本文選題：復(fù)雜型熱性驚厥 + 酸敏感離子通道1a�。� 參考：《第四軍醫(yī)大學(xué)》2014年碩士論文

【摘要】：熱性驚厥是嬰幼兒期最常見(jiàn)的一種抽搐，分為簡(jiǎn)單型和復(fù)雜型兩種。其中復(fù)雜型熱性驚厥不但給患兒帶來(lái)了嚴(yán)重的軀體損害，也常常導(dǎo)致其腦內(nèi)海馬局部回路興奮性增高，以及不同程度的認(rèn)知功能障礙。因此探索復(fù)雜型熱性驚厥的防治成為醫(yī)學(xué)領(lǐng)域亟需解決的重要課題。酸敏感離子通道1（aacid-sensing ion channel1a,ASIC1a）是質(zhì)子門控陽(yáng)離子通道，在神經(jīng)系統(tǒng)具有豐富的表達(dá)，常在酸性環(huán)境下易被激活。目前研究表明ASIC1a參與了諸多疾病的病理生理過(guò)程，阻斷病理?xiàng)l件下ASIC1a的激活具有一定的神經(jīng)保護(hù)性作用。復(fù)雜型熱性驚厥因?yàn)槌榇r(shí)間長(zhǎng)并伴有體溫升高，常常導(dǎo)致顱內(nèi)乳酸堆積以及組織酸化，ASIC1a能否被異常激活并介導(dǎo)細(xì)胞損傷過(guò)程，目前并不清楚。藉此，本研究選用大鼠復(fù)雜性熱性驚厥模型，探討ASIC1a在復(fù)雜型熱性驚厥中的作用，為復(fù)雜型熱性驚厥的防治提供新的靶點(diǎn)。 目的 首先觀察ASIC1a在復(fù)雜型熱性驚厥大鼠海馬部位的表達(dá)，繼而探討ASIC1a拮抗劑阿米洛利不同劑量預(yù)處理對(duì)復(fù)雜型熱性驚厥發(fā)作的影響，以及后處理對(duì)大鼠精神發(fā)育的影響 方法 選用Sprague-Dawley（SD）大鼠（P10），高溫氣浴致強(qiáng)直發(fā)作30分鐘建立復(fù)雜型熱性驚厥模型，在模型建立前或后給予阿米洛利1.3mg/kg和10mg/kg兩劑量腹腔注射，，應(yīng)用免疫組織化學(xué)和蛋白質(zhì)印跡實(shí)驗(yàn)方法，結(jié)合曠場(chǎng)試驗(yàn)、高架十字以及水迷宮等行為學(xué)試驗(yàn)進(jìn)行研究。統(tǒng)計(jì)學(xué)方法采用t檢驗(yàn)與方差分析。 結(jié)果 1．高溫氣浴致大鼠強(qiáng)直發(fā)作30分鐘為復(fù)雜型熱性驚厥模型成功標(biāo)準(zhǔn)。模型建立過(guò)程中無(wú)一只動(dòng)物死亡。通過(guò)免疫組化染色我們發(fā)現(xiàn)復(fù)雜型熱性驚厥后第三天，復(fù)雜型熱性驚厥組SD大鼠海馬CA1和CA3區(qū)錐體細(xì)胞層，以及齒狀回顆粒細(xì)胞層上ASIC1a的表達(dá)量與正常對(duì)照組間無(wú)明顯差異。而復(fù)雜型熱性驚厥組CA1和CA3放射層，以及齒狀回多形細(xì)胞層ASIC1a陽(yáng)性細(xì)胞數(shù)目較正常對(duì)照組明顯增多（p0.01）。Western blot結(jié)果顯示出生后10天的正常SD大鼠腦內(nèi)海馬區(qū)即有ASIC1a蛋白的表達(dá)，且隨著時(shí)間的推移（P11-P17）ASIC1a的表達(dá)量無(wú)明顯變化。復(fù)雜型熱性驚厥后1到7天，ASIC1a總蛋白和膜蛋白表達(dá)量都有所增加，與正常對(duì)照組均有顯著性差異（p0.05）。 2．模型建立前1小時(shí)給予阿米洛利兩種劑量（1.3和10mg/kg）腹腔注射。模型建立前后正常對(duì)照組，1.3阿米洛利組以及10阿米洛利組大鼠體重差的百分比均小于3%，組間無(wú)明顯差異（p0.05）。1.3阿米洛利組和10阿米洛利組中心溫度達(dá)到39.5℃和全身強(qiáng)直性發(fā)作的潛伏期較正常對(duì)照組明顯延長(zhǎng)（p0.01），且阿米洛利兩不同劑量預(yù)處理組間有顯著性差異（p0.05）。阿米洛利預(yù)處理組達(dá)到39.5℃和全身強(qiáng)直發(fā)作的時(shí)間間隔較正常對(duì)照組的明顯延長(zhǎng)（p0.01），且全身強(qiáng)直性發(fā)作的溫度閾值明顯高于正常對(duì)照組（p0.01）。 3.復(fù)雜型熱性驚厥模型建立后1小時(shí)開(kāi)始連續(xù)14天給予阿米洛利（1.3和10mg/kg），1/日，腹腔注射。免疫組化染色及Western blot結(jié)果顯示復(fù)雜型熱性驚厥后GFAP陽(yáng)性的星形膠質(zhì)細(xì)胞較正常對(duì)照組明顯增多（p0.05），而阿米洛利兩劑量后處理組明顯降低這些變化（p0.05）。在水迷宮實(shí)驗(yàn)訓(xùn)練期的第4，5天，復(fù)雜型熱性驚厥組較正常對(duì)照組，1.3阿米洛利組和10阿米洛利組仍需要更長(zhǎng)時(shí)間找到平臺(tái)，結(jié)果有統(tǒng)計(jì)學(xué)差異（p0.05）。第6天測(cè)試期，與正常對(duì)照組相比，其余三組SD大鼠尋找虛擬平臺(tái)較為費(fèi)力，其中復(fù)雜型熱性驚厥組找到虛擬平臺(tái)的次數(shù)較其它三組有明顯統(tǒng)計(jì)學(xué)差異（p0.05）。 結(jié)論 在復(fù)雜型熱性驚厥模型中ASIC1a表達(dá)量明顯增加，ASIC1a拮抗劑阿米洛利能抗熱性驚厥發(fā)作，抑制星形膠質(zhì)活化，改善復(fù)雜型熱性驚厥導(dǎo)致的認(rèn)知功能障礙。提示ASIC1a可能參與了復(fù)雜型熱性驚厥發(fā)作及驚厥后的病理生理變化過(guò)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.

【學(xué)位授予單位】：第四軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R720.597

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