【作者】 尹清；

【导师】 郑健；

【作者基本信息】 第三军医大学 ， 神经病学， 2008， 博士

【摘要】 癫痫是一种由多种病因引起,脑部神经元高度同步化,且常有自限性的异常过度放电所致的突然、反复和短暂的中枢神经系统功能失常,常合并局部结构、代谢及局部脑血流改变,是神经科常见的一组慢性疾病。传统上将非继发于神经系统或其它系统疾病,仅有发作期或/和发作间期脑的电生理学改变者称为“原发性癫痫”。经正规抗癫痫药物治疗,大多数病人的癫痫发作可得到控制,但仍有20-30%的病人癫痫难以控制,最终发展为难治性癫痫,其中60%～70%为颞叶癫痫(temporal lobe epilepsy,TLE),目前颞叶癫痫的发病机制尚未完全阐明。长期反复发作及药物副作用严重威胁着颞叶癫痫病人的日常生活,因此,对于颞叶癫痫发病机制的研究,具有重要意义。海马是颞叶癫痫的主要致痫灶,是其它类型癲痫放电扩散的中继站和放大器。同时海马齿状回(dentate gyrus,DG)及室下区(Subventricular zone,SVZa)也是成年哺乳动物脑内两个神经发生最集中的部位。颞叶癫痫最常见且最主要的病理变化为海马硬化(hippocampus sclerosis),包括神经细胞脱失,胶质增生和突触重组。苔藓纤维芽生(mossy fiber sprouting,MFS)是颞叶癫痫突触重组的主要形式,以齿状回颗粒细胞、内嗅区及CA3区常见。大量颞叶癫痫患者病灶标本和动物模型研究均观察到,癫痫所致的脑损伤发生后,齿状回颗粒细胞轴突(苔藓纤维)出芽,海马神经发生增加,门区异位颗粒细胞形成等现象。海马CA1、CA3区锥体细胞和门区神经元致痫损伤后,内分子层失神经传入同时苔藓纤维也与靶细胞断离,因而触发苔藓纤维出芽进入内分子层并与颗粒细胞建立突触联系。越来越多的研究表明:苔藓纤维出芽与慢性癫痫反复的自发性发作有关,同时又可增强癫痫的反复发作。但新生颗粒细胞增殖、迁移、分化并参与癫痫发作的病理机制及MFS的确切功能,目前尚不清楚。研究表明,在海人酸(Kainic acid,KA)致海马损伤后,海马齿状回下颗粒层神经发生增加,并伴有神经细胞迁移和神经细胞突触可塑性的增加。我们和国外同行近期均观察到颞叶癫痫发病过程中存在海马神经发生异常,主要表现在发生癫痫持续状态后,自发反复性癫痫发作出现前,海马齿状回下颗粒层细胞增殖能力明显增强,新生细胞大多分化为神经元(颗粒细胞),它们大多数向颗粒层迁移,成为成熟颗粒细胞,颗粒细胞产生新的轴突形成MFS。此后在不同的癫痫模型中,均发现癫痫形成伴有齿状回神经发生增加和异位颗粒细胞形成MFS,但不同癫痫模型神经发生增加的程度、时序有所不同。癫痫持续状态后,自发反复性癫痫发作出现前,增强的海马齿状回颗粒细胞为何没有定向迁移到神经元丢失区并补充和替代丢失的细胞,增殖的海马齿状回干细胞的迁移途径如何,最终定位于何处,目前尚不清楚。Noggin与骨成形蛋白4(Bone morphogenetic protein 4,BMP4)是胚胎期神经发生的重要调控分子,在原肠胚期共同参与了神经板与神经管的发生过程,其中BMP4抑制分离的外胚层细胞向神经元分化,促进这些细胞向上皮细胞分化;而Noggin作为BMP4的拮抗结合蛋白,与BMP4的亲和性较高,可阻滞BMP4与其相应的受体结合,从而抑制BMP受体信号通路,促进这些细胞向神经元分化。Noggin对成年中枢神经系统(Central Nervous system, CNS)神经干细胞的增殖、分化、迁移及维持神经元的正常功能有重要的作用。我们还发现,癫痫持续状态增强的海马神经干细胞增殖与海马的BMP4的过表达有关。在KA诱发的癫痫模型,海马CA3、CA4区还存在显著的神经元丢失。在KA致海马损伤后,Noggin在增强的齿状回颗粒细胞增殖中发挥何种作用,目前未见相关报导。为此,本实验采用KA侧脑室注射(intracerebroventricular,I.C.V.)致海马损伤模型,采用原位杂交、免疫组化、细胞培养等技术,观察海马齿状回Nestin阳性细胞的分布与BMP4的表达,探讨神经干细胞的增殖、迁移和分布与癫痫发病机制的关系;同时观察成年大鼠在KA侧脑室注射1天到30天内,海马神经元丢失情况,海马齿状回BrdU标记细胞数与Noggin表达的关系,为阐明Noggin对海马齿状回颗粒细胞增殖与胶质增生的作用提供依据。并通过培养永生化神经干细胞系,在体外实验中进一步证实BMP4/noggin可能对c17.2神经干细胞增殖、分化的作用。主要结果如下:1. KA侧脑室注射诱发SE后,注射侧海马CA3、CA4区神经元丢失明显并维持在整个实验观察阶段,没有细胞数量的恢复迹象,而注射对侧海马神经元丢失不明显。2. KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒下区Nestin阳性细胞异常增殖和迁移,主要分布在海马齿状回门区,同期观察到BMP4-mRNA阳性细胞在该部位有较多分布,提示成年大鼠海马齿状回颗粒细胞异常增殖和迁移可能与BMP4在该区的过表达有关。3.KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞异常增殖,主要分布在齿状回颗粒下层,同期观察到Noggin mRNA阳性细胞主要分布于海马齿状回的门区、颗粒下层、CA3、CA1区;海马Noggin mRNA阳性细胞在3天时升高,7天时下降。提示成年大鼠海马齿状回颗粒下层细胞异常增殖可能与Noggin表达波动有关。4.体外实验证明:BMP4能抑制c17.2神经干细胞增殖及其向神经元分化,noggin通过拮抗BMP4的作用,促进c17.2神经干细胞增殖及其向神经元分化。总之,本实验研究了BMP4/noggin在颞叶癫痫形成过程中在海马的表达,以及在海马神经干细胞异常增殖及分化中可能所起的作用;并在体外实验中观察了BMP4/noggin对于c17.2神经干细胞的增殖和分化的影响。结果表明:KA侧脑室注射致海马损伤后,成年大鼠海马齿状回颗粒细胞异常增殖和迁移,主要分布在海马齿状回门区,可能与BMP4在该区的过表达及Noggin表达波动有关。更多还原

【Abstract】 Epilepsy is caused by multiple etiopathogenisis and characterized by the high degree synchronization of neurons in brain accompanied with an abnormal over-firing-induced self-limited dysfunction of CNS. Epilepsy is a group of common chronic diseases of neurology, typically combined with the alteration of local structure, metabolism and local cerebral blood flow. Idiopathic epilepsy displays the electrophysiological change of the brain during the seizure of onset or/and interphase, which is not secondarily affected from nervous system or other system disorders. Given the regular antiepileptic treatment, seizure in most patients may become under control. However, the seizure of 20%-30% of patients is difficult to be controlled and develops ultimately into intractable epilepsy, 60%-70% of which is temporal lobe epilepsy (TLE). By now, the pathogenesis of TLE is not clear. The long time repeated seizure and drug side-effects pose severe threat to the daily life of TLE patients. Therefore, it is of much consequence to investigate the pathogenesis of TLE.Hippocampus is the main epileptogenic focus of TLE, and is also the relay station and amplifier of the firing diffusion of other types of epilepsy. Meanwhile, both the dentate gyrus (DG) and subventricular zone (SVZ) are the main neurogenesis sites in the adult mammalian brain. The main pathological change of TLE is hippocampus sclerosis, which includes neuron loss, gliosis and synapse reorganization. The main type of TLE synapse reorganization is the mossy fiber sprouting (MFS) in the DG granule cells, entorhinal and CA3 areas. Studies in TLE patients’specimens and animal models have demostrated that the seizure-induced brain injury including MFS in DG granule cells, increased hippocampus neurogenesis and hilar-ectopic granule cells generation. Followed by the seizure-induced injury of the pyramidal cells in the CA1, CA3 and the neurons in the hilus of hippocampus, the inner molecular layer lost the fiber afference and mossy fibers disconnected from the target cells. Consequently, mossy fibers are triggered to sprout into the inner molecular layer and, establish the synaptic connection with granule cells. There is a mount of evidences indicated that the mossy fiber sprouting contributed to the spontaneous repeated seizure of the chronic epilepsy, meanwhile, reinforced the repeated seizures. However, the mechanisms of adult hippocampus neurogenesis under seizure, as well as the function of MFS, are still unclear.Evidences have showed that adult hippocampus DG neurogenesis increased by kainic acid (KA) induced hippocampus injury, and accompanied with increased abnormal neuron migration and synapse plasticity. Recently, both the foreign researchers and our group found that abnormal hippocampus neurogenensis observed in process of TLE onset, which mainly occurred following statural epilepticus and before the spontaneous repeated seizures. Hippocampus DG granule cells proliferation was significantly increased, most of newly born cells differentiated into neuron (granule cells), migrated into granule layer and became the mature granule cells as well as generated new axon to form the MFS. In addition, it has been reported that varies types of animal epilepsy model were accompanied by the seizure generation, the neurogenesis of DG granule cells increased and ectopia granule cells formatted MFS. However, the extent and time curve of increased hippocampus DG neurogenesis are different among all kinds of epilepsy models. After the status epilepticus and spontaneous repeated seizure, the reason why the increased DG granule cells in the hippocampus does not automatically migrate into the neuron-loss zone and supplement or substitute for the lost cells, the route by which proliferative DG stem cells in hippocampus migrated, and the position where those newly born cells located are still under investigation.Noggin and the bone morphogenetic protein 4 (BMP4) are the key regulatory molecules in the embryonic neurogenesis, and contribute to the development of the neural plate and neural tube. BMP4 inhibited the ectoderm cells differentiated into neurons, but facilitated these cells differentiated into epithelial cells. As an antergic binding protein of BMP4, noggin has more affinity with BMP4 and can prevent BMP4 from binding with its receptors. Accordingly, noggin inhibited the signal transduction of BMP receptor, and promoted these cells differentiated into neurons. In the adult CNS, noggin plays an important role in the neural stem cells proliferation and differentiation and maintaining the normal function of neurons. We also demonstreated that KA-induced status epilepticus caused tonic proliferation of hippocampus neural stem cells was related to the hippocampus BMP4 over-expression, and accompanied with mount of neurons in the CA3 and CA4 areas lost. There are still no reports about the function of noggin in the proliferation of the increased DG granule cells followed the KA-induced hippocampus injury.Therefore, to approach the relationship between the epilepsy pathogenesis and proliferation, migration and distribution of the neural stem cells, hippocampus injury animal model induced by KA intracerebroventricular (I.C.V.) injection was used in this study. We observed the expression of nestin and BMP4 in DG of hippocampus by a series of techniques, such as in-situ hybridization, immunohistochemistry and cell culture, etc. Meanwhile, in order to elucidate the effect of noggin on the proliferation of DG granule cells in hippocampus and the gliosis, we detected the neuron loss in the hippocampus of adult rats within 1 day to 30 days after KA I.C.V. injection and disclosed the relationship between the number of BrdU labeled cells in DG of hippocampus and the expression of noggin. Furthermore, we tried to confirm the effect of BMP4/noggin on the proliferation and differentiation of c17.2 neural stem cells in vitro culture of immortalization neural stem cell line. The main results are listed as following:1. Adult rats given KA I.C.V injection induced SE, the neuron loss in the hippocampal CA3 and CA4 areas of the injection side was notable throughout the whole experimental observation phase. There were no detectable cells to recover from injury; otherwise, few cells lost in the opposite side.2. Adult rats given KA I.C.V injection, the nestin positive cells in the DG sub-granular zone were abnormally proliferated and migrated, and those newly born neurons mainly located in the dentate hilus. At the same time, we observed that BMP4 mRNA positive cells increased significantly in this area, which indicated that the abnormal proliferation and migration of the adult DG granule cells may relate to the over-expression of BMP4 mRNA.3. Adult rats given KA I.C.V injection, the DG granule cells in the hippocampus were abnormally proliferated, and those newly born neurons mainly located in the DG sub-granular zone. Meanwhile, we observed that noggin mRNA positive cells distributed in the hippocampal DG hilus, sub-granule layer, CA3, and CA1 areas. The noggin mRNA positive cells in the hippocampus increased 3 days post lesion, while decreased 7 days post-lesion. It suggested that the abnormal proliferation of the DG granule cells in the KA-lesioned hippocampus may be related to the fluctuation of the noggin expression in the hippocampus.4. In vitro study, we demonstrated that the ability of proliferation and differentiation into neurons in the c17.2 neural stem cells was inhibited by BMP4, while noggin facilitated these processes.In conclusion, here we have showed the expression pattern of BMP4/noggin and its potential role in the proliferation and differentiation of hippocampal neural stem cells during the formation process of TLE. Furthermore, in vitro study, we observed the effect of BMP4/noggin on the ability of proliferation and differentiation in the c17.2 neural stem cells. All of these results indicate that KA-lesioned hippocampus promote the DG granule cells abnormally to proliferate and migrate, and those newly born neurons mainly locate in the hippocampus DG hilus area. This may be related to over-expression of BMP4 and the fluctuation of the noggin expression in the hippocampus.更多还原