传学研究因无法严格控制环境变量，关联分析的研究结果不一。生命早期应激（ELS）如何引起成年个体PTSD的易感性增加，其机制应与基因序列改变无关，但更可能与相关基因的表观遗传累积效应有关。Reelin为影响神经可塑性和学习记忆的重要蛋白。ELS有可能通过Reelin的表观遗传调控机制，成为PTSD特征性症状条件性恐惧记忆的危险因子，增加疾病易感性。为验证这一假设，本项目将采用行为药理学、电生理学及分子生物学等技术，对场景条件性恐惧记忆的巩固与再巩固进行研究，分析海马Schaffer-CA1通路的突触可塑性机制与Reelin基因的表观修饰作用，探讨ELS对条件性恐惧记忆表达及其易感性发挥影响的表观遗传调控机制。

Reelin是一种细胞基质外糖蛋白，对神经发育及神经可塑性和学习记忆的易化发挥重大作用。近年来研究发现，reelin基因的表观遗传学调节机制与神经可塑性和学习记忆密切相关。ELS（早期生命应激）可引起成年个体PTSD的易感性增加，reelin基因的表观遗传学调节机制可能参与了此过程的发生和发展。本研究通过药理学、电生理学及分子生物学等技术，对场景条件性恐惧记忆的巩固进行研究，观察海马CA1区reelin基因甲基化与ELS引起成年个体PTSD易感性增加的关系。结果发现：（1）母爱剥夺组（MD组）和母爱分离组（MS组）的成年雄鼠经场景条件性恐惧记忆巩固后僵住行为明显增高，并与雄鼠海马CA1区reelin基因甲基化水平呈负相关。（2）MD组和MS组幼年雄鼠海马CA1区DNMT1、DNMT3A、DNMT3B mRNA表达明显下调，与海马CA1区reelin基因甲基化水平呈正相关。（3）经场景条件性恐惧记忆巩固后，MD组和MS组成年雄鼠海马CA1区reelin蛋白表达明显上调，与海马CA1区reelin基因甲基化呈负相关，但和NMDA 2A比NMDA 2B的蛋白量比值呈正相关。（4）药物干预后Zebularine组成年雄鼠海马CA1区reelin基因甲基化水平显著下调，并与场景条件性恐惧记忆巩固后僵住行为呈负相关。L-methionine组成年雄鼠海马CA1区reelin基因甲基化水平显著上调，并与场景条件性恐惧记忆巩固后僵住行为呈负相关。这些研究结果提示，早期生命应激通过抑制甲基转移酶的表达，使幼年雄鼠海马CA1区reelin基因甲基化水平下调，且这种甲基化状态从幼年一直保持至成年。早期生命应激通过下调成年雄鼠CA1区reelin甲基化水平，促进了场景条件性恐惧记忆巩固过程中reelin蛋白表达并提高了NMDA 2A比NMDA 2B的蛋白量比值，使场景条件性恐惧记忆巩固增强，从而增加了成年雄鼠PTSD的易感性。