酸敏感离子通道1a在复杂型热性惊厥中的作用研究

发布时间：2018-05-11 21:06

本文选题：复杂型热性惊厥 + 酸敏感离子通道1a　；参考：《第四军医大学》2014年硕士论文

【摘要】：热性惊厥是婴幼儿期最常见的一种抽搐，分为简单型和复杂型两种。其中复杂型热性惊厥不但给患儿带来了严重的躯体损害，也常常导致其脑内海马局部回路兴奋性增高，以及不同程度的认知功能障碍。因此探索复杂型热性惊厥的防治成为医学领域亟需解决的重要课题。酸敏感离子通道1（aacid-sensing ion channel1a,ASIC1a）是质子门控阳离子通道，在神经系统具有丰富的表达，常在酸性环境下易被激活。目前研究表明ASIC1a参与了诸多疾病的病理生理过程，阻断病理条件下ASIC1a的激活具有一定的神经保护性作用。复杂型热性惊厥因为抽搐时间长并伴有体温升高，常常导致颅内乳酸堆积以及组织酸化，ASIC1a能否被异常激活并介导细胞损伤过程，目前并不清楚。藉此，本研究选用大鼠复杂性热性惊厥模型，探讨ASIC1a在复杂型热性惊厥中的作用，为复杂型热性惊厥的防治提供新的靶点。目的首先观察ASIC1a在复杂型热性惊厥大鼠海马部位的表达，继而探讨ASIC1a拮抗剂阿米洛利不同剂量预处理对复杂型热性惊厥发作的影响，以及后处理对大鼠精神发育的影响方法选用Sprague-Dawley（SD）大鼠（P10），高温气浴致强直发作30分钟建立复杂型热性惊厥模型，在模型建立前或后给予阿米洛利1.3mg/kg和10mg/kg两剂量腹腔注射，，应用免疫组织化学和蛋白质印迹实验方法，结合旷场试验、高架十字以及水迷宫等行为学试验进行研究。统计学方法采用t检验与方差分析。结果 1．高温气浴致大鼠强直发作30分钟为复杂型热性惊厥模型成功标准。模型建立过程中无一只动物死亡。通过免疫组化染色我们发现复杂型热性惊厥后第三天，复杂型热性惊厥组SD大鼠海马CA1和CA3区锥体细胞层，以及齿状回颗粒细胞层上ASIC1a的表达量与正常对照组间无明显差异。而复杂型热性惊厥组CA1和CA3放射层，以及齿状回多形细胞层ASIC1a阳性细胞数目较正常对照组明显增多（p0.01）。Western blot结果显示出生后10天的正常SD大鼠脑内海马区即有ASIC1a蛋白的表达，且随着时间的推移（P11-P17）ASIC1a的表达量无明显变化。复杂型热性惊厥后1到7天，ASIC1a总蛋白和膜蛋白表达量都有所增加，与正常对照组均有显著性差异（p0.05）。 2．模型建立前1小时给予阿米洛利两种剂量（1.3和10mg/kg）腹腔注射。模型建立前后正常对照组，1.3阿米洛利组以及10阿米洛利组大鼠体重差的百分比均小于3%，组间无明显差异（p0.05）。1.3阿米洛利组和10阿米洛利组中心温度达到39.5℃和全身强直性发作的潜伏期较正常对照组明显延长（p0.01），且阿米洛利两不同剂量预处理组间有显著性差异（p0.05）。阿米洛利预处理组达到39.5℃和全身强直发作的时间间隔较正常对照组的明显延长（p0.01），且全身强直性发作的温度阈值明显高于正常对照组（p0.01）。 3.复杂型热性惊厥模型建立后1小时开始连续14天给予阿米洛利（1.3和10mg/kg），1/日，腹腔注射。免疫组化染色及Western blot结果显示复杂型热性惊厥后GFAP阳性的星形胶质细胞较正常对照组明显增多（p0.05），而阿米洛利两剂量后处理组明显降低这些变化（p0.05）。在水迷宫实验训练期的第4，5天，复杂型热性惊厥组较正常对照组，1.3阿米洛利组和10阿米洛利组仍需要更长时间找到平台，结果有统计学差异（p0.05）。第6天测试期，与正常对照组相比，其余三组SD大鼠寻找虚拟平台较为费力，其中复杂型热性惊厥组找到虚拟平台的次数较其它三组有明显统计学差异（p0.05）。结论在复杂型热性惊厥模型中ASIC1a表达量明显增加，ASIC1a拮抗剂阿米洛利能抗热性惊厥发作，抑制星形胶质活化，改善复杂型热性惊厥导致的认知功能障碍。提示ASIC1a可能参与了复杂型热性惊厥发作及惊厥后的病理生理变化过程。
[Abstract]:Febrile convulsion is the most common type of convulsion in infantile period, which is divided into two kinds of simple and complex type. Complex febrile convulsions not only cause serious physical damage to children, but also lead to higher excitatory in the local loop of the hippocampus in the brain, as well as different degrees of cognitive energy barriers. Therefore, the prevention and treatment of complex thermal convulsions is explored. The acid sensitive ion channel 1 (aacid-sensing ion channel1a (ASIC1a) is a proton gated cation channel, which has a rich expression in the nervous system and is often activated in the acidic environment. The present study shows that ASIC1a has been involved in the pathophysiological processes of many diseases and blocked ASIC1 under pathological conditions. The activation of a has a certain neuroprotective effect. The complex type of febrile convulsion is often caused by the accumulation of lactic acid and the acidification of the tissue because of the long twitching time and the increase of body temperature. It is not clear whether ASIC1a can be activated and mediate the process of cell damage. This study chooses the rat model of complex febrile convulsion to explore ASI The role of C1a in complex febrile seizures is a new target for the prevention and treatment of complex febrile convulsions.
objective
First, to observe the expression of ASIC1a in the hippocampus of complex febrile convulsion rats, and then explore the effect of different doses of ASIC1a antagonist amiloride on complex febrile seizures, and the effect of postprocessing on the mental development of rats.
Method
The Sprague-Dawley (SD) rat (P10) was used to establish a complex thermal convulsion model for 30 minutes with a high temperature gas bath. Before or after the establishment of the model, two doses of amiloride and 10mg/kg were given intraperitoneally. The immunohistochemical and Western blot methods were used to combine the open field test, the elevated cross and the water maze. The t test and ANOVA were used for statistical analysis.
Result
1. high temperature gas bath induced tetanic seizure for 30 minutes was a successful standard for the complex thermal convulsion model. No animal died during the establishment of the model. By immunohistochemical staining, we found the pyramidal cell layer of CA1 and CA3 region of the hippocampus and the granular cell layer of the dentate gyrus of the hippocampus of the complex type febrile convulsion group SD rats, and the complex type of febrile convulsion group. The expression of the upper ASIC1a was not significantly different from that of the normal control group, but the number of CA1 and CA3 radiation layers in the complex thermal convulsion group and the number of ASIC1a positive cells in the dentate gyrus multicell layer were significantly increased (P0.01).Western blot results showed that the expression of ASIC1a protein in the hippocampus of normal SD rats at 10 days after birth, and the expression of ASIC1a protein in the hippocampus of normal SD rats after birth, and There was no obvious change in the expression of (P11-P17) ASIC1a with time. 1 to 7 days after the complex febrile convulsion, the total expression of ASIC1a protein and membrane protein all increased, and there was a significant difference from that of the normal control group (P0.05).
2. 1 hours before the establishment of the model, two doses of amiloride (1.3 and 10mg/kg) were administered intraperitoneally. The percentage of the normal control group before and after the model establishment, the 1.3 amiloride group and the 10 amiloride group were less than 3%, and there was no significant difference between the groups (P0.05) and the center temperature of the.1.3 amiloride group and the 10 amiloride group at 39.5 and the whole body. The latent period of tetanic attack was significantly longer than that of the normal control group (P0.01), and there was a significant difference between the amiloride two preconditioning groups (P0.05). The time interval between the amiloride preconditioning group and the whole body tetanus was significantly longer than that of the normal control group (P0.01), and the temperature threshold of the generalized tetanic seizure was significantly higher. In the normal control group (P0.01).
3. complex thermal convulsion model was established 1 hours after the establishment of amiloride (1.3 and 10mg/kg), 1/ day, intraperitoneal injection. Immunohistochemical staining and Western blot results showed that GFAP positive astrocytes were significantly increased after complex febrile convulsions (P0.05), while two doses of amiloride decreased after two doses of amiloride. Lower these changes (P0.05). In the 4,5 day of the water maze test, the complex thermal convulsion group was more than the normal control group, the 1.3 amiloride group and the 10 amiloride group still needed a longer time to find the platform, and the results were statistically different (P0.05). The sixth day test period, compared with the normal group, the other three groups of SD rats were more expensive to find virtual platforms. In the complex febrile convulsion group, the number of virtual platforms was significantly different from that of the other three groups (P0.05).
conclusion
In the complex thermal convulsion model, the expression of ASIC1a was significantly increased. Amiloride, a ASIC1a antagonist, could resist the seizures of febrile convulsion, inhibit astroglia activation and improve the cognitive dysfunction caused by complex febrile convulsion, suggesting that ASIC1a may be involved in the pathophysiological changes of complex febrile seizures and convulsions.

【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R720.597

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