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。     

SH3GL2在无病灶性难治性癫痈患者脑组织中的表达

目的：分析无病灶性难治性癫痂患者脑组织中SH3GL2 mRNA与蛋白的表达水平，探讨可能的发病机制。方法：运用逆转录-聚合酶链式反应（RT—PCR）和Western blot分别检测10例无病灶性难治性癫痫患者及10例对照组脑组织SH3GL2mRNA及蛋白的表达。结果：RT—PCR检测显示，无病灶性难治性癫痂患者脑组织SH3GL2 mRNA表达的相对水平为（102．23±54．68），对照组为（44．59±17．12），两者差异有显著统计学意义（P〈0．01）；Western blot分析表明，无病灶性难治性癫痫患者脑组织的SH3GL2蛋白表达相对水平（229．54±89．82）高于对照组（143．88±59．69），两者差异也有显著统计学意义（P〈0．05）。结论：SH3GL2 mRNA与蛋白表达水平在无病灶性难治性癫痂患者脑组织中明显增强，提示表达于中枢神经系统的SH3GL2可能在人类难治性癫痫的发病机制中起了一定作用。     

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。     

基因芯片检测难治性癫脑组织中sh3gl2的表达

目的：运用基因芯片和RT-PCR技术检测难治性癫脑组织中sh3gl2 mRNA表达,从分子水平探讨难治性癫可能的发病机制。方法：在应用基因芯片对难治性癫患者手术切除的颞叶组织与对照组行基因表达谱分析研究的基础上,筛选出目的基因后用RT-PCR对芯片扫描结果进行验证。结果：候选基因sh3gl2在难治性癫患者脑部颞叶组织中出现高表达,与对照组相比差异有显著统计学意义（P〈0.01）;RT-PCR结果与芯片结果一致。结论：sh3gl2在难治性癫颞叶皮质中的表达增加,提示了其可能是难治性癫发生发展中的一个重要因素。     

cDNA芯片检测耐/非耐苯妥英钠癫痫鼠脑线粒体基因的差异表达

目的：通过了解耐/非耐苯妥英钠（PHT）癫痫鼠与人类同源线粒体基因的差异表达来探索难治性癫痫的分子病理机制。方法：建立PHT耐药和非耐按照癫痫鼠模型，取脑组织常规抽提Mr-NA，逆转录生成cDNA并标记后，与含有4096条人类已知基因的cDNA表达谱芯片杂交，检测线粒体内37个基因及线粒体外相关基因在两者间的差异表达。结果：发现耐PHT鼠脑线粒体37条基因中有12条基因表达异常。结论：脑细胞线粒体基因表达异常可能是难治性癫痫的分子病理基础，能量代谢障碍和神经元凋亡在难治性癫痫形成，尤其是后期的发生和发展起着重要作用。     

发育及DNA损伤反应调节基因1mRNA在癫痫患者脑组织中的表达及临床意义

目的观察REDD1mRNA在癫痫患者脑组织中的表达及临床意义。方法将50例癫痫患者分成难治性(42例)和非难治性癫痫(8例)两组,用基因芯片和免疫荧光定量PCR分别检测脑组织中REDD1mRNA基因表达,并与对照组进行比较。结果基因芯片扫描提示REDD1mRNA在癫痫患者脑组织中与对照组比较表达明显增加,难治性癫痫组和非难治性癫痫组没有明显的表达差异,免疫荧光定量PCR的结果与基因芯片结果一致。结论 REDD1mRNA在癫痫患者脑部中存在高表达,这种高表达可能通过雷帕霉素哺乳动物靶标系统导致神经元坏死与凋亡。     

MCP-1在难治性癫痫患者脑组织中的表达

目的了解难治性癫癎患者脑组织中单核细胞趋化蛋白-1(monocyte chemoattractant protein-1, MCP-1)的表达,探索其在难治性癫癎发病机制中的作用。方法在基因芯片扫描获阳性结果基础上,用免疫组织化学方法研究40例难治性癫癎患者脑组织中MCP-1的表达,并与对照组进行比较。结果基因芯片扫描显示MCP-1 mRNA表达上调,免疫组化结果进一步证实MCP-1在难治性癫癎患者脑组织中的表达较对照组明显增多(P<0．05)。结论MCP-1在难治性癫癎的发病机制中可能起着重要作用,阻断MCP-1有可能改变难治性癫癎的预后。     

MCP-1在难治性癫(癎)患者脑组织中的表达

目的 了解难治性癫(癎)患者脑组织中单核细胞趋化蛋白-1(monocyte chemoattractant protein-1,MCP-1)的表达,探索其在难治性癫(癎)发病机制中的作用.方法在基因芯片扫描获阳性结果基础上,用免疫组织化学方法 研究40例难治性癫(癎)患者脑组织中MCP-1的表达,并与对照组进行比较.结果 基因芯片扫描显示MCP-1 mRNA表达上调,免疫组化结果进一步证实MCP-1在难治性癫(癎)患者脑组织中的表达较对照组明显增多(P＜0.05).结论 MCP-1在难治性癫(癎)的发病机制中可能起着重要作用,阻断MCP-1有可能改变难治性癫(癎)的预后.     

药物难治性癫痫患者脑内多药耐药相关蛋白1表达的研究

目的 研究药物难治性癫痫患者脑内皮层多药耐药相关蛋白1（multidrug resistant-associated protein 1，MRP1）表达的情况。方法 选择12例药物难治性癫痫患者癫痫切除灶与12例正常对照脑组织标本．用逆转录聚合酶链反应（RT-PCR）、免疫组化及免疫蛋白印记（Western blot）方法，分析比较MRP1基因在各组的表达。结果 药物难治性癫痫患者组脑内MRP1的表达显著高于正常对照组（P〈0．01）。在癫痫病灶内广泛分布的MRP1免疫阳性细胞主要为毛细血管内皮细胞和星形胶质细胞。结论 脑内高表达的MRP1参与了难治性癫痫的耐药机制。     

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。     

Investigation of changes in global gene expression in the frontal cortex of early-weaned and socially isolated piglets using microarray and quantitative real-time RT-PCR

We hypothesize that early-weaned piglets experience aberrant expression of stress-responsive genes in the frontal cortex, a key brain area involved in cognitive function and behavior organization. To test this hypothesis, female early-weaned piglets (EW; n = 6) were weaned 10 days after birth, while non-weaned piglets (NW; n = 6) were left with their dams. Half of EW (n = 3) and NW (n = 3) animals were socially isolated (SI) for 15 min at 12 days of age, when all animals (n = 12) were euthanized and tissue collected. The effects of EW and SI were examined by gene expression profiling using cDNA microarray hybridizations, generated from a porcine brain cDNA library. A total of 103 genes were differentially expressed (P < 0.05, fold change >1.25) among four direct comparisons. Forty-two genes had known functions, from which 24 showed relevant brain-related functions. Quantitative real-time polymerase chain reaction (Q-RT-PCR) was used to confirm regulation of expression of a subset of 6 genes with important brain functions, selected from the microarray outcomes. In non-weaned animals, a significant suppression of mRNA abundance for carboxypeptidase E, 14-3-3 protein and phosphoprotein enriched in astrocytes 15 kDa was observed in response to SI. Also, in early-weaned animals, diazepam binding inhibitor and actin-related protein 2/3 complex mRNA levels were suppressed in response to SI. Results suggest that social isolation of non- and early-weaned piglets may impact expression of genes involved in regulation of neuronal function, development, and protection in the frontal cortex of young pigs.     

小脑变性相关蛋白2 mRNA在癫痫患者脑组织中的表达及其临床意义

目的了解小脑变性相关蛋白2(CDR2)mRNA在癫痫(EP)患者脑组织中的表达及其临床意义。方法将48例EP患者分成耐药(40例)和非耐药(8例)两组,用基因芯片扫描和FQ-PCR分别检测脑组织中CDR2基因表达,并与对照组比较。结果基因芯片扫描提示CDR2 mRNA在EP患者与对照组中的表达差异有统计学意义(P<0.05)。FQ-PCR结果与基因芯片扫描一致。结论EP患者脑组织中有CDR2 mRNA表达,由于其表达产物可通过血-脑脊液屏障进入脑脊液(CSF)和血液中,因而检测CDR2 mRNA在血或CSF中的表达产物有可能成为诊断EP的一个重要指标。     

The use of real-time PCR analysis in a gene expression study of Alzheimer's disease post-mortem brains

The measurement of gene expressions in brains with neurodegenerative diseases is a major area of brain research. The objective of our research was to determine whether quantitative real-time PCR could measure messenger RNA (mRNA) expression in brains with post-mortem intervals beyond 12h. In the present paper, we examined the quality of RNA from brain specimens of both Alzheimer's disease (AD) patients (n = 13) and non-demented normal control subjects (n = 6). To determine a unregulated endogenous reference gene in AD, we measured mRNA expressions of the commonly used reference genes beta-actin, 18S rRNA, and GAPDH. In addition, we determined whether post-mortem interval, brain weight, or age at death influences mRNA expression. Our real-time PCR analysis results indicate that mRNA expression can be detected in all brain specimens for beta-actin, 18S rRNA, GAPDH, and also synaptophysin, a known marker for AD. Further, using real-time PCR analysis, we found that beta-actin and 18S rRNA are differentially expressed in the brain specimens of both AD and control subjects, while GAPDH is similarly expressed in AD and control brain specimens. These findings suggest that GAPDH can be used as a endogenous reference gene in the study of AD brains. A comparative gene expression analysis also suggests that synaptophysin is down-regulated in AD brain specimens compared to control brain specimens.     

Gene expression profiling during the embryonic development of mouse brain using an oligonucleotide-based microarray system

We analyzed gene expression profiles in embryonic day 12, 15, 18 and postnatal day 0 mouse brains by utilizing a GeneChip microarray. Significant differential expression was observed in 1413 of 12,422 (11.4%) represented on the chip. Then, 397 genes known to be related to neural development and functions were selected and analyzed in more detail. Clustering of the differentially expressed genes in terms of gene function and their temporal expression patterns indicated an aspect of the genetic foundation that underlies cellular events. Moreover, we identified a novel gene that encodes a putative protein kinase, Ebr kinase, which is differentially expressed in the developing brain.     

Bilateral induction of the S-100A9 gene in response to spreading depression is modulated by the cyclooxygenase-2 activity

Cyclooxygenase-2 (COX-2) was reported to be induced in the infarcted human brain. Spreading depression (SD) is thought to play a role in this induction. In this study, we correlated the expression of SD-associated genes with COX-2 production in brains after SD. Rats were divided into 3 groups: rats that did not undergo SD (group I saline controls, n=7), rats that underwent unilateral SD as a result of KCl application (group II, n=9), and rats that were pretreated with the selective COX-2 inhibitor, JTE-522 3 h before the induction of SD (group III, n=7). The expression of the SD-associated genes, S-100A9, and mitogen-activated proteinkinase phosphatase (cpg21) was analyzed 2 h later using a cDNA array. In group II, COX-2 and cpg21 mRNA expression, as determined by RT-PCR, were significantly upregulated in the hemisphere undergoing SD. While the expression of S-100A9 mRNA was bilaterally upregulated in these animals, this expression was significantly reduced in group III, and was accompanied by reduced bilateral production of PGE(2). Thus, the bilateral induction of expression of the S-100A9 gene in response to SD was associated with COX-2 activation.     

Evidence for induction of the ornithine transcarbamylase expression in Alzheimer's disease

To more rapidly identify candidate genes located within chromosomal regions of interest defined by genome scan studies in Alzheimer's disease (AD), we have developed a customized microarray containing all the ORFs (n=2741) located within nine of these regions. Levels of gene expression were assessed in total RNA from brain tissue of 12 controls and 12 AD patients. Of all genes showing differential expression, we focused on the ornithine transcarbamylase (OTC) gene on Xp21.1., a key enzyme of the urea cycle which we found to be expressed in AD brains but not in controls, as confirmed by RT-PCR. We also detected mRNA expression of all the other urea cycle enzymes in AD brains. Immunochemistry experiments revealed that the OTC expression was strictly restricted to vascular endothelial cells in brain. Furthermore, OTC activity was 880% increased in the CSF of probable AD cases compared with controls. We analysed the association of the OTC -389 G/A and -241 A/G promoter polymorphisms with the risk of developing AD. We observed that rare haplotypes may be associated with the risk of AD through a possible modulation of the methylation of the OTC promoter. In conclusion, our results suggest the involvement of a new pathway in AD brains involving the urea cycle.