难治性颞叶癫痫颞叶脑组织中uPAR蛋白的表达及临床意义

目的研究难治性颞叶癫痫患者脑组织中的uPAR的表达,探讨其在难治性癫痫发病中的意义。方法从第四军医大学唐都医院神经外科建立的难治性癫痫患者脑组织库中随机抽取30例难治性颞叶癫痫患者术后脑组织,用免疫组织化学、免疫印迹(Western blot)检测uPAR的蛋白表达产物,并与15例对照组进行比较。结果uPAR蛋白在难治性颞叶癫痫患者颞叶脑组织中的表达量与对照组相同部位比较明显增高。结论难治性颞叶癫痫患者颞叶脑组织中uPAR蛋白产物表达增高可能与难治性颞叶癫痫发病过程中病理生理学改变有重要关联,可能为难治性颞叶癫痫的治疗提供新的靶点。     

匹罗卡品致痫诱发大鼠齿状回颗粒细胞GAP-43 mRNA表达

目的 研究边缘系统癫痫发作后海马颗粒细胞生长相关蛋白（GAP-43）基因表达变化。方法 建立匹罗卡品急、慢性癫痫模型,用原位杂交方法定量检测不同时间点海马颗粒细胞GAP-43mRNA表达。结果 对照组颗粒细胞几乎不表达GAP-43mRNA,匹罗卡品致病后6～12h颗粒细胞表达GAP-43mRNA增高,15～30d呈现第2次高峰。结论 成年大脑海马颗粒细胞在致痫后发生可塑性变化,GAP-43mRNA表达是癫痫大鼠大脑结构性重组（颗粒细胞苔藓纤维出芽）的重要分子机制。     

耐药性癫(癎)患者脑组织中热休克蛋白27相关蛋白1的表达

目的研究热休克蛋白27相关蛋白1(heat shock 27 000 associated protein 1,HSPBAP1, GenBank:AK096705)在耐药性癫痫患者脑组织中的表达,探讨其在耐药性癫痫发病机制中的作用。方法从重庆医科大学附属第一医院神经内科建立的耐药性癫痫脑库中随机抽取36例耐药性癫痫患者术后脑组织,用荧光定量聚合酶链反应(FQ-PCR)和免疫组化法对HSPBAP1的表达进行检测,并与8例对照进行比较。结果耐药性癫痫患者脑组织中HSPBAP1 mRNA的相对表达量是对照组的34．11倍;HSPBAP1蛋白在耐药性癫痫患者颞叶(0．0507±0．0003,P<0．01)和海马(0．0488±0．0196,P<0．05)中的表达量,与对照组相同部位比较明显增加。结论耐药性癫痫患者脑组织中HSPBAP1表达增高,能抑制热休克蛋白27(HSP27)的神经元保护作用,可能是导致神经元凋亡及海马苔藓纤维发芽的一个重要原因。     

铁调节转运体1 mRNA与耐药性癫痫

目的观察耐药性癫痫患者脑组织中铁调节转运体1（IREG1）mRNA的表达,探讨其在耐药性癫痫发病中的作用。方法收集耐药性癫痫患者术后脑组织,首先用基因芯片进行差异基因表达的筛选,然后用RT-PCR从基因水平进行验证,并与对照组比较。结果含有4097条人类全长基因的芯片扫描显示有142个基因表达有差异,37个基因表达下调,105个基因表达上调。目的IREG1 mRNA在耐药性癫痫患者颞叶中表达明显增加（P〈0.05）,Cy3和Cy5信号比值为2.75。RT-PCR在实验组和对照组的灰度值分别为2.6138,1.7708,变化趋势与芯片扫描结果相同。结论IREG1 mRNA可能参加了耐药性癫痫的形成,并在耐药性癫痫的发病机制中起着一定作用。     

多药耐药相关蛋白-1在难治性颞叶癫痫患者脑组织中的表达及丙磺舒的干预作用

目的 观察难治性颞叶癫痫患者脑组织中多药耐药相关蛋白1( MRPl)及应用MRP1拮抗剂丙磺舒干预后的表达,探讨MRP1与难治性颞叶癫痫多药耐药的关系.方法 应用免疫组化检测难治性颞叶癫痫患者脑组织实验组和对照组MRP1的表达情况,同时应用免疫蛋白印记(Western blot)方法检测实验组、丙磺舒干预组和对照组MRP1的表达.结果 免疫组化结果显示MRP1在难治性颞叶癫痫患者脑组织中表达增强,与对照组比较差异有显著性(P＜0.05).Western blot结果显示丙磺舒干预组MRP1蛋白水平较实验组明显较少,差异有显著性(P＜0.05).结论 脑内高表达的MRP1参与难治性颞叶癫痫的耐药机制,丙磺舒可以降低脑组织内MRP1的表达.     

难治性颞叶癫痫患者脑组织中Vav3的表达

目的研究难治性颞叶癫痫患者脑组织中Vav3的表达,探讨其在难治性颞叶癫痫形成中的意义。方法应用免疫组织化学法检测难治性颞叶癫痫患者脑组织和正常脑组织中Vav3蛋白的表达。光学显微镜下观察Vav3蛋白表达结果,并对免疫组织化学结果进行分析。结果免疫组化检测发现Vav3蛋白在难治性颞叶癫痫患者脑组织中阳性表达率明显高于正常脑组织,两组相比差异具有统计学意义（x^2=15．841,P〈0．015。实验组患者海马组中Vav3染色评分2分和3分占总体的76．93％（10／13）,而在颞叶组中Vav3染色评分1分和2分占总体的64．71％（11／17）,可见在难治性颞叶癫痫患者海马组织较颞叶组织神经元中Vav3的表达明显增强。结论Vav3在难治性颞叶癫痫患者的海马和颞叶脑组织中表达增强,提示其有可能参与了难治性颞叶癫痫的形成。     

8溴-环磷酸腺苷对脑缺血再灌注大鼠PKA及GAP-43蛋白表达的影响

目的:观察脑室注射8-溴-环磷酸腺苷(8-B-cAMP)对脑缺血再灌注大鼠大脑皮质蛋白激酶A(PKA)及生长相关蛋白43(GAP-43)表达的影响。方法:采用线栓法制作大鼠脑缺血再灌注模型,将45只大鼠分为假手术对照组,缺血组(单纯脑缺血再灌注组)和8-B-cAMP组(脑缺血再灌注并脑室注射8-B-AMP)。用放免法测缺血周边区脑组织cAMP的含量,Westernblot(免疫印迹法)检测蛋白激酶A(PKA)及生长相关蛋白43(GAP-43)的表达。结果:脑缺血组6h、24h cAMP的含量下降,GAP-43及PKA蛋白表达减少;8-B-cAMP治疗组脑组织GAP-43蛋白表达较缺血组增加,且这种变化与cAMP的含量及PKA蛋白表达增加相一致。结论:8-B-AMP能够增加PKA及GAP-43的表达从而促进脑缺血再灌注后的轴突再生。     

硫化氢／3-巯基丙酮酸转硫酶在癫痫患者颞叶中的表达

目的探讨硫化氢(H_2S)/3-巯基丙酮酸转硫酶(3-MST)在癫痫患者颞叶中的表达。方法难治性癫痫患者(实验组)外侧颞叶标本15例,高颅压患者(对照组)内减压切除的正常外侧颞叶脑组织标本15例。检测外侧颞叶脑组织中H_2S含量,采用免疫荧光及Western blotting技术检测3-MST表达,观察H_2S/3-MST在癫痫患者外侧颞叶及高颅压患者正常外侧颞叶脑组织中的表达变化。结果实验组外侧颞叶脑组织细胞中H_2S/3-MST表达显著高于对照组,差异均具有统计学意义(均P0.05)。结论在癫痫的病理生理过程中,H_2S/3-MST的高表达可能与其在癫痫中神经保护作用相关。     

海人酸诱导颞叶癫癎大鼠海马中GAP-43和NCAM的表达变化以及托吡酯的干预作用

目的研究海人酸（KA）对大鼠海马中生长相关蛋白-43（GAP-43）和神经细胞粘附分子（NCAM）表达的影响，以及托吡酯（TPM）对其的干预作用。方法将48只大鼠随机分成生理盐水（NS）组、4mgKA组、10mgKA组和10mgKA＋TPM组（n=12），建立KA诱导颞叶癫癎大鼠模型和TPM干预模型，观察大鼠行为学改变，并通过RT-PCR和WesternBlot的方法测定各组大鼠海马中GAP-43及NCAM的mRNA和蛋白表达水平。结果大鼠建模成功；4mgKA组、10mgKA组大鼠GAP-43及NCAM的mRNA和蛋白表达水平明显高于NS组（P〈0．01），且10mgKA组高于4nagKA组（P〈0．01）；10mgKA＋TPM组大鼠的GAP-43和NCAM表达明显低于10mgKA组（P〈0．01）。结论KA能够诱导致癎大鼠海马GAP-43和NCAM表达上调，且与KA剂量有关，而TPM能够抑制KA诱导的GAP-43和NCAM的表达上调。     

大鼠杏仁核电刺激癫痫持续状态后颞叶癫痫模型海马区神经生长相关蛋白43的表达

目的 探讨神经生长相关蛋白43(GAP-43)在大鼠杏仁核电刺激癫痫持续状态后颞叶癫痫(SE)模型海马区的表达及意义.方法 健康雄性Wistar大鼠60只,分成4组:空白对照组、未刺激组、未发作组和发作组.空白对照组:不植入电极；未刺激组:植入电极未行电刺激；发作组:植入电极电刺激后15d内、30 d内能观察到稳定的自发性反复发作(SRS)的大鼠归为发作组,其余归为未发作组.分别在15d、30 d时,将标本应用RT-PCR及免疫荧光检测方法检测大鼠海马GAP-43的表达.结果 致痫15d,发作组、未发作组和未刺激组的GAP-43表达高于空白对照组,并依次降低(P＜0.05).致痫30 d,发作组和未发作组GAP-43表达仍高于空白对照组,差异有统计学意义(P＜0.05)；未刺激组与空白对照组水平相当,二者差异无统计学意义(P＞0.05).结论 GAP-43参与了神经元损伤修复和突触重塑,其可能是颞叶癫痫的病理基础——突触重塑的重要分子机制.     

Upregulation of liprin-α1 protein in the temporal neocortex of intractable epileptic patients and experimental rats

Intractable epilepsy (IE) patients have synaptic dysfunction. However, the exact mechanism of synaptic function needs further elucidation. The aim of this study was to use immunohistochemistry, immunofluorescence, and Western blotting to investigate the expression of the Liprin-α1 protein, one of the synapse-associated proteins, in human IE brain tissues and experimental rats and to discuss the possible role of Liprin-α1 in IE. We selected 30 temporal neocortical tissue samples from patients with intractable temporal lobe epilepsy (TLE) and 10 histologically normal temporal lobes from controls. Fifty-six Sprague-Dawley rats were divided randomly into seven groups; one control group and six groups with epilepsy induced by lithium-pilocarpine administration. Temporal lobe tissues were taken from controls and from rats at 1, 3, 7, 14, 30, and 60 days postseizure. Liprin-α1 was mainly expressed in neurons of human controls and TLE patients and was significantly higher in TLE patients than in controls. Liprin-α1 was also expressed in neurons of control and experimental rats and it was significantly higher in experimental rats than in the control group. The expression of Liprin-α1 in animals in the experimental group gradually increased from Days 1 to 30 postseizure induction and reached a stable level when spontaneous recurrent seizures (SRS) appeared. These results suggest that an increased expression of Liprin-α1 in the brain may be associated with human IE.     

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。     

Molecular profiling of temporal lobe epilepsy: comparison of data from human tissue samples and animal models

The advent of gene chip technology and the era of functional genomics have initially been accompanied by huge anticipations to quickly unravel the molecular pathogenesis of multifactorial diseases. Expectations have, today, given way to some concerns about this non-hypothesis driven approach. However, the careful and controlled application of expression microarrays in concert with refined bioinformatic tools may provide novel insights in major disorders particularly of highly complex organs such as the central nervous system (CNS). Epilepsies are among the most frequent CNS disorders affecting approximately 1.5% of the population worldwide. In temporal lobe epilepsy (TLE), the seizure origin typically involves the hippocampal formation, a structure located in the mesial temporal lobe. Many TLE patients develop pharmacoresistance, i.e. seizures can no more be controlled by antiepileptic drugs. In order to achieve seizure control, surgical removal of the epileptogenic focus has been established as successful therapeutic strategy. Hippocampal biopsy tissue of pharmacoresistant TLE patients represents an excellent substrate to analyze molecular mechanisms related to structural and cellular reorganization in epilepsy. The complexity of alterations in TLE hippocampi suggests numerous genes and signaling cascades to be involved in the pathogenesis. By microarrays, genome wide expression profiles can be constituted from TLE tissues. However, hippocampi of pharmacoresistant TLE patients represent an advanced stage of the disease. Early stages of epilepsy development are not available for functional genome analysis in humans. Animal models of TLE appear particularly helpful to study molecular mechanisms of highly dynamic processes such as the development of hyperexcitability and pharmacoresistance. In this review, we summarize recent data of gene expression profiles in human and experimental TLE and discuss the relevance of novel tools for bioinformatic analysis and data mining.     

耐药性癫(癎)患者伴有(癎)样放电的脑组织中SCN8A基因产物的异常表达

目的观察电压门控钠通道SCN8A(sodium channel voltage-gated type VIII alpha,SCN8A)基因在耐药性癫患者脑组织中的表达,探讨其与耐药性癫的关系。方法在基因芯片研究的基础上,采用免疫组织化学、免疫荧光组织化学技术,分析比较40例耐药性癫患者手术切除脑组织标本中SCN8A基因编码的产物钠通道蛋白Nav1.6的表达水平,并与20名对照组进行比较。结果发现SCN8A在耐药性癫患者脑组织中表达上调,与对照组比较有统计学意义(P<0.05)。结论SCN8A基因在耐药性癫脑组织中表达上调,可能与耐药性癫中样放电的产生和(或)传播有关。     

Altered expression of Dscam in temporal lobe tissue from human and experimental animals

Down syndrome cell adhesion molecule (Dscam) is a neural adhesion molecule that plays an essential role in the establishment of neural circuits. Considerable evidence suggests that Dscam is required for axon guidance and dendritic arborization. Our aim was to investigate the expression of Dscam in the temporal lobes of patients with intractable epilepsy (IE) and of experimental animals. In this study, we used immunohistochemistry, immunofluorescence, and western blotting to examine Dscam expression in thirty-five surgical samples from brains of IE patients and 15 control brain samples. We also measured the levels of Dscam during the entire epileptic process in a rat model of temporal lobe epilepsy. Dscam expression in IE patients was significantly higher compared with that in the controls. In addition, Dscam was also highly expressed in the rat brain during the different phases of the epileptic process. It is the first time to find abnormal expression of Dscam in the brain tissues in patients with IE. And this finding provides an experimental evidence for the study of neuronal circuit remodeling and synaptic plasticity in IE, furthermore, our results also suggest that Dscam may be involved in the generation and the development of IE.     

癫痫患者脑组织PSD-93 mRNA表达上调

目的研究癫痫患者脑组织中突触后密度-93(PSD-93)mRNA的表达,探讨其在癫痫形成中的作用。方法将56例癫痫患者分成耐药和非耐药两组,首先用基因芯片对其术后标本进行扫描,并与对照组比较,随后对目标基因PSD-93用逆转录聚合酶链反应(RT-PCR)技术进行验证。结果基因芯片检测发现与依赖N-甲基-D-天门冬氨酸受体(NMDARs)-一氧化氮(NO)信号有关的基因PSD-93在癫痫患者中表达上调,RT-PCR实验结果与基因芯片一致。RT-PCR电泳图的灰度值均数在对照组为23.577、非难治性癫痫组为56.931、难治性癫痫组为51.607,非难治性、难治性癫痫组与正常对照组灰度的比值中位数分别为2.415、2.189(P<0.05),而非难治性与难治性癫痫组灰度值比值为1.103(P>0.05)。结论脑细胞膜相关鸟苷酸激酶家族(MAGUK)蛋白信号转导复合体的分子接头蛋白基因PSD-93mRNA表达增加,其可能通过信号传导异常及神经元坏死参与了癫痫的形成。     

Down-regulation Synaptic Vesicle Protein 2A in the Anterior Temporal Neocortex of Patients with Intractable Epilepsy

Synaptic vesicle protein 2A (SV2A) involvement has been reported in the animal models of epilepsy. The aim of this study was to investigate the expression of SV2A in human intractable epilepsy (IE) brain tissue. Using immunohistochemistry, immunofluorescence, and Western blot, we detected SV2A expression in tissue samples from the anterior temporal neocortex of 33 patients who had been surgically treated for IE. We compared these tissues with nine histologically normal anterior temporal lobe samples from controls. SV2A immunoreactive staining was 0.1651 ± 0.0564 in patient group and 0.2347 ± 0.0187 in the control group (p < 0.05) using immunohistochemistry, and this finding was consistently observed with Western blot analysis (0.1727 ± 0.0471 versus 0.3976 ± 0.0983, p < 0.05). Immunofluorescence staining showed that SV2A was mainly accumulated in neurons. Our findings demonstrate that down-regulation of SV2A is present in patients with temporal lobe epilepsy.     

Rnd1在难治性癫痫患者脑组织中的表达

目的观察Rnd1在难治性癫痫颞叶脑组织中的表达情况,探讨Rnd1在难治性癫痫发病的作用。方法收40例难治性癫痫患者脑组织颞叶(实验组)以及10例正常颞叶脑组织(对照组),用免疫组化、免疫荧光、Western-blot方法检测患者颞叶脑组织Rnd1的表达,并比较实验组与对照组颞叶脑组织中Rnd1的表达情况。结果免疫组化显示实验组Rnd1的平均灰度(127.62±6.23)与对照组(171.79±3.84)相比差异有统计学意义(t=29.86,p<0.01);实验组Rnd1的阳性细胞数(30.20±3.42)与对照组(11.18±0.67)相比差异有统计学意义(t=21.36,p<0.01)。免疫荧光显示Rnd1的阳性表达区域与特异性神经无性烯醇化酶(NSE)的分布基本重叠。结论 Rnd1主要表达于神经元,可能参与了癫痫的形成并在难治性癫痫形成的过程中发挥了重要作用。