【作者】 周颖；

【导师】 肖波； 黄志凌；

【作者基本信息】 中南大学 ， 神经病学， 2008， 博士

【摘要】 研究背景和目的:颞叶癫痫(temporal lobe epilepsy,TLE)是难治性癫痫的常见类型。目前认为颞叶癫痫难治的重要原因是神经环路重建以及兴奋性异常的神经网络形成,但其机制欠清楚。因此,探索神经网络兴奋性异常的原因具有重要意义。业已证实,癫痫发病与兴奋性谷氨酸能突触传递效能增高密切相关,其机制涉及突触前神经递质的释放增加、突触间隙谷氨酸盐代谢障碍、突触后受体的敏感性增加3个方面。以往的研究多侧重于突触前和突触间隙调节机制,而对突触后调节机制研究不多。突触后致密物(postsynaptic density,PSD)作为兴奋性突触后膜的超级信号复合体,可调节突触后受体的敏感性,改变神经信号传递的下游效应,势必最终决定兴奋性突触的传递效能,这可能是癫痫神经网络兴奋性异常形成的核心所在,亟待深入探讨。但PSD与癫痫的关系国内外研究甚少,只见于数量有限的几种PSD蛋白质表达变化,迫需全面深入探讨PSD蛋白与TLE发病的关系。为更快、更深入、更全面地探讨PSD蛋白质在颞叶癫痫形成中的作用机制,本研究采用高通量、高敏感的蛋白质组学技术,构建颞叶癫痫大鼠全脑组织PSD蛋白质的双向凝胶电泳图谱,首次对颞叶癫痫相关PSD蛋白进行筛选和鉴定,为深入研究PSD蛋白与癫痫的关系提供线索,为寻找TLE防治新靶点提供依据。研究方法:建立氯化锂—匹罗卡品大鼠颞叶癫痫模型。实验分为三组,即TLE自发组(spontaneous recurrent seizure,SRS)、TLE无自发组(non-spontaneous recurrent seizure,NSRS)、正常对照组(Norm组)。采用蔗糖密度梯度离心联合膜顺序提取法分别提取三组大鼠全脑组织的PSD蛋白成分。利用蛋白质组学技术对提取的PSD蛋白进行比较筛选,鉴定出TLE发作相关蛋白,并采用western免疫印迹法进一步验证部分TLE相关PSD蛋白的表达情况。研究结果:1、氯化锂—匹罗卡品大鼠颞叶癫痫模型:致痫成功率为91.4%,模型成功率为71.4%,死亡率为20%。2、PSD纯度的检验:检测发现突触后标志分子NR1和PSD-95在P2成分、突触体和PSD蛋白三组成分中均呈免疫印迹阳性,而突触前标志分子SynPhy在PSD成分中呈阴性。3、PSD蛋白质的双向凝胶电泳:通过PDQuest软件进行比较分析,结果显示:One-PSD图谱约有286±25个蛋白质点,Two-PSD图谱有720±17个蛋白质点,两组比较有显著差异性(P＜0.01)。与对照组相比较,TLE自发组(SRS)表达上调的有12个,表达下调的有58个;TLE无自发组(NSRS)表达上调的有15个,表达下调的有38个。与TLE无自发组比较,TLE自发组表达上调的蛋白点有15个,表达下调的有25个。其中发现仅在TLE自发组特异表达的有4个。4、部分差异PSD蛋白点的质谱分析:鉴定的26个差异蛋白质点根据功能可分为6类:①细胞骨架蛋白:微管蛋白tubulin alpha和tubulin beta,internexin-alpha,actin;②运输蛋白:Sorting Nexin 3(SNX3);③能量代谢相关酶蛋白:肌酸激酶(creatine kinase,CK),果糖二磷酸醛缩酶(fructose-bisphosphate aldolase A,FBA),甘油醛三磷酸脱氢酶(glyceraldehydes-3-phosphate dehydrogenase,GAPDH),琥珀酸辅酶A连接酶(Succinate-coenzyme A ligase,SCOL),乌头酸水合酶(Aconitate hydratase,ACO);④分子伴侣:热休克蛋白-27(heat shockprotein-27,HSP-27);肽酰脯胺酸顺反异构酶(peptidyl-prolyl cis-transisomerase A,PPIase);⑤信号转导分子:甲状腺素受体相互作用蛋白6(Thyroid receptor-interacting protein 6,TRIP6);⑥其它:髓鞘碱性蛋白S(myelin basic protein S,MBP),LIM结构域(LIM domain)。5、Western验证结果与蛋白质组学分析和鉴定基本一致,HSP27、SNX3、tubulin-alpha三种蛋白质均在PSD中呈免疫阳性,且它们在SRS、NSRS和Norm三组中的表达强度呈现出递增或递减现象,即HSP27:SRS＞NSRS＞Norm;tubulin-alpha:SRS＜NSRS＜Norm;SNX3:SRS＜NSRS＜Norm。研究结论:1、采用蔗糖梯度离心联合膜顺序提取法可获得高纯度和高溶解性的PSD蛋白,为深入研究PSD蛋白提供了技术基础。2、构建出大鼠脑组织PSD蛋白的2-D图谱,为PSD蛋白与相关疾病的研究奠定了基础。3、本研究筛选和鉴定出的TLE相关PSD蛋白,可能是促/抗TLE发病的重要候选蛋白分子。更多还原

【Abstract】 Background and Objective:Temporal lobe epilepsy(TLE) is a common type of refractory epilepsy. At present, studys show that the important reason of refractory epilepsy is reconstruction of neural circuit loop and abnormal excitability of neural network. But its concrete pathogenic mechanisms are still poorly understood up to now.Therefore, exploration of neural network abnormal excitability is of great significance. As we know,excitatory glutamatergic neurotransmission has been considered to be related with the pathogenesis of epilepsy, including that dilivery of presynaptic neurotransmitter; dysmetabolism of glutamate in synaptic cleft; sensitivity of postsynaptic receptor. In the past, study of postsynaptic regulatory mechanism is rare. Postsynaptic density(PSD) is considered to be a tightly packed protein complex.As the important material foundation of postsynaptic regulatory mechanism, PSD can change downstream effects of neural signal transmission by regulating sensitivity of postsynaptic receptor. So this may be a core of abnormal excitability of neural network,which need to be elucidated. At present, research reports about PSD and epilepsy are not often seen.To explore the mechnism of PSD in TLE, we will first construct 2D electrophoregram of PSD fractions by use of high-throughput and high-sensitive proteomic techniques.Subsequently, PSD proteins related with temporal lobe epilepsy will be screened out and identified,which can provide new cue for further studying the relationship of PSD and TLE,and find new target for preventing and treating TLE.Methods:In this study,the Sprague-Dawley rats were randomly enrolled in SRS group、NSRS group、Normal group.The lithium-pilocarpine model of epilepsy was established. PSD fractions were gradually obtained by using sucrose gradient centrifugation and membrane sequence abstraction. we respectively isolated PSD proteins of SD rats from three groups by using two-dimensional gel electrophoresis.Then we analyzed some differential protein spots in three groups,and identified 26 proteins using MALDI-TOF-MS. In addition, the expression of three identified PSD proteins was detected in the PSD fractions by western blotting.Results:1. The lithium-pilocarpine modle of TLE: Status epilepticus were induced at the achievement ratio of 91.4%.The achivement ratio of the TLE animal model is 71.4%.The mortality rate is 20%.2. The analysis of PSD purity: Two postsynaptic mark molecules NMDA receptorl and PSD-95 were expressed in P2,synaptosome and PSD fractions,but synaptophysin(presynaptic mark) was not expressed in PSD. 3. Two-dimensional gel electrophoresis of PSD: We analyze these 2-DE images by PDQuest software.A total of 720±17 protein spots were detected in the two-PSD fraction,and a total of 286±25 protein spots were detected in the one-PSD.There is a significant difference between the two fractions(P<0.01). Compared with normal group, the expressive levels of 12 proteins were upregulated and 58 proteins were downregulated in SRS; the expression levels of 15 proteins were upregulated and 38 proteins were downregulated in NSRS.In addition,we found that there were 4 proteins only in SRS group.Compared with NSRS,the expressive levels of 15 proteins were upregulated and 25 proteins were downregulated in SRS.4. The mass spectrographic analysis of serveral differential PSD proteins: The 26 differential protein spots were identified by MALDI-TOF-MS. And these proteins consisted of members of proteins with diverse functions, including proteins related to cytoskeletal、transport、signal transduction、chaperone、energy metabolism.5. Three identified proteins (HSP27、SNX3、tubulin-alpha) were detected in PSD fractions by western blotting. The results were consistent with that of proteomic analysis and identify. The expressive levels of three proteins in three groups showed as follows, HSP27 : SRS>NSRS>Norm ; tubulin-alpha : SRS<NSRS<Norm ; SNX3 : SRS<NSRS<Norm. Conclusions:1. The method of sucrose gradient centrifugation plus membrane sequence abstraction could guarantee the purity of PSD and improve the dissolubility of PSD fractions,which might serve as an important technique for studying PSD proteins.2. The construction of 2-D electrophoregram of PSD proteins in rat brains might provide foundation for further studying PSD proteins related with disease.3. The identified PSD proteins might be important candidate molecules,which might promote or inhibit development of TLE.更多还原