左乙拉西坦对颞叶癫痫大鼠c-fos与Calretinin表达的影响

发布时间：2018-01-12 21:11

本文关键词：左乙拉西坦对颞叶癫痫大鼠c-fos与Calretinin表达的影响　出处：《河南大学》2015年硕士论文　论文类型：学位论文

更多相关文章： 颞叶癫痫 左乙拉西坦 c-fos Calretinin

【摘要】：背景癫痫是一种神经系统慢性疾病,影响人群广泛且其发病率逐年增加。颞叶癫痫(Temporal lobe epilepsy,TLE)是最常见的难治性癫痫,其发病机制复杂且尚未阐明确切病因。目前TLE治疗手段有限,只有手术治疗可以根治癫痫,但抗癫痫药物(Antiepileptic drugs,ADE)是目前控制癫痫发作的主要手段。左乙拉西坦(Levetiracetam,LEV)作为新型抗癫痫药物的代表,比传统ADE更安全。随机对照试验的荟萃分析证明左乙拉西坦对各类型癫痫(包括癫痫持续状态(SE))均可一线用药,对成人、儿童、婴幼儿甚至孕妇的安全性均较高。有效的生物标志物,对疾病诊断、病程进展、药物的治疗的结果预测,以及避免药物副作用的识别是在医学兴趣的新兴领域。即早基因(immediate early genes,IEGs)是一类通过第二信使诱导对外界刺激能够快速应答原癌基因家族的基因。即早基因(IEGs)c-fos是中枢神经系统(CNS)神经元激活活化间接的标记之一。即早基因表达变化情况在神经细胞损坏、死亡、缺血再灌注等方面都有重要意义。钙离子(Ca2+)、钙结合蛋白是维持脑细胞正常生理重要因子之一,钙结合蛋白通过调节众多的Ca2+依赖酶和参与了有关的细胞功能。钙结合蛋白在神经细胞中有保护作用,钙结合蛋白表达变化可反应脑组织病变部位和程度。目的研究现公认的颞叶癫痫模型(氯化锂-匹罗卡品大鼠模型),使用SD(Sprague Dawley)大鼠构建该模型,并对该模型中抗癫痫药物左乙拉西坦(LEV)作用后大鼠的大脑海马组织中即早基因c-fos和钙结合蛋白Calretinin表达改变情况进行研究分析,为进一步明确颞叶癫痫的发病机制和病理生理改变,以期指导癫痫的诊断和临床治疗。方法建立SD大鼠颞叶癫痫急性(EP3d)、慢性(EP3d)模型,根据随机数字表分组为对照组(Control组)、模型组(EP组)和药物组(LEV组),对大鼠行为学表现、HE染色、Fluoro-Jade B染色以验证模型构建成功;对c-fos和Calretinin在海马组织的表达在基因水平(Real-time RCR实验)、蛋白水平(Western blot实验)和组织水平(免疫组化和免疫荧光实验)进行检验,并用SPSS 21.0进行单因素ANOVA分析和作图以明确c-fos和Calretinin在EP组和LEV组的表达变化。结果共有156只大鼠,其中15只为Control组,其他使用毛果芸香碱构建癫痫模型,成功构建122只,建模成功率为86.5%。HE染色显示海马区细胞损伤,Fluoro-Jade B染色显示EP组有大量Fluoro-Jade B(+)细胞聚集,且损伤程度与时间正相关。LEV组HE染色、Fluoro-Jade B染色亦出现海马区损伤,但和EP组比较,损伤程度稍轻。c-fos在基因、蛋白和组织水平上分别检测,可见对照组c-fos表达量低,EP组在EP急性期(4h、6h)表达量猛增,在慢性期又恢复正常。LEV组在EP急性期(4h、6h)表达量有所增高,但之比于EP组,LEV组c-fos表达量是降低,组间比较有统计学意义(P0.05)。对照组Calretinin高表达,EP组在癫痫慢性期(7d、14d、28d)表达量迅速降低,EP急性期下降不明显。而LEV组Calretinin在慢性期(7d、14d、28d)表达量有所下降,比EP组表达量有所增高,且组间比较有统计学意义(P0.05)。即早基因c-fos和钙结合蛋白Calretinin通过免疫荧光双标记实验验证二者之间无共定位关系。结论1.成功构建颞叶癫痫大鼠模型。2.颞叶癫痫大鼠海马组织中即早基因c-fos在癫痫急性期(4h、6h)高表达,钙结合蛋白Calretinin在EP慢性期(14d、28d)表达量下降明显。3.左乙拉西坦可一定程度降低即早基因c-fos在癫痫急性期(4h、6h)高表达,但和Control组相比,c-fos的表达量仍增高。左乙拉西坦可一定程度增高钙结合蛋白Calretinin在癫痫慢性期表达量。但和Control组相比,Calretinin的表达量仍有所下降。4.左乙拉西坦(LEV)可降低大鼠脑细胞损伤。5.即早基因c-fos和钙结合蛋白Calretinin在癫痫大鼠大脑中无共定位关系。
[Abstract]:Background epilepsy is a chronic disease of the nervous system, affecting population wide and its incidence increased year by year. Temporal lobe epilepsy (Temporal lobe, epilepsy, TLE) is the most common type of refractory epilepsy, its pathogenesis is complicated and has not yet been clarified. At present the exact cause of TLE treatment is limited, only surgery can cure epilepsy, but anti epileptic drugs (Antiepileptic, drugs, ADE) is currently the primary means of control of epilepsy. Levetiracetam (Levetiracetam, LEV) as the representative of new antiepileptic drugs, more secure than traditional ADE. The meta-analysis of randomized controlled trials that levetiracetam on various types of epilepsy (including status epilepticus (SE)) children can be a first-line drug, for adults, higher safety of infants and even pregnant women. Effective biomarkers, the progression of disease diagnosis, drug treatment, the prediction results, and avoid the side effects of drugs The recognition is in the emerging field of medical interest. Early gene (immediate early genes, IEGs) is a second messenger induced rapid response to external stimuli to proto oncogene gene family. Early gene c-fos (IEGs) is the central nervous system (CNS) neuron activation marker of early indirect. Gene expression changes in nerve cell damage and death, have important significance in ischemic reperfusion. Calcium, calcium binding protein (Ca2+) is one of the important factors to maintain normal physiological brain cells, calcium binding protein by regulating Ca2+ dependent enzymes and numerous cellular functions involved in the protective effect of calcium binding protein. In nerve cells, calcium binding protein expression changes in brain tissue lesion and extent of reaction. Objective to study the model of temporal lobe epilepsy is recognized (lithium pilocarpine rat model, using SD (Sprague Daw) Ley) to construct the rat model, and the model of antiepileptic drug levetiracetam (LEV) in hippocampus tissue of rats after immediate early gene c-fos and calcium binding protein Calretinin expression analysis, to further clarify the pathogenesis and pathophysiology of temporal lobe epilepsy in order to change. The diagnosis of epilepsy and to guide clinical treatment. Methods SD rats with temporal lobe epilepsy (EP3d) in acute, chronic (EP3d) model, according to randomly divided into control group (Control group), model group (EP group) and treatment group (group LEV), findings on rat HE staining. Fluoro-Jade B staining to verify the successful model; on c-fos and Calretinin in the hippocampus in gene expression level (Real-time RCR) and protein level (Western blot test) and tissue level (immunohistochemistry and immunofluorescence test) test, and with SPSS 21 for single ANOVA analysis and mapping to identify c-fos and Calretinin in EP group and LEV group. The changes of the expression of the results of a total of 156 rats, 15 of them as Control group, the other using pilocarpine to construct epilepsy model, successfully constructed 122, modeling success rate of 86.5%.HE staining showed that hippocampal cell damage, Fluoro-Jade B EP staining showed that the group had a large number of Fluoro-Jade (+) B cell aggregation, and the damage degree and time is positively related to.LEV group HE staining, Fluoro-Jade B staining also showed hippocampus injury, but compared with the EP group, damage to a lesser extent in.C-fos gene, protein and tissue levels were detected and observed in control group the expression of c-fos the low EP group in acute period of EP (4h, 6h) expression increased,.LEV group returned to normal in the acute period of EP in chronic phase (4h, 6h) expression was increased, but the ratio in the EP group, LEV group the expression of c-fos is decreased, the difference was statistically significant (P0.05). For Compared with the high expression of Calretinin, EP in the chronic phase of epilepsy group (7d, 14d, 28d) expression in acute period of EP rapidly decreased, no significant decline. While the LEV group of Calretinin in chronic phase (7d, 14d, 28d) expression decreased than group EP expression was increased, and the comparison among groups of statistics significance (P0.05). Early gene c-fos and calcium binding protein Calretinin test between the two co localization by immunofluorescence double labeling experiments. Conclusion 1. construction in hippocampus of temporal lobe epilepsy rat model of temporal lobe epilepsy rat.2. immediate early gene c-fos in acute epilepsy (4h, 6h) high expression and the calcium binding protein Calretinin in chronic phase EP (14d, 28d) significantly decreased.3. expression of levetiracetam can be reduced to some extent of immediate early gene c-fos in acute epilepsy (4h, 6h) high expression, but compared with Control group, the expression of c-fos was increased. Lacita left B increased calcium combination Protein Calretinin expression in the chronic phase of epilepsy. But compared with group Control, the expression of Calretinin still decreased..4. levetiracetam (LEV) could reduce brain cell injury in rats..5., early gene c-fos and calcium binding protein Calretinin had no co localization in the brain of epileptic rats.

【学位授予单位】：河南大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R742.1

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