【作者】 余巨明；

【导师】 蒋雨平；

【作者基本信息】 复旦大学 ， 神经病学， 2003， 博士

【摘要】 癫痫是神经系统常见病，临床表现以慢性、自发性和反复性发作为特征。其病因及发病机制复杂，至今尚无根治性治疗药物。动物实验已证实，一次长时或反复的癫痫发作可引起海马等易损区神经元坏死、反应性胶质细胞增生及苔藓纤维发芽等病理改变，并可能是癫痫发作得以长期维持的基础。已经证实，有些细胞因子和生长因子促进了以上病理变化的发生；有些则参与了对抗痫性脑损伤及癫痫发作。提示细胞因子和生长因子在癫痫后的病理机制中具有重要作用，并可能成为今后癫痫更有效治疗的切入点。活化素是转化生长因子β超家族成员之一。它有活化素A、活化素B和活化素AB三种，分别由βA-βA、βB-βB及βA-βB两个β亚单位组成。近年发现，缺血缺氧、局部机械性海马损伤及海人藻酸毒性损伤模型的海马组织中活化素βA mRNA表达明显上调，而活化素βB mRNA表达无变化；外源性活化素A能保护体内外神经元免受损伤。最新研究还发现，碱性成纤维细胞生长因子的神经元保护作用是通过诱导活化素βA mRNA的表达增加起作用的。因此活化素A有可能是脑保护与修复的关键性因子。然而，活化素βA 及活化素受体mRNA在癫痫发作模型海马组织中表达变化的研究鲜见报道；癫痫发作后活化素βA mRNA的变化是否导致活化素A蛋白水平的相应改变尚不清楚；活化素A是否对癫痫发作及海马病理改变有影响亦未见研究。本研究以匹鲁卡品（pilocarpine,PC）诱导癫痫持续状态（status epilepticus,SE）小鼠模型。发作行为按Racine标准分为0-Ⅴ级，以出现Ⅱ级以上发作并持续1h为癫痫持续状态(status epilpticus,SE)成模标准，成模后即刻腹腔注射安定终止发作，并以成模时定为SE后1h；无明显抽搐或呈非持续状态（non- status epilepticus ,NSE）发作小鼠列为NSE组，亦在与SE组相当时间腹腔注射安定；对照组以生理盐水取代PC，其余处理与PC给药鼠相同。首先应用RT-PCR方法同时观察了成模鼠（SE）与非成模鼠（NSE）海马组织活化素βA mRNA表达的时程变化；应用原位杂交及焦油紫尼氏染色方法分别观察了SE小鼠海马组织活化素βA mRNA表达的分布与海马形态结构的病理变化；接着采用非还原型SDS-PAGE和Western blotting观察了SE与NSE小鼠海马组织活化素A与抑制素A蛋白表达的动态变化，并同时应用RT-PCR分析了活化素受体ⅡA mRNA的表达；最后通过发作程度评分、脑电图记录、焦油紫尼氏染色及组织原位凋亡检<WP=7>测等方法观察了预先脑室注射重组人活化素A对PC致痫作用与海马病理变化的影响。结果显示：1、SE开始前小鼠海马活化素βA mRNA呈一过性明显下调，SE后1h上升至对照水平，SE后3h表达显著增高，6h达高峰，24h开始下降，48h下降至稍高于正常水平。而NSE小鼠海马活化素βA mRNA表达无明显改变。2、海马活化素βA mRNA表达阳性细胞为神经元，最早于3h时出现在CA2及DG区，6h达高峰时CA3区出现强阳性表达，24h时开始下降，48h时仅CA2区与对照相比仍有统计学差异，但CA1区始终未出现明显阳性表达；焦油紫尼氏染色显示，海马CA2区神经细胞受损相对较轻，CA1、CA3区损害严重。3、PC诱导SE引起的活化素βA mRNA表达上调能引起活化素A蛋白水平的相应上调，但其表达略微滞后，于6h增高达显著水平，24h达高峰，48h仍维持在较高水平；抑制素A蛋白水平及活化素受体ⅡA mRNA表达与对照组相比无明显差异。4、预先1h脑室注射rhACT与注射PBS比，腹腔注射PC后，rhACT组（16只）小鼠无死亡、癫痫持续状态及全身强直-阵挛发作，仅见全身颤抖或前肢阵挛发作；而PBS组（20只）4只严重发作致死（20%），11只出现癫痫持续状态（55%），2只出现全身强直-阵挛发作（10%），仅3只表现全身震颤而无抽搐发作（15%）。两组比较，发作程度有非常显著的差异（P＜0.001）；rhACT组脑电图无明显改变，或痫样放电显著减少，波幅明显降低；而PBS组脑电图表现阵发或持续高幅棘波、棘慢波放电；rhACT组24h及48h后海马各区未见明显结构性改变与神经元凋亡。而PBS组24h及48h后海马存在明显病理改变与神经元凋亡，并以48h为重。结论：1、PC诱导的SE能明显上调海马活化素βA mRNA及活化素A蛋白的表达，但对活化素受体ⅡA mRNA及抑制素A蛋白表达无明显上调作用。2、海马病理损害的轻重可能与活化素βA mRNA表达早晚及持续时间长短有关。3、外源性活化素A可以抑制痫样放电，减轻癫痫发作程度及海马神经元的损伤。表明SE后内源性活化素A蛋白表达的增加很可能起对抗癫痫性脑损伤与癫痫发作的作用。更多还原

【Abstract】 Epilepsy is a common neurological disease which characterized by chronic and spontaneous recurrent seizures.The cause and pathogenesis of epilepsy are very complex,and no radical drugs exist until now.Animal experiments have demonstrated that prolonged and recurrent seizure activity can result in a series of pathological changes including hippocampal neuronal cell death ,reactive gliosis and mossy fiber sprouting,which may be a basis for chronic and recurrent seizures of epilepsy.It has been proved that some cytokines and growth factors facilitate the genesis of these pathological changes,and some have an effect of anticonvlusion and neuroprotection.These results suggested that cytokines and growth factors may have a important role in the mechanisms underlying seizure-induced neuropathological changes and hopefully contribute to future therapeutic interventions.Activin is a member of the transforming growth factor-β superfamily.Activin is classified into activin A,activin B and activin AB,which are formed by two β-subunits of βA-βA,βB-βB and βA-βB respectively.Recent works indicated thatβA mRNA of activn was strikingly upregulated in the hippocampi following hypoxic-ischemic injury,focal mechanical brain injury and kaninc acid-induced excitoxic brain injury,butβB mRNA of activin showed no detectable changes. It was also found that exogenous application of activin A can protect neurons both in vitro and in vivo against injuries.Very recent study showed that induction of activin A is an essential step in the signaling cascade of basic fibroblast growth factor required for neuroprotection.Therefore,it is possible that activin A may play a critical role in neural protection and repair after all kinds of brain damage.But to date,reports on hippocampal expression of activin βA and activin receptor mRNA in convlusive animal models are very rare；no information is available on changes in activin and inhibin protein levels in the mouse hippocampus after seizures ,nor are there reports on the effects of activin A exogenous<WP=9>application on seizures, epileptiform discharges and related neuropathology. In this study,pilocarpine(PC) was used to induce status epilepticus(SE) in mice and the behavioral seizures were classified as 0～Ⅴ stages according to Racine’s standard. Animals that exhibited seizures above Ⅱ stage and lasted uninterruptedly for 1 hour served as SE group.Animals that did not have seizures or did not develop SE 1? h after PC administration served as non-status epilepticus group(NSE). Animals that administered nature saline(NS) other than PC served as control group. All animals were given an intraperitoneal(ip) injection of diazepam to suppress seizure activity immediately after experiencing 1 h of SE ,or 1? h after PC or NS administration for those animals in NSE and control groups. The time that animal had experienced 1h of SE was defined as 1h post-SE. Firstly, RT-PCR was used to study the changes of time course of hippocampal activinβA mRNA expression in SE and NSE mice；in situ hybridization was used to observe the distribution of activinβA mRNA in the hippocampus；cresyl violet staining was used to study the changes of hippocampal pathology. Secondly, western blotting was used to detect the dynamic changes of activin A and inhibin A proteins, and RT-PCR was used to detect the dynamic changes of activin receptorⅡA mRNA. Finally, using seizure score, EEG recording, cresyl violet and TUNEL staining ,the effects of recombinant human activin A(rhACT),which was injected intracerebroventricularly(icv) 1h before intraperitoneal injection of pilocarpine ,on PC-induced seizures and pathological changes of hippocampus were observed.The results showed that: 1) Prior to the beginning of SE（0h），the activin βA mRNA expression was transiently significantly lower than that of normal and NSE mice，but went up to control levels at 1h post SE. Fron then on, the level of activinβA mRNA still went up，and increased significantly at 3h post SE. The peak arrived at 6h post SE and could be更多还原