颞叶内侧癫(癎)海马CA1区CytC、caspase-9、caspase-3蛋白的表达研究

目的 探讨凋亡相关蛋白细胞色素C(CytC)、caspase-9、caspase-3在颞叶内侧癫(癎)(MTLE)病人致(癎)海马CA1区神经元内的表达及意义.方法 选择30例典型MTLE病人为实验组,6例颢叶深部血管畸形病人为对照组.采用苏木精-伊红染色、原位末端标记(TUNEL)方法在光镜下观察MTLE病人致(癎)海马CA1区神经元凋亡的情况.采用免疫组化S-P法检测并比较MTLE海马CA1区与对照组海马组织神经元内凋亡相关蛋白CytC、caspase-9、caspase-3的表达.结合病史资料,分析caspase-3表达水平与癫(癎)病程和发作频率的相关性.结果 MTLE组致(癎)海马CA1区光镜下可观察到神经元退变、噬神经元现象,TUNEL染色发现凋亡神经元18例,对照组则为阴性;CytC、caspase-9、caspase-3蛋白在MTLE组海马CA1区均有明显表达,对照组中则呈轻微表达,差异显著(P<0.01).caspase-3蛋白表达水平与发作频率呈正相关,而与病程长短无关.结论 MTLE致(癎)海马神经元中存在外源性凋亡途径,这可能是癫(癎)发展的重要因素之一.     

Bcl-2、fas蛋白在颞叶癫痫病灶内的表达与神经元凋亡

海马硬化是颞叶癫痫最常见的病理类型，其主要表现为神经元脱失和胶质细胞增生。以往研究认为，神经元坏死是导致海马硬化的主要原因；近年来一些的动物实验表明，神经元凋亡可能参与了这一病理过程。但目前尚有争议，有学者在动物实验中研究发现癫痫发作导致神经细胞坏死而非凋亡。为进一步探讨海马硬化与神经元凋亡的关系，本文采用颞叶癫痫病人手术切除的颞叶病灶，联合应用光镜、电镜及原位末端标记（TUNEL）方法检测神经细胞凋亡，同时应用免疫组化的方法检测凋亡相关基因bcl-2及fas蛋白的表达。     

颞叶癫痫患者致痫灶中凋亡调控基因Bcl-2和Bax的表达

目的 探讨神经元凋亡与颞叶癫痫患者海马硬化的关系.方法 取16例颞叶癫痫患者手术切除标本,用光镜、电镜及原位末端标(TUNEL)方法检测神经细胞凋亡;应用免疫组化方法检测细胞凋亡相关基因表达产物Bax和Bcl-2蛋白的表达.结果 对照组患者光镜、电镜检查及TUNEL染色均未发现凋亡的神经细胞;癫痫组患者光镜检查及TUNEL染色未发现凋亡的神经细胞,但在电镜检查的3例标本中,1例有少量早期凋亡征象的神经元.免疫组化染色结果表明,对照组患者脑组织内Bcl-2蛋白无表达,癫痫组患者脑内Bcl-2蛋白表达明显增强(P<0.001);Bax蛋白在癫痫组与对照组中均微弱表达,两者差异无统计学意(P>0.05).结论 神经元凋亡部分参与了癫痫患者海马硬化的形成,Bcl-2和Bax 蛋白在这一过程中可能发挥了作用.     

颞叶癫(癎)海马神经元凋亡机制的研究进展

颞叶癫(癎)的海马病理变化分子机制一直是神经学科研究的重点.在发现海马致(癎)灶中存在天冬氨酸特异性半胱氨酸蛋白酶3(caspases-3)后,学者们推测凋亡机制可能对颞叶癫(癎)海马病理的形成具有一定作用.近年来,许多学者建立了相应的实验模型或活体标本,以凋亡基础理论为依据,从不同角度对颞叶癫(癎)中海马神经元损伤的凋亡机制进行研究,相继发现了凋亡级联效应途径中其他分子的显著表达,包括死亡受体、凋亡效应酶、凋亡调节蛋白等,甚至在基因水平找到了凋亡存在的证据,同时也引发了抗凋亡的治疗研究,为颞叶癫(癎)海马神经元损伤分子机制的进一步研究及颞叶癫(癎)的药物治疗研究提供了理论基础.本文对此进行综述.     

雌激素对癫痫持续状态大鼠海马CA3区神经元保护作用的研究

目的研究雌激素对癫癎持续状态大鼠海马CA3区神经元保护作用.方法利用尼氏染色和免疫组化染色技术,观察癫癎发作前后给予雌激素治疗对癫癎发作造成神经元凋亡及Bcl-2、Bax表达的影响.结果癫癎发作前给予雌激素治疗可明显减轻持续癫癎发作造成的神经元缺失并可抑制Bax表达,但对Bcl-2表达无明显影响.结论雌激素对癫癎持续状态导致的海马神经元损伤有保护作用,对凋亡促进基因Bax表达的影响可能参预了其神经保护作用.     

海马神经元癫癎样放电模型中神经元凋亡的研究

目的通过检测体外海马神经元癫癎样放电后的细胞凋亡,旨在探讨颞叶癫癎病人海马神经元丢失的机制.方法首先制备Sombati神经元癫癎样放电模型,然后采用Tunel标记、荧光染色以及流式细胞技术对模型中的凋亡神经元作了定性和定量检祆测.结果发现神经元癫癎样放电后出现细胞凋亡,且随着放电时间的延长,凋亡细胞增加.结论反复癫癎样放电导致神经元凋亡,其发生可能与癫癎样放电的兴奋性毒性有关.     

耐药性癫(癎)患者颞叶中细胞周期依赖性蛋白激酶5的表达

目的 研究细胞周期依赖性蛋白激酶5(cyclin dependent kinases 5,CDK5)在耐药性癫(癎)患者颞叶中的表达,探索其在耐药性癫(癎)发病机制中的作用.方法 收集耐药性癫(癎)患者术后脑组织,用荧光定量PCR、免疫组化和Western blot 3种检测方法从基因和蛋白水平分别测定CDK5在耐药性癫(癎)患者颞叶中的表达,并与对照组进行比较.结果 荧光定量PCR发现CDK5 mRAN比对照组明显增加,免疫组化检测显示这种基因的蛋白表达产物主要分布在神经元轴突和胶质细胞中,Western blot检测在相对分子质量35 000处有一蛋白条带,并且可见实验组(颞叶和海马中分别为1.4293±0.1839和2.0733±0.4738)高于对照组(颞叶和海马中分别为0.9680±0.4147和1.403±0.6163,P＜0.05).结论 CDK5在耐药性癫(癎)患者颞叶中表达增强,提示他们可能参与了耐药性癫(癎)的形成.     

凋亡调控基因在难治性颞叶癫痫患者脑中表达的研究

目的:探讨神经元凋亡与难治性癫痫患者海马硬化关系。方法:16例颞叶癫痫患者手术标本,用光镜、电镜及原位末端标记(TUNEL)方法检测神经元凋亡;应用免疫组化方法检测Bax、Bcl-2蛋白的表达。结果:对照组未发现凋亡神经元;癫痫组在电镜检查的3例标本中,1例有少量早期凋亡征象。对照组Bcl-2蛋白无表达,癫痫组Bcl-2蛋白表达明显增强(P<0.001);Bax蛋白在癫痫组与对照组中均微弱表达,两者差异无统计学意义(p>0.5)。结论:凋亡基因Bcl-2、Bax参与癫痫过程。     

难治性颞叶癫(癎)的相关基因表达谱

目的 筛选难治性颞叶癫(癎)相关基因表达谱,并通过对表达谱筛选所发现的差异基因进行验证,从分子水平探讨颞叶癫(癎)可能的发病机制.方法 在利用基因芯片对难治性颞叶癫(癎)患者手术切除颞叶组织与对照间行基因表达谱分析研究的基础上,扩大样本量,应用RT.PCR方法,验证SH3GL2、BTN2A2、KCNJ4基因的mRNA在颞叶癫(癎)患者脑内的表达差异.结果 芯片研究结果显示,颞叶癫(癎)脑组织标本与对照组相比,表达趋势一致的已知基因包括:免疫相关因子、离子通道相关因子、信号转导相关基因、细胞增殖周期因子、细胞凋亡相关基因等;经RT.PCR证实,颞叶癫(癎)组突触重建调控因子SH3GL2的mRNA表达水平(1.022±0.547)明显高于对照组(0.446±0.171,t=-3.181),免疫调控基因BTN2A2的mRNA表达在颞叶癫(癎)组(0.481±0.196)同样高于对照组(0.243±0.111,t=3.351),而编码内向整流钾通道亚单位基因KCNJ4 mRNA的表达水平在颞叶癫疴组(0.438±0.178)则明显低于对照组(0.795±0.112),差异均有统计学意义(P<0.05).结论 在颞叶癫(癎)发生发展各个阶段均有基因的参与,是一个复杂的调控过程;芯片试验结果对进一步探讨疾病可能的发病机制以及为探求疾病治疗的新靶点提供了依据.     

颞叶癫癎21例手术治疗分析

目的探讨颞叶癫癎的外科治疗方法及效果.方法 21例颞叶癫癎患者,术前均行EEG、MRI检查,其中6例行PET检查,经定侧定位后,行手术治疗.其中13例行标准前颞叶切除术,5例行病灶切除+致癎灶切除,3例行选择性海马杏仁核切除.术中应用皮层电极或深部电极进行监测;神经导航下海马钙化切除1例.结果术后无明显并发症,均取得满意近期效果.结论海马硬化是颞叶癫癎发生的主要原因;手术是治疗颞叶癫癎的重要手段,且疗效满意.     

低频经颅磁刺激对颞叶癫痫大鼠颞叶和海马细胞凋亡的影响

目的　 探讨低频经颅磁刺激对颞叶癫痫大鼠病灶区颞叶和海马的细胞凋亡有无抑制作用。 方法　 制作氯化锂 匹罗卡品所致的慢性期颞叶癫痫大鼠动物模型 ,分为两组 ,一组给予磁场强度 0 .4T ,刺激时程 0 .2ms的低频 (0 .5Hz)经颅磁刺激 ,另一组给予“假性”刺激。以原位末端标记法 (TUNEL)标记DNA片段 ,电子显微镜检测两组颞叶和海马的细胞凋亡情况 ,并分别与对照组进行比较。结果　 在慢性期颞叶癫痫大鼠的颞叶和海马均可见大量凋亡细胞。在给予适量的低频经颅磁刺激后 ,相应脑部的凋亡细胞数均明显减少 (P <0 .0 5 )。 结论　适量的低频经颅磁刺激能抑制颞叶癫痫大鼠的细胞凋亡 ,对癫痫所致的脑部损伤有修复作用 ,是一种有前途的治疗癫痫的新疗法。     

Neuronal apoptosis in the resected sclerotic hippocampus in patients with mesial temporal lobe epilepsy.

To further confirm at the molecular level that neuronal apoptosis occurs in mesial temporal sclerosis (MTS), the main substrate of mesial temporal lobe epilepsy (MTLE), 24 resected sclerotic hippocampi from 24 patients with drug-resistant MTLE associated with MTS were studied microscopically, electronmicroscopically and immunohistochemically, with detection of expression of apoptosis-associated genes including bcl-2, p53, bax, fas and caspase-3. Early apoptosis changes were found morphologically in hippocampi from three patients with MTLE using transmission electron microscopy. Positive immunostained neurons for bcl-2, p53, fas and caspase-3 were found in the sclerotic hippocampi of 19/24, 14/24, 22/24 and 20/24 patients respectively, which was statistically different from controls. Correlative analysis showed the expression of p53, fas and caspase-3 were positively correlated with seizure frequency. Apoptosis may contribute to MTS, and seizures may induce apoptosis, and thus contribute to neuronal loss in MTS.     

Evidence of tumor necrosis factor receptor 1 signaling in human temporal lobe epilepsy

Seizures, particularly when prolonged, may cause neuronal loss within vulnerable brain structures such as the hippocampus, in part by activating programmed (apoptotic) cell death pathways. Experimental modeling suggests that seizures activate tumor necrosis factor receptor 1 (TNFR1) and engage downstream pro- and anti-apoptotic signaling cascades. Whether such TNFR1-mediated signaling occurs in human temporal lobe epilepsy (TLE) is unknown. Presently, we examined this pathway in hippocampus surgically obtained from refractory TLE patients and contrasted findings to matched autopsy controls. Western blotting established that total protein levels of the TNFR1 proximal signaling adaptor TNFR-associated protein with death domain (TRADD), cleaved initiator caspase-8 and apoptosis signal-regulating kinase 1 (ASK1) were higher in TLE samples than controls. Intracellular distribution analyses revealed raised cytoplasmic levels of TNFR1, TRADD and the caspase-8 recruitment adaptor Fas-associated protein with death domain (FADD), and higher levels of TRADD and cleaved caspase-8 in the microsomal fraction, in TLE samples. Immunoprecipitation studies detected TRADD-FADD binding, and fluorescence microscopy revealed TRADD co-localization with FADD in TLE hippocampus. These data suggest that TNFR1 signaling is engaged in the hippocampus of patients with refractory temporal lobe epilepsy.     

Heat shock protein 70 expression in epilepsy suggests stress rather than protection

Although heat shock protein 70 (HSP70) has been suggested to be a stress marker or to play a protective role in brain injury, the relevance of its pathological expression in epilepsy is unclear. We investigated the expression of HSP70 in brain tissue from human temporal lobe epilepsy (TLE) patients and from kainic acid (KA)-induced seizure-related neuronal damage in vivo and in vitro. The human TLE tissue showed severe neuronal loss and gliosis in hippocampal CA3 area. The KA-induced neuronal damage was similar to pathological changes of the TLE hippocampus. An increased number of TUNEL-positive cells were observed at day 5 when compared with day 2 after seizure induction. Intense HSP70 immunofluorescence was observed in hippocampal CA3 pyramidal neurons of rat, 2 days following KA administration, which then declined in labeling by day 5. No HSP70 expression was found in Fluoro-Jade B positive dying neurons by double staining. Western blot analysis showed an increased level of p53 and Bax expression following KA treatment. In vitro, there was no apparent difference in the degree of apoptosis between HSP70 siRNA- and control empty vector-transfected primary neurons following KA treatment. Our results revealed that HSP70 was a useful indicator of stressed neurons in acute phase of epilepsy, but not associated with neuronal death, thereby suggesting that HSP70 played no role in neuroprotection during an epileptogenic state.     

Caspase-3 cleavage and nuclear localization of caspase-activated DNase in human temporal lobe epilepsy.

Programmed cell death (apoptosis) signaling pathways have been implicated in seizure-induced neuronal death and the pathogenesis of human temporal lobe epilepsy (TLE). End-stage DNA fragmentation during cell death may be mediated by nucleases including caspase-activated DNase (CAD), apoptosis-inducing factor (AIF) and endonuclease G. In the present study, we investigated the subcellular localization of these nucleases in resected hippocampus from TLE patients and autopsy controls. Subcellular fractionation determined levels of CAD were significantly higher in the nuclear fraction of TLE samples compared with controls, and semiquantitative immunohistochemistry revealed cleaved caspase-3 positive cells in TLE sections but not controls. While mitochondrial levels of AIF and endonuclease G were higher in TLE samples than controls, nuclear localization of AIF was limited and restricted to cells that were negative for cleaved caspase-3. Nuclear accumulation of endonuclease G was not found in TLE samples. These data support ongoing caspase-dependent apoptosis signaling in human TLE and suggest that interventions targeting such pathways may have potential as adjunctive neuroprotective therapy in epilepsy.     

水通道蛋白-4在颞叶癫痫儿童致痫灶组织中表达的初步研究

目的探讨水通道蛋白-4(AQP-4)在颞叶癫痫儿童致痫灶组织中的表达及其在癫痫发作中的意义。方法 2010年1月至2011年6月手术治疗颞叶癫痫儿童患者13例和同期脑深部肿瘤儿童患者7例,分别取切除的致痫灶脑组织和正常脑皮层组织行AQP-4表达的免疫组化染色检测,比较它们AQP-4表达水平的差异。结果正常组脑组织中灰质、白质均有AQP-4表达,主要表达在血管周围星形细胞足突膜上,表达分布存在一定规律性,即集中在细胞与血管接触面;6例表达强度为(++),1例表达强度为(+++)。致癫痫脑组织中AQP-4主要在发育不良神经元周围的胶质细胞中表达,分布无明显规律性;3例AQP-4表达强度为(++),10例表达强度为(+++)。两组AQP-4表达强度有明显差异(P<0.05)。结论 AQP-4在颞叶癫痫儿童致痫灶脑组织中表达强度明显高于正常大脑皮层。AQP-4可能介导了儿童颞叶癫痫的发生。     

难治性癫痫患者脑组织中Eml 5mRNA表达及其意义

目的 运用基因芯片和荧光定量PCR技术检测难治性癫痫患者脑组织中Eml5（EMAP like protein5，Eml5）mRNA表达，探讨其表达异常在难治性癫痫患者中的意义。方法 收集36例难治性癫痫患者和8例非难治性癫痫患者术后脑组织，用含有14109条人类基因的芯片对其扫描，筛选出目的基因后，用荧光定量PCR对芯片扫描结果进行验证，并与8例正常脑组织进行比较。结果 发现候选基因End5mRNA在难治性癫痫患者脑部海马和颞叶中出现高表达，荧光定量PCR结果与芯片结果一致。结论 End5mRNA表达上调可能是难治性癫痫发生发展中的一个重要因素，其可能通过加强神经微管组装，促进神经元轴突延伸，触发海马齿状回颗粒细胞苔藓纤维异常发芽，参与了病理性神经网络重组和兴奋性突触重构。     

难治性颞叶癫痫患者颞叶皮层GRP78、caspase4的表达

目的观察难治性颞叶癫痫患者脑组织内质网应激相关分子GRP78、caspase4的表达,探讨内质网应激在难治性颞叶癫痫脑损伤中的作用。方法应用免疫组化、Western blot方法检测32例难治性颞叶癫痫患者手术切除的颞叶皮层脑组织GRP78、caspase4的表达,并与性别、年龄相匹配的对照组进行比较。结果与对照组相比,GRP78、caspase4在癫痫组表达明显上调(P0.05)。结论癫痫诱发了内质网应激,caspase4可能参与了癫痫后脑损伤,很可能为癫痫脑损伤的治疗提供新的靶点。