本文選題：顱腦創(chuàng)傷 + 創(chuàng)傷性癲癇��； 參考：《天津醫(yī)科大學》2017年碩士論文

【摘要】：目的:癲癇是一種慢性神經(jīng)系統(tǒng)疾病,具有一過性、刻板性、突發(fā)突止的特點。創(chuàng)傷性腦損傷(traumatic brain injury,TBI)是癥狀性癲癇的重要病因之一。癲癇發(fā)作引起的腦缺血缺氧反應(yīng)使大腦神經(jīng)元受損,產(chǎn)生大量自由基,造成線粒體損傷,而線粒體損傷引起的細胞凋亡和壞死,使得癲癇發(fā)作進一步加重。本研究通過建立創(chuàng)傷性癲癇(posttraumatic epilepsy,PTE)大鼠模型,觀察線粒體及氧化應(yīng)激反應(yīng)相關(guān)指標的變化,并通過檢測腦組織細胞凋亡及相關(guān)凋亡蛋白的表達,探討創(chuàng)傷性癲癇下線粒體功能的變化并觀察應(yīng)用抗氧化劑α-硫辛酸(α-lipoic acid,α-LA)后各相關(guān)指標的改變,進一步探索神經(jīng)細胞凋亡和超微結(jié)構(gòu)損傷變化在創(chuàng)傷性癲癇的發(fā)病機制中的作用,以及抗氧化劑對這一病理重構(gòu)過程的影響。方法:取45只雄性Wistar大鼠隨機分成三組(對照組、創(chuàng)傷性癲癇組、α-硫辛酸干預(yù)組),每組15只。建立Fe Cl2所致顱腦創(chuàng)傷后癲癇大鼠模型,參考立體定位圖譜埋藏不銹鋼電極,記錄各組大鼠腦電圖(EEG)。采用尼氏染色法觀察大鼠皮層及海馬神經(jīng)元情況,應(yīng)用Western-Blot法檢測與分析Caspase-3蛋白質(zhì),并觀測線粒體中丙二醛(MDA)和一氧化氮(NO)的含量,檢測Na+K+-ATP酶、Ca2+Mg2+-ATP酶、總ATP酶、超氧化物歧化酶(SOD)、谷胱甘肽過氧化物酶(GSH-Px)活力及線粒體膜電位的變化,進一步了解神經(jīng)元凋亡和超微結(jié)構(gòu)損傷在PTE的發(fā)病機制。結(jié)果:1.正常對照組大鼠皮層電極腦電圖(EEG)均未見叢集性棘波放電,創(chuàng)傷性癲癇組皮層電極EEG均描記到叢集性棘波放電,通過α-硫辛酸干預(yù)后,叢集性棘波放電現(xiàn)象明顯改善。2.創(chuàng)傷性癲癇組大鼠出現(xiàn)癲癇發(fā)作,表現(xiàn)為(1)耳、面部痙攣性抽動,包括眨眼、動須、節(jié)律性咀嚼,雙耳顫動等;(2)渾身顫動,節(jié)律性點頭;(3)動物前肢陣發(fā)性抽搐,頻率隨時間增加;(4)雙前肢抬起,呈半直立位,隨后全身及四肢肌肉陣攣,不能直立,后傾跌倒;(5)動物四肢抽動,無法站立,出現(xiàn)全身強直陣攣發(fā)作。按Racine標準均達到中重度發(fā)作。發(fā)作結(jié)束后大鼠精神萎靡,活動及進食量減少。對照組大鼠則無癲癇發(fā)作,而α-硫辛酸干預(yù)組癲癇發(fā)作程度明顯減輕。3.對各組大鼠大腦皮層與海馬進行尼氏染色,創(chuàng)傷性癲癇組海馬區(qū)錐體細胞的超微結(jié)構(gòu)有明顯改變,表現(xiàn)為細胞核發(fā)生變形、固縮,染色質(zhì)固縮凝集在核膜的內(nèi)表面,碎裂成碎片,在核膜附近形成多個密集的斑塊,核膜不清晰;α-硫辛酸干預(yù)組神經(jīng)元形態(tài)結(jié)構(gòu)基本正常,核膜基本清晰,無染色質(zhì)聚集;胞質(zhì)內(nèi)有少許水腫空白區(qū)。4.Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、總ATP酶活性在創(chuàng)傷性癲癇組與對照組比較均有顯著的下降,而α-硫辛酸干預(yù)組則有明顯的改善,兩組比較有顯著的統(tǒng)計學差異。線粒體細胞膜電位在癲癇組有明顯下降,通過α-硫辛酸干預(yù),能夠抑制其下降。5.SOD與GSH-PX兩指標在創(chuàng)傷性癲癇組濃度降低,MDA與NO兩指標則升高,證實在癲癇發(fā)作時,氧化應(yīng)激狀態(tài)的存在。而α-硫辛酸干預(yù)組這些指標則有顯著的改善,兩者比較有明顯的統(tǒng)計學差異。6.在創(chuàng)傷性癲癇組,細胞凋亡明顯增多,凋亡相關(guān)蛋白Caspase-3表達明顯上調(diào),而α-硫辛酸干預(yù)組則細胞凋亡現(xiàn)象有顯著的下降,同樣Caspase-3表達水平也下降明顯。結(jié)論:1.在Fe Cl2所致創(chuàng)傷性癲癇大鼠癲癇模型中,MDA、NO水平顯著升高,Na+-K+-ATP酶、Ca2+-Mg2+-ATP酶、總ATP酶、SOD、GSH-Px活力則明顯降低;提示在創(chuàng)傷性癲癇大鼠模型中的腦細胞線粒體內(nèi)會產(chǎn)生大量的自由基,腦細胞發(fā)生氧化應(yīng)激損傷,導(dǎo)致抗氧化防御功能明顯下降;腦細胞線粒體中的能量供應(yīng)受到相應(yīng)的阻礙。2.α-硫辛酸對創(chuàng)傷性癲癇大鼠線粒體氧化應(yīng)激損傷有保護作用。其機理可能與提高了腦組織中線粒體內(nèi)抗氧化的相關(guān)酶的活性,使腦組織線粒體生物膜的脂質(zhì)過氧化水平降低有關(guān)。3.在FeCl2所致創(chuàng)傷性癲癇大鼠模型中,氧化應(yīng)激反應(yīng)誘導(dǎo)神經(jīng)細胞凋亡以及神經(jīng)元超微結(jié)構(gòu)的改變,α-硫辛酸保護的作用途徑之一可能為抑制氧化應(yīng)激誘導(dǎo)的神經(jīng)細胞凋亡。
[Abstract]:Objective: epilepsy is a chronic nervous system disease, which is characterized by an excessive, stereotyped and abrupt abrupt characteristic. Traumatic brain injury (TBI) is one of the important causes of symptomatic epilepsy. The cerebral ischemia and hypoxia response caused by epileptic seizures causes the brain neurons to be damaged and produces a large number of free radicals, causing mitochondrial damage and line damage. In this study, the changes in mitochondrial and oxidative stress related indexes were observed by establishing the posttraumatic epilepsy (PTE) rat model, and the traumatic epilepsy was explored by detecting the apoptosis of the brain tissue and the expression of the associated apoptotic proteins. The changes in the function of the mitochondria and the changes of the related indexes after the application of antioxidant alpha -lipoic acid (alpha -LA) were observed. The effects of apoptosis and ultrastructural damage on the pathogenesis of traumatic epilepsy were further explored, and the effects of antioxidants on this pathological process. Methods: 45 males were taken. Sex Wistar rats were randomly divided into three groups (control group, traumatic epilepsy group, alpha lipoic acid intervention group) and 15 rats in each group. The model of epileptic rats after Fe Cl2 was established. The electroencephalogram (EEG) of each group was recorded in the stereotaxic map, and the neurons in the cortex and hippocampus of rats were observed by Nissl staining. Caspase-3 protein was detected and analyzed by Western-Blot, and the content of malondialdehyde (MDA) and nitric oxide (NO) in mitochondria was observed. The changes of Na+K+-ATP enzyme, Ca2+Mg2+-ATP enzyme, total ATP enzyme, superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) activity and mitochondrial membrane potential were detected. The apoptosis and ultrastructure of neurons were further understood. Results: 1. the cortex electrode electroencephalogram (EEG) of the normal control group had no cluster spinous discharge, and the cortical electrode EEG of the traumatic epilepsy group was traced to the cluster spinous wave discharge. The cluster spinous discharge phenomenon obviously improved the epileptic seizures in the.2. traumatic epileptic group by the prognosis of the alpha lipoic acid. 1. (1) ears, facial spasmodic movement, including blinking, moving whiskers, rhythmic chewing, double ear tremor, (2) trembling, rhythmic nods, (3) the frequency of the forelimb spasms, frequency increased with time; (4) the double forelimbs were raised in a vertical position, with the posterior body and limbs muscle clonus, unable to erect, backward dip; (5) animal limbs twitching, (5) the animals' extremities twitching, unable to Standing, generalized tonic clonic seizures. Moderate and severe seizures were reached according to the Racine standard. After the end of the seizure, the rats were depressed and the activity and food intake decreased. The control group had no epileptic seizures, and the degree of epileptic seizures in the intervention group of alpha lipoic acid significantly alleviated the Nissl staining and traumatic epilepsy in the cerebral cortex and hippocampus of rats in each group. The ultrastructure of pyramidal cells in the hippocampus of the group was changed obviously, which showed that the nuclei were deformed and fixed, and the chromatin was condensed and condensed on the inner surface of the nuclear membrane, and the fragmentation became fragments. There were many dense patches near the nuclear membrane, and the nuclear membrane was not clear. The morphological structure of the neurons in the alpha lipoic acid intervention group was basically normal, and the nuclear membrane was basically clear and no chromatin was found. There was a significant decrease in the activity of.4.Na+-K+-ATP enzyme, Ca2+-Mg2+-ATP enzyme and total ATP enzyme in the traumatic epileptic group and the control group, but there was a significant improvement in the alpha lipoic acid intervention group. The two groups had significant statistical differences. The mitochondrial membrane potential decreased significantly in the epileptic group, through alpha. The intervention of lipoic acid could inhibit the decrease of the concentration of.5.SOD and GSH-PX two in the traumatic epileptic group, and the increase of MDA and NO two index, which confirmed the presence of oxidative stress during epileptic seizures, and these indexes were significantly improved in the alpha lipoic acid intervention group, and there was a significant statistical difference between the two groups,.6. in the traumatic epilepsy group, Apoptosis was significantly increased, apoptosis related protein Caspase-3 expression was obviously up-regulated, while alpha lipoic acid intervention group had a significant decrease in apoptosis, and the same level of Caspase-3 expression decreased obviously. Conclusion: 1. in the epileptic model of traumatic epileptic rats induced by Fe Cl2, the level of MDA, NO is significantly increased, Na+-K+-ATP enzyme, Ca2+-Mg2+-ATP enzyme, total A. The activity of TP, SOD and GSH-Px decreased significantly, suggesting that a large number of free radicals produced in the mitochondria of the brain cells in the rat model of traumatic epilepsy, the oxidative stress in the brain cells was damaged and the antioxidant defense function decreased obviously. The energy supply in the mitochondria of the brain cells was affected by the corresponding hindering.2. alpha lipoic acid to the traumatic epileptic rats Mitochondrial oxidative stress damage has protective effect. Its mechanism may improve the activity of mitochondrial antioxidant related enzymes in brain tissue, reduce the lipid peroxidation level of mitochondrial biofilm in brain tissue related to.3. in FeCl2 induced rat model of traumatic epilepsy, and induce neuronal apoptosis and neuronal ultrastructure induced by oxidative stress reaction. One of the protective pathways of alpha lipoic acid may be to inhibit oxidative stress induced neuronal apoptosis.
【學位授予單位】：天津醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R742.1

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本文編號：2022458