【作者】 宋琦琳；

【导师】 罗建红；

【作者基本信息】 浙江大学 ， 神经生物学， 2007， 硕士

【摘要】 创伤后应激障碍（Post-traumatie stress disorder，PTSD）是指对亲身经历或目睹的异乎寻常的威胁性、灾难性事件的剧烈反应。人们对创伤后应激障碍的认识已有100余年历史，曾被冠名为“炮弹休克”、“战争神经症”等。在美国精神障碍诊断与统计手册第三版（DSM-Ⅲ）中首次定义PTSD，并描述了其重新体验创伤、回避、过度警觉等三组主要症状。重新体验创伤症状包括梦魇、闪回记忆、创伤幻觉重现；回避症状包括故意遗忘、不愿谈论或回想；过度警觉症状包括严重的惊恐反应、睡眠障碍、注意力难以集中等。目前研究PTSD最多的是采用脑功能成像技术，主要用PET和fMRI。有的研究认为PTSD患者的海马体积变小，且与PTSD症状的严重程度成正相关；而有的认为PTSD患者海马体积没有变化。还有的研究发现PTSD患者晚期左侧杏仁核变小但活动增强，而早期右侧杏仁核反应降低。PTSD患者的前额叶体积似乎更小，在相关的认知任务试验中反应低下。谷氨酸受体家族分为离子型谷氨酸受体和代谢型谷氨酸受体两大类。离子型谷氨酸受体根据选择性激动剂的不同又可分为NMDA受体、AMPA受体和KA受体。离子型谷氨酸受体介导中枢神经系统的兴奋性突触传递。NMDA受体是由NR1、NR2或NR3亚单位组成的异聚体分子。已知NR1亚单位是NMDA受体的必需组分，不同的NR2亚单位与NR1装配形成了具有不同通道特征的NMDA受体亚型。NMDA受体是一类电压依赖的配体门控型离子通道，通道开放不仅需要谷氨酸和甘氨酸的共同结合，还需解除Mg²⁺对通道阻滞作用。AMPA受体主要是由GluR1、GluR2、GluR3和GluR4四种亚单位组成，目前认为AMPA受体是由四个亚单位组成的四聚体，既可以形成同聚体也可以形成异聚体。AMPA受体主要介导了中枢神经系统快速的兴奋性突触传递。AMPA受体激活引起细胞膜部分去极化，当去极化达到一定程度时解除Mg²⁺对NMDA受体通道的阻滞作用。研究表明，NMDA受体通道的激活有助于AMPA受体插入到树突棘的膜表面，从而使突触传递效能明显增强。目前没有治疗PTSD的特效药，仅限于用其他精神疾病的药物来替代治疗。先前的药理学研究表明阻断AMPA受体可以缓解PTSD的症状，但是具体哪个或者哪几个脑区参与其中，以及可能的分子机制尚不清楚。根据功能影像学研究，PTSD患者可能存在杏仁核、海马、前额叶皮层几个脑区结构和功能的改变。已知谷氨酸受体在突触可塑性和学习记忆方面起着重要作用，而PTSD与恐惧记忆密切相关。因此，在本研究中我们通过研究PTSD大鼠模型谷氨酸受体在海马、前额叶皮层、杏仁核这三个脑区的表达变化，来探究PTSD敏感化症状的分子机制，为临床治疗PTSD提供一定的理论依据。到现在为止，还没有能完全模拟人类PTSD疾病的动物模型，只能模拟PTSD的某个症状。我们根据Rau V，DeCola JP的研究建立了PTSD大鼠模型（该模型能有效模拟PTSD重要特征之一—敏感化）；将大鼠断颈处死，立即取脑，在冰上分离出海马、前额叶皮层、杏仁核；根据经典的方法提取脑组织总蛋白和膜蛋白；免疫印迹反应检测NR1、GluR1的含量。我们发现：（1）在海马部位，PTSD组膜蛋白GluR1含量显著升高，且维持在高水平。提示PTSD可能影响了海马的突触结构和功能，从而导致了行为敏感化。（2）在前额叶皮，PTSD组和Control组神经元细胞膜表面AMPA受体和NMDA受体含量均显著低于Naive组，这个结果提示应激可能引起前额叶皮层的活动降低，与影象学的结果一致。（3）在杏仁核，PTSD组NR1含量较Control组升高，PTSD组总蛋白中NR1的增加可能与其恐惧记忆相关。更多还原

【Abstract】 Post-traumatic stress disorder （PTSD） is a debilitating anxiety disorder that may develop after an individual has experienced or witnessed a severe traumatic event. It has been know over a hundrand years, ever been named as "shell shock"or "war neurosis". PTSD is characterized in the DSM-III by a phenomenological triad incorporating the symptoms of re-experiencing, avoidance and hyperarousal. The re-experiencing symptoms of PTSD include nightmares, intrusive memories and flashbacks of the trauma. The avoidance symptoms Neurobiology of posttraumatic stress disorder include amnesia for the trauma or a reluctance to discuss or think about the trauma. Finally, the hyperarousal symptoms include an exaggerated startle response, fitful sleep and poor concentration.Most functional brain imaging studies in PTSD have used either PET or fMRI. Imaging studies in PTSD patients have demonstrated volume reductions in the hippocampus that appear correlated with illness severity and the degree of cognitive deficit, although there is evidence of decreased hippocampal volume predating PTSD. PTSD subjects also showed a small but significant enhancement in left amygdala activity, most apparent during the late phase, but reduction in Early right amygdala response. In contrast, prefrontal cortex appears to be volumetrically smaller and is hyporesponsive during symptomatic states and the performance ofcognitive tasks in PTSD.Glutamate receptors are classified into two groups: ionotropic and metabotropic receptor families. There are three major types of ionotropic glutamate receptors （iGluRs）, which are named after reasonably selective agonists, thus, called N-methyl-D-aspartate （NMDA）, α-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid （AMPA） and 2-carboxy-3-carboxymethyl-4-isopropenylpyrrolidine （kainate） receptors. Ionotropic glutamate receptors mediate the vast majority of fast excitatory synaptic transmission in the CNS. NMDA receptors are heteromeric complexes incorporating different subunits within a repertoire of three subtypes: NR1, NR2 and NR3. It is widely recognized that the NR1 subunit is essential to the functional NMDA receptor channels, while various combinations of NR2 and NR1 subunits could endow NMDA receptor channel with different functional properties. NMDA receptor is a voltage-dependent ligand-gated ion channel. Activation of NMDA receptor not only requires to bind with glutamate and glycine, but also need to remove Mg²⁺ from NMDA receptor channel by membrane depolarization. AMPA receptors can exist as either homomeric or heteromeric assemblies of GluR1-4 receptor. AMPA receptors are the principal molecular units for fast excitatory synaptic transmission in the central nervous system. Activation AMPA receptor cause local membrane depolarization, which can release Mg²⁺ from nearby NMDA channel. Previous studies indicated, activation of NMDA receptor can induce AMPA receptors to insert into cell membrane of dendrite spine and enhance the synapse transmission efficiency.Until now, there is no wonder drug which can be used for PTSD medications and other mental disorder is always clinically used instead. Pevious pharmacological studies indicated that inhibition of AMPA receptor can ameliorate the symptom of PTSD, whereas the brain regions and mechanisms which are involved in PTSD are unknown. According to brain imaging studies, the brain regions involved in PTSD may include hippocampus, prefrontal cortex and amygdala, which may change functionally and structurally. It is well known that glutamate receptors play a key role in synaptic plasticity. In additon, PTSD is associated with fear memory andsynaptic plasticity in these brain regions. Therefore, in this study, to explore the sensitization mechanism of PTSD, we examined the expression level of the glutamate receptors in the hippocampus, prefrontal cortex and amylgada regions of PTSD model. It may aid to understand the underlying mechanism of PTSD sensitization and provide value for clinical therapy.So far, there is no any animal model of PTSD which can mimic human symptom. According to Rau V’s study, we established an animal model of PTSD, which successfully mimic the sensitizaiton, one of the important symptoms of PTSD. Exposure therapy （extinction） and amnestic （NMDA antagonist） treatment have no effect on this PTSD model. After treatment described as above, animals were sacrificed immediately. Hippocampus, prefrontal cortex and amygdala were dissected on ice. Total protein and membrane protein extraction were carried out using techniques described previously. Then, we examined the expression of NR1 and GluRl by Semi-quantitative Western blotting analysis. We found that hippocampal GluRl in membrane fraciton of PTSD group increased and maintained at hight level compared with control group. It suggests that trauma event may effect on synaptic structure and founction in hippocampus, thus result in behavioral sensitization. Moreover, our results indicate that, in stress groups, expression of NR1 and GluRl decreased compared with Naive group, which suggests stress reduced activity, and is consistent with neruoimaging. Finaly, NR1 amygdaloid totl lysis of PTSD group is increased. It suggests that NR1 increase may be related with fear memory of PTSD.更多还原