【作者】 孟繁东；

【导师】 鞠躬；

【作者基本信息】 第四军医大学 ， 神经生物学， 2004， 博士

【摘要】 神经元之间信息传递的基本结构是突触，突触分为兴奋性突触与抑制性突触。中枢神经系统兴奋性突触主要释放谷氨酸，通过突触间隙，与突触后膜存在的多种谷氨酸受体相结合，引发突触后神经元兴奋性改变。谷氨酸受体根据激活拮抗剂特点可分三种类型，即NMDA受体、AMPA受体、KA受体等。谷氨酸与突触后膜相应受体结合后，改变神经元细胞膜的离子通透性，导致去极化，引发神经电活动。 NMDA受体与AMPA受体同属离子通道型受体，主要集中在突触后致密区(PSD)，都由数个亚基组成，但亚基的类型及作用完全不同，并有着不同的结合调控蛋白，从而发挥不同的作用。NMDA受体在突触后膜分布相对稳定，而AMPA受体可随着突触活动强度而发生变化。AMPA受体调节突触后神经元的快速去极化，NMDA受体启动突触的可塑性调节，这两种受体在突触的锚定有着不同的调控机制。突触膜上的受体类型、数量及在膜上的定位是进行有效信息传递不可或缺的。有一个大的蛋白家族能与它们相互作用并形成复合体，以调节它们的分布、活性，从而影响突触功能及神经元之间的信息传递。在它们的合成与运输过程中也受某些蛋白的调控。 AMPA受体与NMDA受体在兴奋性突触后膜上有大量的共存，但它不与第四军医大学博士学位论文PSD一95蛋白家族发生作用，而是通过其亚基GluRZ/3特异性地与其它的PDZ蛋白质家族成员结合，包括谷氨酸受体反应蛋白(glut田爪ate reeeptor一interacting proteinG孙pl)，G犯PZ(ABp一L)及激酶C反应蛋白(protein interacting with C kinaselPICKI)等。GRlpl与GRIpZ都各有7个PDZ结构域，PDZ结构域在受体的定位、分布及功能上有重要的作用。AM队受体C一末端(一ESVKI)能选择性地与GRIPI的4、5结构域及GRJpZ的3、5、6结构域结合。GRIpZ(又称ABp一L ABp一like protein)实际上与ABp(AMPA reeeptor binding protein)是同源的，它有第7个PDz结构域，而ABP没有。G班Pl与GRIPZ都是分子量在1 30kDa左右的蛋白，在中枢神经系统中有丰富的表达，其中以大脑的皮层、海马及嗅球中的表达量较高，二者之间有大量的共存，主要存在于兴奋性突触的胞膜上及突触后致密区(PSD)结构中。G犯Pl在胚胎早期就有表达，而G斑PZ表达出现较晚，与AMPA受体表达基本同步。G班Pl在抑制性突触(GABA能)及GABA能神经纤维中也有表达。他们可与AMPA受体结合形成复合体，影响突触后膜AMPA受体的分布，并能调节AMPA受体的功能，从而影响神经元的兴奋性信息传递。 同样GRIPI与G犯PZ能与驱动蛋白(丸nesin)结合，调节神经元树突的物质运输。在神经元，驱动蛋白由两条重链(KHC:KIFS)及两条轻链(KLC)组成，不管是在轴突中的物质运输(如:s”apsin，GAP并3)还是树突中的物质运输(如:信使RNA，AMpA受体)都是由这种多功能的转运蛋白进行的，调控着物质的运输方向。所有的KJFS同工酶(EJFSA，KIFSB，KIFSC)都含有最小的GR[P1结合位点。说明它的重链可以直接与G犯 Pl结合，可以与G班Pl及GluR形成大的复合体，对GRIPI在神经元及突触上的分布是必须的，同样也影响了A加[PA受体的功能。 谷氨酸受体(GluR)可以调节中枢神经系统的兴奋性突触传递，并在神经发育、神经兴奋性毒性及突触可塑性方面起一定作用。在缺血、缺氧等急性神经元损伤中，谷氨酸起着关键性作用。正常情况下，由于细胞外兴奋性氨基酸第四军医大学博士学位论文(excitatory alnino acids，EAA)的浓度受神经元和胶质细胞的高亲和性摄取系统所控制，不会积累到使神经元损伤的程度，在脑缺血等病理情况下，细胞外EAA的浓度由于神经元释放增加、重摄取减少及死亡细胞内EAA大量溢出而大幅度增加。EAA不仅通过NMDA受体导致细胞变性死亡，而且AMPA受体也参与了此过程。由于EAA所造成的神经损伤可进一步使谷氨酸释放增加，因此谷氨酸的神经毒性作用具有正反馈的性质。由于G班Pl与G犯PZ不仅能与Kinesin结合影响受体的合成运输，并能与AM队受体结合调节受体的分布与功能，所以在这一过程当中，GRIPI与GRIPZ可能起着重要的作用，搞清楚在损伤过程中GRIPI与G班PZ的功能变化及作用机制，有可能为寻找相应的损伤拮抗剂并及早抑制神经损伤的进一步发展提供可靠的科学依据。具有较大的临床治疗意义。 本研究采用成熟的大鼠脑缺血模型，根据脑缺血损伤后的不同时间点分为6个组，首先采用免疫组织化学的方法，分别用抗G班P1与G犯PZ抗体进行免疫标记，分析脑缺血后不同时间点缺血坏死的特征，研究发现在大鼠脑缺血损伤发生后损伤灶周围有不同程度的坏死出现，在未发生坏死皮层出现G租Pl及GRIPZ的阳性标记细胞与神经纤维，损伤后6h和ld组的阳性标记较多，说明在损伤发生后G班Pl与G班PZ表达增强，可能参与了损伤或损伤修复过程。由于AMPA受体在神经毒性中的作用，我们进一步研究缺血损伤发生后G班Pl与GRJPZ与A加于A受体的关系。通过免疫组织化学双标记研究，证实了AMPA受体亚基Gl证Rl、GluRZ/3与GRIPI、G犯PZ在神经元存在双标记，有明显的共存现象，共存多出现在损伤后6h和ld，说明GRIPI、GRIPZ在缺血损伤或修复过程中起了一更多还原

【Abstract】 The basic communication between neurons is synape,and it was divided inio excitatiory and inhibitory synapse.In excitatory synapes ,a signal-transmitting neuron secretes glutamate (Glu) -a chemical messenger that diffuses across the synaptic cleft to bind to glutamate receptors (GluR) concentrated in the postsynaptic membrane of the receiving neuron. GluR can be subdivided based on their pharmacology into three major classes: AMPA, kainate, and NMDA receptors et al.,. AMPA receptor in the postsynaptic membrane can bind with Glu,which can induce the depolarization.NMDA receptor and AMPA receptor are all ionotropic glutamate receptors. They concentrate at postsynaptic sites and largely colocalized in excitatory synapses. Both of them are heteromeric complexes composed of some homologous subunits that differentially combine to form a variety of AMPA or NMDA receptor subtypes and play different roles. NMDA receptor are relatively fixed, AMPA receptors cyclesynaptic membraneson and off. AMPA receptors mediate rapid excitatory synaptic transmission whereas NMDA receptors initiate synaptic plasticity. .Mechanisms for controlling synaptic expression of these two receptors classes are different. Synaptic localization and clustering of ion channels and neurotransmitter receptors are necessary for normal synaptic transmission . There is a large family of interacting proteins regulate AMAP receptors turnover at synapses , they can form a complex to regulate the distribution and function of the receptors, and have effects on the neuroal transmission.Ionotropic glutamate receptors of the AMPA largely colocalize with NMDA receptors in excitatory synapses at postsynaptic site, but they do not interact with PSD-95 family proteins. Instead, the AMPA receptor subunits GluR2/3 bind specifically to other PDZ proteins, termed glutamate receptor-interacting protein (GRIP), AMPA receptor-binding protein (ABP), and protein interacting with C kinase 1 (PICK1). Both GRIP1 and GRIP2 contain seven PDZ domains and no other recognizable motif, ABP resembles GRIP in primary sequence; it differs from GRIP most notably in lacking the C-terminal seventh PDZ domain. The C-terminal sequence (-ESVKI) shared by AMPA receptor GluR2/3 subunits is reported to bind selectively to the forth and fifth PDZ domains (PDZ4/5) of GRIP and the third, fifth, and sixth PDZ domains of ABP. The GRIP1 and GRIP2 antibodies specifically detected GRIP1 and GRIP2 as 135 and 130 kDa proteins, respectively. The GRIP1 protein was expressed in brain and testis but was not detected in heart, spleen, lung, liver, skeletal muscle, kidney, and intestine, whereas the major forms of GRIP2 were selectively expressed in brain (Fig. 2a). GRIP1 and GRIP2 had similar distributions in rat brain regions and were enriched in the olfactory bulb, cortex, and hippocampus and were colocalized in many regions. GRIP1 expression in rat brain was detected in earlyembryonic stages, peaked at approximately postnatal day 6-8, in contrast, GRIP2 expression was relatively low early in development and increased postnatally, reaching a peak at postnatal day 14, similar to what is observed for GluR2. we could also find enrichment of GRIP in GABAergic neurons and in GABAergic nerve terminals. GRIPl and GRIP2 are AMPA receptor binding proteins potentially involved in the targeting of AMPA receptors to synapses. GRIPl also may play functional roles at both excitatory and inhibitory synapses, as well as in early neuronal development.GluR can regulate the excitatory synaptic transmission in the CNS, they play roles in neuronal development, excitotoxicity, and synaptic plasticity. During acute injury of neural anoxia or ischemia, Glutamate are necessary .In normal condition, the density of extracellular EAA(excitatory amino acids) is mediated by the ingestion of neurons and glia cells , and can not run up to hurt the neurons. When anoxia or ischemia occur , the density of extracellular EAA increase dramatically because of the increasing of releasing ,the decreasing of ingestion and the overflowing of in更多还原