【作者】 刘备；

【导师】 高国栋； 张华；

【作者基本信息】 第四军医大学 ， 外科学， 2013， 博士

【摘要】 癫痫是一种神经系统疾病，通常是指大脑神经元突然异常放电，导致各种临床表现反复发作的慢性疾病。难治性癫痫也称顽固性癫痫，一般情况下，每月发病超过1次，正确用药超过3种，正规治疗时间超过2年仍无疗效者，可视为难治性癫痫[1]。大约30%的癫痫患者属于难治性癫痫[2]。我国癫痫患病率约为5‰，其中难治性癫痫患者有上百万名，给家庭和社会带来了沉重的经济负担和精神负担。几十年来，利用体外及体内癫痫发作模型努力探讨自发性癫痫反复发作（癫痫病患者的大脑的决定性标志）的分子和细胞机制。尽管我们在理解癫痫发生中获得巨大的进步，但是调查人员仍然没有制定可靠致痫病灶的生物标志物或替代标志物。目前对于导致癫痫发生的病理机制知之甚少，尤其是累积事件比如头部外伤，炎症，或长期热性惊厥所触发的癫痫[3]。癫痫治疗面临的主要挑战是癫痫固有的复杂性和异质性以及癫痫发作表现出的不同的遗传易感性。因此，癫痫发作共享一个基本病理生理机制是不可能的。过去几十年来，抗癫痫治疗的总目标是影响或者阻止原发性癫痫发作的急性过[4-6]。目前癫痫治疗一方面是急需要针对疾病发展变化的治疗，全面或部分阻止癫痫发作的反复发作。癫痫治疗的另一方面是减少或阻止癫痫发作所致的并发症。比如认知障碍，心理障碍所造成的危害。迄今为止，在动物模型中验证的癫痫发病过程中的分子和细胞改变包括：神经元损伤和细胞死亡，轴突和树突的可塑性，突触前和突触后的修饰，神经发生，神经炎症，胶质细胞活化，血管损伤和血管生成，细胞外基质破裂，离子通道结构和功能的改变[7]。近年来，研究发现癫痫发生和能量代谢失衡关系密切，能量代谢物质不平衡可以诱发癫痫发生。比如谷氨酸是中枢神经系统最主要的兴奋性神经递质，广泛分布于中枢神经系统，多种致痫机制通过谷氨酸递质及其受体途径导致癫痫发生。相关文献报道乳酸及乳酸转运体在动物模型的癫痫发生中起到重要作用，转运体的活动不仅直接反映突触信息的传递，当转运体的重摄取功能异常，就可以导致神经递质在突触间隙的浓度变化，从而引起病理生理改变，如果突触前递质质量异常，不仅突触后的受体出现数量改变，而且突触前的转运体也会发生代偿性变化，这种转运体的改变要比受体的改变更敏感、更直接。近年来一些研究发现转运体对癫痫的发生和抗癫痫药的作用机理、新药开发都出现了新的看法。基于此，我们进行以下研究：第一部分MCT4在癫痫动物模型和临床癫痫样本表达分析目的：研究MCT4在癫痫动物模型和临床癫痫样本表达情况。方法：从第四军医大学唐都医院神经外科标本库中随机选取30例颞叶癫痫组织标本作为实验组，12例外伤组织标本作为对照组。将大鼠分为生理盐水对照组、SE4h、SE12h、SE1d、 SE3d、SE7d、SE14d、SE28d、SE60d组，每组8只。建立大鼠氯化锂-匹罗卡品模型，在模型建立成功后在不同时间点收集大鼠颞叶及海马组织。利用西式印迹检测MCT4在癫痫大鼠皮层及海马组织表达，利用西式印迹和免疫组织化学检测MCT4在癫痫样本中表达情况，同时对临床癫痫样本进行甲基化PCR分析。结果：在西式印迹实验中，MCT4在癫痫模型颞叶皮层中急性期开始表达降低，在潜伏期表达达到最低水平，MCT4在海马的表达和颞叶皮层基本一致。在临床癫痫样本中MCT4表达降低。利用甲基化PCR方法证实MCT4在癫痫样本中表达减少与MCT4启动子区过渡甲基化有关。结论：无论是在癫痫模型还是临床癫痫样本中，MCT4表达均降低，研究还发现MCT4在癫痫样本中表达减少与MCT4启动子区过渡甲基化有关，这提示MCT4可能与癫痫发病机制密切相关。第二部分MCT4在癫痫发病中的作用分析目的：研究MCT4在癫痫发病中的作用。方法：建立大鼠皮层星形胶质细胞原代培养模型，在模型建立成功后将含有MCT4shRNA的慢病毒颗粒感染原代培养星形胶质细胞，然后通过细胞免疫荧光方法检测鉴定。利用MTT方法验证干涉MCT4对大鼠星形胶质细胞增殖能力的影响。同时采用流式细胞仪技术，验证干涉MCT4对大鼠星形胶质细胞凋亡及细胞周期影响。利用MCT4阻断剂阻断MCT4信号，观察阻断MCT4信号对神经元电活动影响。结果：成功构建MCT4干涉的原代培养的星形胶质细胞模型，MTT结果显示原代星形胶质细胞干涉MCT4后，细胞增殖能力显著减弱。流式结果证实原代细胞中干涉MCT4后，显著促进细胞凋亡。流式结果也证实原代细胞中干涉MCT4后导致细胞发生G1期阻滞。膜片钳结果证明：给予癫痫大鼠海马MCT4信号阻断剂后，神经元兴奋性增加。结论：在大鼠的原代培养星形胶质细胞中干涉MCT4可以导致细胞增殖能力减弱，流式细胞仪技术分析显示干涉MCT4促进细胞凋亡并且导致细胞发生G1期阻滞。而且干涉MCT4后促使神经元兴奋性增高。这些结果提示MCT4在正常星形胶质细胞的增殖、凋亡以及细胞周期方面扮演重要作用，并且MCT4表达对维持正常神经元兴奋性表达起到重要作用，因此MCT4在癫痫患者病灶中表达减少，可能是癫痫反复发作的一个重要原因。第三部分MCT4在癫痫发病中调控机制研究目的：研究MCT4在癫痫发病中调控机制研究。方法：建立大鼠皮层星形胶质细胞原代培养模型，在模型建立成功后将含有MCT4shRNA的慢病毒颗粒感染原代培养星形胶质细胞，然后收集蛋白利用西式印迹方法检测EAAT1表达情况。利用免疫共沉淀检测星形胶质细胞中MCT4是否和EAAT1相互结合。同时对所用癫痫样本进行免疫组织化学染色，检测MCT4和EAAT1在癫痫样本中表达情况。在原代培养的星形胶质细胞中加入放线菌酮，观察干涉MCT4后对EAAT1稳定性是否有影响。结果：大鼠星胶质细胞中干涉MCT4后可以导致EAAT1表达降低，免疫共沉淀证明了在原代培养星形胶质细胞中MCT4和EAAT1是相互结合的。免疫组织化学证实癫痫病灶中MCT4和EAAT1表达具有临床相关性。放线菌酮实验证实，在原代培养的星形胶质细胞中干涉MCT4可以导致EAAT1稳定性减弱。结论：星形胶质细胞中干涉MCT4可以导致EAAT1表达减少，并且免疫共沉淀也证实了在星形胶质细胞中EAAT1和MCT4相互作用，这提示在星形胶质细胞中MCT4可能调控EAAT1表达。斯皮尔曼等级相关性分析证实了MCT4和EAAT1在癫痫病灶中的表达具有临床相关性。放线菌酮实验进一步证实了在星形胶质细胞中干涉MCT4可以影响EAAT1转录后的稳定性。以上实验从多方向说明了MCT4和EAAT1密切相关，可能调控EAAT1表达，为临床上以MCT4为新的药物靶点开发新的抗癫痫药物提供充分的证据。更多还原

【Abstract】 Epilepsy is characterized by a long term risk of recurrent seizures.Epilepsy isusually controlled, but not cured, with medication. However, more than30%ofpeople with epilepsy do not have seizure control even with the best availablemedications. Intractable epilepsy is a seizure disorder in which a patient’s seizuresfail to come under control with treatment. For several decades, both in vitro and invivo models of seizures and epilepsy have been employed to unravel the molecularand cellular mechanisms underlying the occurrence of spontaneous recurrentseizures (SRS) the defining hallmark of the epileptic brain. However, despitegreat advances in our understanding of seizure genesis, investigators have yet todevelop reliable biomarkers and surrogate markers of the epileptogenic process.Sadly, the pathogenic mechanisms that produce the epileptic condition, especiallyafter precipitating events such as head trauma, inflammation, or prolonged febrile convulsions, are poorly understood. A major challenge has been the inherentcomplexity and heterogeneity of known epileptic syndromes and the differentialgenetic susceptibilities exhibited by patients at risk.Therefore, it is unlikely thatthere is only one fundamental pathophysiologic mechanism shared by all theepilepsies.Identification of antiepileptogenesis targets has been an overarching goalover the last decade, as current anticonvulsant medications appear to influence onlythe acute process of ictogenesis. Clearly, there is an urgent need to develop noveltherapeutic interventions that are disease modifying therapies that eithercompletely or partially prevent the emergence of SRS.An important secondary goalis to develop new treatments that can also lessen the burden of epilepsycomorbidities (e.g., cognitive impairment, mood disorders) by preventing orreducing the deleterious changes during the epileptogenic process. So far, themolecular and cellular changes reported during the presumed epileptogenesisprocess in animal models include neuronal injury and cell death, axonal anddendritic plasticity, presynaptic and postsynaptic modifications, neurogenesis,neuroinflammation, glial cell activation, vascular damage and angiogenesis,disruption of extracellular matrix integrity, as well as structural and functionalchanges in ion channels properties. In recent years, a variety of studies have foundthat the incidence of epilepsy and energy metabolism imbalances are closely related,the the energy metabolites imbalance can induce epileptic occurs.Glutamate is the primary excitatory neurotransmitter in the central nervous system(CNS), mechanisms that promote glutamatergic neurotransmission could playimportant roles in the development of epilepsy. Increased levels of extracellularglutamate in fact have been observed in human epileptic serum. In vivomicrodialysis studies also show elevated extracellular concentration of glutamate inthe epileptogenic cortex as compared to the nonepileptogenic cortex in patientswith temporal lobe epilepsy (TLE). These reports suggest that dysfunctionalextracellular glutamate cycling and reuptake may play a vital role in the genesisand maintenance of focal epileptic activity.Glutamate transporters, also referred toas excitatory amino acid transporters (EAAT), represent the sole mechanism of active reuptake of glutamate into the astrocytes and neurons and are essential todampen neuronal excitation following glutamate release at synapses.Monocarboxylate transporters (MCT) facilitates the transport of monocarboxylatefuels (lactate and pyruvate) and acidic drugs, such as valproic acid, across cellmembranes. A resent research shows that MCT1is deficient on microvessels in theepileptogenic hippocampal formation in patients with medicationrefractoryepilepsy. To further define the role of MCT in the pathophysiology ofmedicationrefractory epilepsy, we used immunohistochemistry, western blot andwhole cell clamp analysis to localize and quantify the transporters in thehippocampal formation in the novel and highly relevant rat model ofedicationrefractory epilepsy and in nonepileptic control animals. We were also todetermine the expression and distribution of MCT in tissue samples from therefractory cortex of patients who had been surgically treated for refractory epilepsy.We compared these tissues with histologically normal samples from controls. Inour present study, we found MCT4was lost on hippocampus and piriform corticesin the lithium-pilocarpine model. In addition, we were able tocoimmunoprecipitate MCT4and EAAT1in primary rat astrocytes. We also foundRNAi-mediated inhibited of MCT4can decrease the expression of EAAT1inprimary rat astrocytes. It is possible that EAAT1is a substrate for MCT4, implyingMCT4may directly modulate EAAT1in primary rat astrocytes. Therefore, wehypothesize that the loss of MCT in brain is mechanistically involved in thepathophysiology of intractable epilepsy, and propose that re-expression of MCTmay represent a novel therapeutic approach for this disease.This study is supposedto confirm the above hypothesis. The results from this study will shed light on newstrategic treatment for epilepsy. The following research has been conducted on thisbasis.Part1The expression of monocarboxylate transporter4in epilepsy animalmodels and clinical epilepsy sampleObjective： The purpose of our research is to explore the expression of monocarboxylate transporter4in epilepsy animal models and clinical epilepsysample. Methods：The cases included in the study were obtained from the files ofthe Department of Neurosurgery of Tangdu Hospital of the Fourth Military MedicalUniversity. We examined30specimens obtained from patients undergoing surgeryfor medically intractable epilepsy. The control samples obtained from12patientswho underwent neurosurgical intervention for increased intracranial pressure due tohead trauma. The li-pilo model has been established. The rats were randomlydivided into a normal control and eight Li-pilo model (SE4h、SE12h、SE1d、 SE3d、SE7d、SE14d、SE28d、SE60d) groups. After the success of the model, Rattemporal lobe and hippocampal tissue collected at different time points. The epilepsyrat cortex and hippocampus expression of MCT4was studied by Western blot. Theexpression of MCT4in the cortex of patients with intractable epilepsy was studiedby western blot and immnohistochemistry. Meanwhie, these clinical samples wasstudied by methylation-specific PCR. Results: In western blot test, the temporalcortex MCT4protein level was shown to be low in the acute phase of the epilepsymodel, the expression of MCT4protein decreased dramatically in the latent phase ofthe epilepsy model. The expression of MCT4in the hippocampus is consistent withthe expression in temporal cortex. In brain tissue, decreased MCT4proteinexpression has been found in the cortex of patients with intractable epilepsy. We alsofound hypermethylation of MCT4Promoter Contributes to the down-regulation ofMCT4in human epileptic brian tissue. Conclusion: Both in the epilepsy model orclinical epilepsy sample found MCT4expression reduced dramatically. The studyalso found that hypermethylation of MCT4Promoter Contributes to the decreasedexpression of MCT4in epileptic tissue.Based on the phenomenon of decreasedMCT4expression in the cortex of patients with intractable epilepsy, we suggest thatMCT4may play a role in intractableepilepsy.Part2The role Of MCT4in epileptogenesisObjective: To explore the role of MCT4in epileptogenesis. Methods: The model of primary culture rat cortical astrocytes has been established. Primary culturecells were transduced with the lentiviral particles containing MCT4shRNAsequences, these cells were identified by immunofluorescence. Measurement ofthese cells growth by Methyl Thiazolyl Tetrazolium Assay (MTT). Apoptosis andcell cycle of these cells were analyzed by flow cytometry. Moreover, the MCT4blockers are used to observe the effects of the blockers on the electrical activity ofneurons. Results: The model of MCT4shRNA primary culture rat corticalastrocytes was successfully established. In our study, the cell growth curvesshowed that the growth of MCT4shRNA cells was notably inhibited in atime-dependent manner. We also found the decreased cell number was attributableto apoptosis induced by MCT4-targeting shRNA. To explore the potentialcontribution of MCT4shRNA to cell cycle progression, we used flow cytometry toevaluate the cell cycle distribution. The results showed that MCT4shRNA cellsaccumulated in G0/G1phase, but the cell numbers in G2/M phase were reducedsharply. The patch clamp results show that the MCT4signal blockers CHCincreased neuronal excitability. Conclusion: In the current study, we demonstratedthat suppression of MCT4inhibited primary culture rat cortical astrocytes growth.In the model of primary culture rat cortical astrocytes, we found suppression ofMCT4inhibited proliferation and induced apoptosis. We also demonstrated thatsuppression of MCT4increased neuronal excitability. Because CHC increasedneuronal excitability and that depletion of MCT4in primary culture rat corticalastrocytes suppressed proliferation and induced apoptosis, MCT4may play animportant role in epileptogenesis.Part3The potential molecular mechanisms of MCT4in epileptogenesisObjective: To explore the potential molecular mechanisms of MCT4inepileptogenesis. Methods: The model of primary culture rat cortical astrocytes hasbeen established. Primary culture astrocytes were transduced with the lentiviralparticles containing MCT4shRNA sequences, Western blot analyses wereperformed as described above. Whole primary culture rat cortical astrocytes lysates were obtained by resuspending cell pellets in RIPA buffer. Lysates were incubatedovernight with EAAT1antibody before being absorbed with proteinA/G PLUS agarose beads. Precipitated immunocomplexes were released by boilingwith2×SDS electrophoresis sample buffer and were prepared for Western blotanalysis. For immunohistochemical analysis, clinical sample sections were stainedand evaluated. Primary culture rat cortical astrocytes stably transfected with thelentiviral particles containing MCT4shRNA sequences were treated with20mg/mlcycloheximide (CHX) for0,1,3,6, and12h. After treatments, Western blotanalyses were performed. Results: Western blot results demonstrated decreasedexpression levels of EAAT1in MCT4silence astrocytes, compared with controlcells. We found MCT4was coimmunoprecipitated with EAAT1when anti-EAAT1was used to pull down EAAT1protein and its associated proteins. Wedemonstrated that the expression of MCT4and EAAT1has clinical relevance inclinical epilepsy sample. We also found the decrease in the EAAT1protein levelfor MCT4silence astrocytes correlated with a decrease in its stability. Conclusion:In the current study, we demonstrated that suppression of MCT4contributes to thedecreased expression of EAAT1in primary culture rat cortical astrocytes. Inaddition, we were able to coimmunoprecipitate MCT4and EAAT1in imary culturerat cortical astrocytes. A clinical relevance comparison of the expressions of MCT4and EAAT1demonstrated that EAAT1expression in clinical epilepsy sample wassimilarly associated with MCT4expression.CHX treatment revealed that MCT4bindingEAAT1could stabilize EAAT1expression in primary culture rat corticalastrocytes. Taken together, these results provide the biological basis for MCT4as acandidate therapeutic target for antiepileptic drugs.更多还原