【作者】 吴丽文；

【导师】 尹飞；

【作者基本信息】 中南大学 ， 儿科学， 2010， 博士

【摘要】 研究背景与目的：癫痫(epilepsy)是小儿神经系统常见疾病之一,发病率为3.5‰-6.6‰。内侧颞叶癫痫(mesial temporal lobe epilepsy, MTLE)是最常见的难治性癫痫之一,以海马硬化为其突出的临床病理特征。迄今为止,MTLE的治疗一直是癫痫治疗的难点,药物治疗及手术治疗都未能取得令人满意的疗效。因此,有关海马硬化的MTLE综合征的发病机制、对抗癫痫药物治疗的反应以及耐药机制等仍是近几十年来癫痫研究的热点与难点。儿童时期的癫痫以及癫痫综合征与成年期有很大不同。发育期的中枢神经系统由于其结构与功能以及与癫痫有关的神经递质、受体尚未完全成熟,痫性发作对未成熟大脑发生发展的影响以及未成熟大脑对抗癫痫药物治疗反应都随着大脑发育各阶段的不同而有很大的差异。大量研究表明,在伴有海马硬化性颞叶癫痫的成人患者中有相当高比例可追问到儿童早期有长时间痫性发作史,尤其是热性惊厥史。幼年早期长时间痫性发作后大脑可塑性发生的改变对个体能产生长远的影响,与MTLE的形成密切相关。因此,研究发育阶段幼鼠痫性发作后大脑各阶段的结构和功能与癫痫发生发展的关系以及对抗癫痫药物治疗的反应对于MTLE病理生理研究有着重要的意义。近年来有关MTLE的研究多集中在神经再生和突触可塑性、神经细胞损伤、细胞代谢和蛋白合成、神经传递和信号传导、细胞骨架的改变、离子通道等各个方面,但绝大多数研究均局限于某一个时间点的某一个方面,缺乏系统、连续的观察。而癫痫的发生是不断发展和变化的,因此,本研究运用蛋白质组学方法,分离鉴定幼鼠MTLE模型发生发展的各个时间段差异表达的蛋白质,并运用生物信息学技术,将各个阶段的差异表达蛋白质进行全景式比较分析,功能归类,从一个整体、连续发展的角度探讨MTLE发病机制。在此基础上,运用广谱抗癫痫药物丙戊酸(valproate, VPA)在早期对幼鼠模型进行干预,探讨VPA早期干预对幼鼠MTLE模型的疗效以及相关治疗机制,并收集人难治性MTLE术后海马标本进行临床验证,从多个角度探讨难治性MTLE的发病机制以及VPA早期干预的治疗机制,为今后通过药物治疗难治性MTLE提供一个新的视角。研究方法：本研究分为三部分：1.分别用10mg/kg、30mg/kg、50mg/kg、100mg/kg四种剂量的匹罗卡品对3周龄Sprague-Dawley大鼠进行诱导出癫痫发作,寻求匹罗卡品的最佳致痫剂量；致痫后连续观察8周,比较MTLE模型鼠的行为学、脑电图的改变,尼氏染色以及Timm染色观察海马的形态学改变,对慢性MTLE模型进行鉴定。2.根据MTLE发生发展过程取三个时间点(急性期、潜伏期、慢性期)一共分为6个组：急性对照组、急性模型组、潜伏对照组、潜伏模型组、慢性自发发作组、慢性无发作组。采用二维凝胶电泳技术分离各组大鼠海马总蛋白,PDQuest软件识别各组间差异表达蛋白质,基质辅助激光解吸电离飞行时间质谱进行鉴定,生物信息学软件对差异表达蛋白质进行比较分析、功能归类,构建与幼鼠MTLE发生发展相关的差异蛋白相互作用网络。Western blot技术对部分差异蛋白的表达进行印证。3.在自发发作开始出现的潜伏期末用VPA对MTLE模型持续干预2周,观察其对治疗的反应,尼氏染色和Timm染色观察治疗后的海马形态学改变并进行疗效评估；同时,对治疗与未治疗两组的大鼠海马蛋白表达谱进行分析比较,结合MTLE模型各阶段之间的差异表达蛋白谱,寻找与VPA早期干预密切相关的蛋白质并运用westernblot技术印证其表达情况。进一步地,收集人难治性MTLE术后海马标本,western blot检测其表达情况进行临床验证。研究结果：1.匹罗卡品10 mg/kg组的诱导成功率为0%,30mg组诱导成功率为30%,50mg/kg组的诱导成功率为60%,100 mg/kg组的诱导成功率为10%,其最佳致痫剂量为50 mg/kg;慢性MTLE模型成功率为63.3%。慢性MTLE模型鼠出现自发部分运动性发作,脑电图呈现典型癫痫样放电,尼氏染色显示有神经元脱失以及变性、胶质细胞增生；Timm染色可见到异常苔藓纤维出芽,异常兴奋环路形成。2.建立了幼鼠慢性MTLE模型发生发展各个阶段的海马二维凝胶电泳图谱。共鉴定了各个阶段差异表达的蛋白质共110个,这些蛋白主要参与的功能有：细胞骨架调节、细胞连接、能量代谢、突触形成和神经递质释放、热休克蛋白、信号通路调节分子等；通过比较分析发现部分蛋白在癫痫发生发展的各个阶段有不同表达。并用Western blot技术对部分差异表达蛋白进行了验证,其结果与比较蛋白质组学研究的结果基本一致。3.发现VPA早期干预可完全控制幼鼠MTLE模型的癫痫发作。尼氏染色和Timm染色的结果表明VPA具有神经保护作用,能阻止神经元脱失和异常苔藓纤维出芽,并发现突触相关蛋白synapsin-1、dynamin-1和neurogranin在VPA治疗组与未治疗组中有差异表达。Western blot技术检测synapsin-1、dynamin-1和neurogranin在大鼠模型和临床样本中的表达水平与比较蛋白质组学研究的结果一致。研究结论：1.本研究成功诱导出具有自发发作的幼鼠慢性MTLE模型,在发作形式、脑电图以及海马形态学改变等均符合人类MTLE的特征。为今后研究幼年期痫性发作对成年慢性MTLE的影响以及药物干预等提供了一个比较理想的实验性动物模型。2.首次建立了幼鼠慢性MTLE模型海马的连续、动态的凝胶电泳图谱；共鉴定了各阶段差异表达的蛋白质110个,这些蛋白可能与MTLE发病机制密切相关,为全面探讨慢性颞叶癫痫的病理生理机制及寻求最佳的药物作用靶点提供新的思路。3.丙戊酸早期干预幼鼠MTLE模型可完全控制癫痫发作,为今后临床上药物控制难治性MTLE提供了可能的最佳干预时间点；突触相关蛋白synapsin-1、dynamin-1、neurogranin与慢性MTLE模型发病机制、丙戊酸早期干预的治疗机理相关,可能是今后关于难治性MTLE研究的一个重要靶点。更多还原

【Abstract】 Background and objective:Epilepsy is one of the common seen neurological disorders in children, with prevalence and incidence of 3.5‰-6.6‰. Mesial temporal lobe epilepsy (MTLE) is the most common epilepsy syndrome with pharmacologically intractable partial-onset seizures. The pathological substrate is hippocampal sclerosis (HS). To date, most of the patients with MTLE do poorly responds to anti-epileptic drugs (AEDs) and surgically interruption, which is a headachy problem in the world. So, the studies about the pathogenesis of MTLE associated with HS, the responds to AEDs and mechanisms of drug-resisitence, are the focus and key fields about epilepsy in the recent decades.The epilepsy in children is quite different with the epilepsy in adults. In early development of the central nervous system, the structure and function, the neurotransmitters and receptors related to epilepsy had enormous room for growth. The influence of seizures to the immature brain and the effect of the AEDs are various in the different stages of the brain’s development. Quite a lot of research data shown that, a fairly high ratio of MTLE adult patients with HS can be pursued long history of epileptic seizures, particularly febrile convulsion in childhood. The brain plasticity change after long epileptic seizures in early childhood can produce long-term effects in the individual. MTLE with HS is closely related to the abnormal brain plasticity formation. Under go developmental changes from infancy to adult after the influence of seizures forms, plasticity and learning also contributes to the pathophysiology of epilepsy.The current molecular researches related to MTLE have been focused on regeneration and synaptic plasticity, neurons damage regulation, cellular metabolism and protein synthesis, neurotransmission and signal transduction, cytoskeleton changes and ion channels changes and ect. But a great part of these studies were limited to a certain aspect of a certain time, lacking a systematic and consecutive research. So, considered epileptogenesis’s unceasing development and changes, this study aimed to explore the pathogenesis and development of MTLE from a general and consecutive perspective, by using proteomics method to separate and identify the differentially expressed proteins in the different stages of immature rat models with MTLE, combined with bio-informatics technology to perform a global comparative analysis and function classification with the differentially expressed proteins. Then, the wide-broad AEDs valproate (VPA) was administrated in early stage of MTLE development to observe its therapeutic effects and to explore the related mechanisms. Furthermore, to approach the mechanisms and impact of VPA’s administration in different aspects, the expression levels of partial significant proteins in the postoperative hippocampus from patients with drug-resistant MTLE were validated, to explore new strategies and targets for clinical intervention in the future.Methods:The research includes three parts:1. For seizure induction, the 3-week-old Sprague-Dawley rats were intraperitoneally injected pilocarpine with different doses of 10 mg/kg,30 mg/kg,50 mg/kg and 100mg/kg, to obtain the most suitable dosage of pilocarpine. The surviving animals were continuously monitored for 8 weeks to observe the spontaneous seizures. After the chronic spontaneous MTLE induced, EEG was recorded to observe the epileptic discharges, nissl and Timm staining were performed to evaluate neurons loss and mossy fiber sprouting, so as to validate the chronic MTLE models.2. The MTLE rats were divided into 6 groups based on different stages (acute stage, latent stage, chronic stage):acute control group, acute seizure group, latent control group, latent seizure group, chronic spontaneous seizure group, chronic no spontaneous seizure group. Proteins in the hippocampus were separated by two-dimensional polyacrylamide gel electrophoresis (2-DE) technology. PDQuest software was used to analyze 2-DE images, and MALDI-TOF-MS was used to identify the differentially expressed proteins. Then the differentially expressed proteins were analyzed and classified globally and then the protein interaction network was construction by applying bio-informatics software. Western blot techniques were used to determine the differential expression levels of the partial proteins.3. VPA was administrated at or before the onset of spontaneous seizures for 2 weeks to observe the MTLE rats’responds to AEDs. Nissl and Timm staining were performed to evaluate changes of the hippocampal morphology, so as to evaluate VPA’s therapeutic effect. Proteins in hippocampus of VPA-treated and un-treated rats were separated and identified. The differentially expressed proteins were analyzed and classified globally by bio-informatics software to explore the interest proteins related to the VPA’s treatment. Furthermore, the expression levels of partial proteins in the postoperative hippocampus from patients with drug-resistant MTLE were detected, to validate the results clinically.Results:1.The seizure rate in 10mg/kg pilocarpine group was 0%, in 30mg/kg group was 30%, in 50mg/kg group was 60%, in 100mg/kg group was 10%. The most suitable dosage of pilocarpine was 50 mg/kg. The chronic MTLE rate was 63.3%. In chronic MTLE rats, there were spontaneous focal motor seizures observed; the EEG shown typical epileptiform discharge; a widespread cell loss and degeneration, reactive gliosis and plasticity of neurons were noted in the hippocampus by nissl staining, and Timm staining found abnormal mossy fiber terminal staining increased in inner molecular layer of dentate gyrus.2. The 2-DE patterns of different stage’s hippocampus with chronic MTLE of immature rats were established.110 differentially expressed proteins were identified, which could be divided into several main groups based on their functions:cytoskeleton modification, cell junction, energy metabolism, synaptic plasticity and neurotransmitter release, heat shock proteins, signal pathway regulatory molecules, and etc. The expression levels of several differentially expressed proteins were verified by western blot technique, and the results were coincidence with the proteome analysis.3. The administration of VPA in the early stage could control the spontaneous seizures in the immature MTLE rats. The results of nissl and Timm staining shown that VPA could prevent the neurons loss and the abnormal mossy fiber sprouting which conformed the neuroprotective effects of VPA. The synapse-related proteins synapsin-1, dynamin-1 and neurogranin were differentially expressed between the VPA-treated group and un-treated group. The western blot results both in the MTLE rats and human hippocampus were similar to the changes noted in 2-DE.Conclusion:1. This study successfully established a chronic MTLE model of immature rat, which was well coincidence with human chronic temporal epilepsy in seizure pattern, EEG recording and hippocampus pathology. This may conduce to further studies involving etiology or possible intervention of early stage’s hippocampus damage induced adult chronic MTLE2. The consecutive and dynamic 2-DE patterns of hippocampus in immature rats with MTLE were established firstly in this study. The 110 differentially expressed proteins identified by 2-DE in combination with MALDI-TOF-MS may be closely related to the mechanism of temporal lobe epilepsy, which provided important clues to elucidate the pathophysiological mechanisms of chronic MTLE and to identify an optimum medication intervention for the development of pharmacological therapies targeted at MTLE.3. The administration of VPA in the early stage could obtain seizure free based on the immature MTLE rat models, what provided an optimum medication intervention time of anti-epileptic drugs. The synapse-related proteins synapsin-1、dynamin-1 and neurogranin involved in the pathogenesis of chronic MTLE and the pharmacological effects of VPA, which probable developed into the focus of the study related to intractable epilepsy in the future.更多还原