本文選題：難治性癲癇 + 皮質(zhì)發(fā)育障礙��； 參考：《第三軍醫(yī)大學(xué)》2017年碩士論文

【摘要】：研究背景:腦的發(fā)育是一個(gè)復(fù)雜而序貫的過(guò)程:包括神經(jīng)前體細(xì)胞(Neural Precursor Cells,NPCs)的增殖、遷移、分化,神經(jīng)突觸的形成,神經(jīng)遞質(zhì)的合成以及最終整合為具有完整功能的神經(jīng)網(wǎng)絡(luò)。在胚胎發(fā)育期,由于各種內(nèi)在和外部刺激因素導(dǎo)致NPCs的異常增殖、遷移和分化則形成皮質(zhì)發(fā)育障礙(Malformation of cortical dysplasia,MCD),并表現(xiàn)為腦組織結(jié)構(gòu)和功能的異常。目前的研究發(fā)現(xiàn),MCD內(nèi)存在具有起搏性質(zhì)的結(jié)構(gòu)異常神經(jīng)元,同時(shí)伴有抑制性神經(jīng)遞質(zhì)減少、興奮性神經(jīng)遞質(zhì)增加及膠質(zhì)細(xì)胞活化增生,這些因素可導(dǎo)致神經(jīng)元興奮性增高并產(chǎn)生自發(fā)性放電,最終引起臨床癲癇發(fā)作。已有多個(gè)研究顯示,在難治性癲癇手術(shù)標(biāo)本中,MCD發(fā)生率在25%-53%,在3歲以前的嬰幼兒難治性癲癇,MCD的發(fā)生率高達(dá)80%。因此,MCD成為難治性癲癇的重要病因,研究其發(fā)生及形成機(jī)制成為目前癲癇研究的重要內(nèi)容。目前,MCD動(dòng)物模型多采用冰凍損傷、致畸劑(如BCNU、MAM等)、輻射照射等方式模擬外源性損傷、化學(xué)致畸和放射照射干擾NPCs發(fā)育而誘導(dǎo)MCD的形成,這些方式能夠部分地模擬外源性致畸因素誘導(dǎo)MCD的過(guò)程,并被廣泛應(yīng)用于目前的MCD研究中。與外源性損傷誘導(dǎo)MCD不同的是,在胚胎期還存在內(nèi)源性的MCD誘導(dǎo)方式,包括遺傳改變、缺血、缺氧、感染、炎癥和內(nèi)環(huán)境改變等,這些因素導(dǎo)致的內(nèi)源性腦損傷及繼發(fā)的炎癥反應(yīng)(內(nèi)源性炎癥反應(yīng))是胚胎期MCD形成重要原因。大量的研究表明,高遷移率族蛋白B1(High Mobility Group Box 1,HMGB1)是各種內(nèi)源性損傷產(chǎn)生的共同結(jié)果和重要的內(nèi)源性炎癥反應(yīng)誘導(dǎo)劑,作為固有免疫的主要配體,能啟動(dòng)Toll樣受體(Toll Like Receptors,TLRs)和晚期糖基化終末產(chǎn)物受體(Receptor For Advanced Glycation Endproducts,RAGE)介導(dǎo)的免疫反應(yīng)。HMGB1是一種具有高度保守性的非組蛋白染色體結(jié)合蛋白,主要分布于細(xì)胞核,能與DNA特異性結(jié)合,生理情況下參與基因轉(zhuǎn)錄和修復(fù),除自然分泌外,在細(xì)胞損傷、死亡情況下大量釋放到細(xì)胞外,具有很強(qiáng)的致炎活性,能啟動(dòng)并放大炎癥反應(yīng)。已有的研究顯示,胚胎期損傷和內(nèi)源性炎癥能干擾NPCs的增殖、遷移及分化過(guò)程,而HMGB1啟動(dòng)的TLRs、RAGE受體激活是其中重要的起始信號(hào)。因此,本研究擬在腦發(fā)育的關(guān)鍵時(shí)期(即胚胎早期),通過(guò)注射HMGB1模擬內(nèi)源性在體損傷,激活系列炎癥反應(yīng),觀察其對(duì)腦發(fā)育和神經(jīng)行為學(xué)的影響,為建立內(nèi)源性MCD動(dòng)物模型和MCD發(fā)生研究提供新的思路。目的:通過(guò)孕期腹腔注射HMGB1模擬內(nèi)源性損傷,明確HMGB1誘導(dǎo)子鼠腦發(fā)育和神經(jīng)行為異常的最佳時(shí)間及最佳劑量;研究HMGB1干預(yù)對(duì)子鼠腦發(fā)育的病理學(xué)改變;研究HMGB1干預(yù)對(duì)子鼠的癲癇易感性和電生理改變;研究HMGB1干預(yù)對(duì)子鼠情緒、認(rèn)知等神經(jīng)行為改變。方法:1.分別對(duì)妊娠7天、妊娠14天(分別以E7、E14表示)的SD大鼠予以腹腔注射HMGB1,劑量分別為1μg/2ml PBS、5μg/2ml PBS、10μg/2ml PBS,對(duì)照組予以腹腔注射2ml PBS。2.觀察子鼠的一般行為學(xué)表現(xiàn);子鼠于第90天齡予BrdU(100mg/kg)連續(xù)腹腔注射3天后取材,采用琉瑾-吉姆薩染色(尼氏染色)、免疫組化(Neu N)觀察腦組織病理學(xué)改變;DCX-Brd U、NeuN-Brd U免疫熒光雙標(biāo)技術(shù)檢測(cè)海馬神經(jīng)發(fā)生情況;子鼠于第90天齡用匹魯卡品(270mg/kg)誘發(fā)癲癇發(fā)作,并予以腦電圖記錄,以研究子鼠的癲癇易感性和電生理改變。3.子鼠于第90天齡進(jìn)行Open Field Test實(shí)驗(yàn)、新舊物體識(shí)別實(shí)驗(yàn)和Morris水迷宮實(shí)驗(yàn)(定位航行實(shí)驗(yàn)和空間探索實(shí)驗(yàn)),研究HMGB1干預(yù)對(duì)子鼠抑郁焦慮、學(xué)習(xí)記憶等情緒、神經(jīng)行為的影響。結(jié)果:一、孕期HMGB1腹腔注射影響子鼠腦發(fā)育的研究1.對(duì)照組、E7各組及E14-1μg組子鼠一般行為學(xué)無(wú)異常表現(xiàn);E14-5μg組、E14-10μg組子鼠表現(xiàn)出活動(dòng)量少、易激惹、興奮躁動(dòng)、“洗臉樣”動(dòng)作頻繁等一般行為學(xué)改變。2.形態(tài)學(xué)顯示對(duì)照組、E7各組子鼠雙側(cè)大腦飽滿對(duì)稱、皮質(zhì)表面光滑,腦組織體積、重量無(wú)明顯差異。E14-1μg組出現(xiàn)皮層表面呈顆粒狀改變;E14-5μg組出現(xiàn)腦組織畸形,雙側(cè)大腦萎縮、皮層呈顆粒狀改變;E14-10μg組腦組織畸形更明顯,雙側(cè)大腦半球變小、四疊體過(guò)度外露伴上下丘過(guò)度增生、小腦巨大畸形、枕葉部分缺失變形。E14各組子鼠腦組織體積減小、重量減輕,隨著劑量增加,腦組織體積越小、重量越輕。3.尼氏染色、免疫組化(Neu N標(biāo)記)顯示對(duì)照組、E7各組子鼠腦組織皮層層狀結(jié)構(gòu)清楚、細(xì)胞排列有序,海馬形態(tài)正常;E14-1μg組子鼠表現(xiàn)為皮層層狀結(jié)構(gòu)紊亂,海馬齒狀回顆粒細(xì)胞排列稀疏;E14-5μg組、E14-10μg組子鼠均顯示皮層變薄、層狀結(jié)構(gòu)消失、細(xì)胞排列紊亂、伴異形神經(jīng)元出現(xiàn),海馬齒狀回顆粒細(xì)胞及神經(jīng)元丟失、寬度變窄;其中E14-10μg組皮層和海馬均出現(xiàn)團(tuán)塊狀增殖結(jié)節(jié)。4.免疫熒光雙標(biāo)技術(shù)(DCX-Brd U,Neu N-BrdU)顯示對(duì)照組、E7各組子鼠海馬齒狀回神經(jīng)發(fā)生無(wú)明顯差異;E14-1μg組、E14-5μg組、E14-10μg組子鼠齒狀回新生神經(jīng)元減少,且劑量越高,其增殖、分化能力越低。5.匹魯卡品誘發(fā)癲癇檢測(cè)癲癇易感性,顯示對(duì)照組、E7各組、E14-1μg組子鼠癲癇發(fā)作的起始時(shí)間及癥狀無(wú)明顯差異,均無(wú)自發(fā)性癲癇;E14-5μg組、E14-10μg組子鼠癲癇發(fā)作的起始時(shí)間短、癥狀重,且E14-10μg組出現(xiàn)癲癇持續(xù)狀態(tài),癲癇點(diǎn)燃后出現(xiàn)自發(fā)性癲癇和癇性放電。二、孕期HMGB1腹腔注射對(duì)子鼠神經(jīng)行為的影響1.OFT實(shí)驗(yàn)顯示對(duì)照組子鼠活躍,中央活動(dòng)及周邊活動(dòng)路程多;E7各組、E14-1μg組子鼠中央活動(dòng)路程減少,周邊活動(dòng)增加;E14-5μg組和E14-10μg組子鼠更喜好于周邊活動(dòng),表現(xiàn)為周邊活動(dòng)的路程及時(shí)間明顯增多,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。2.新舊物體識(shí)別實(shí)驗(yàn)顯示E7各組子鼠物體分辨指數(shù)(Discriminationhidex,DI)與對(duì)照組無(wú)明顯差異;E14各組子鼠DI值均低于對(duì)照組,其中E14-10μg組DI值最低且統(tǒng)計(jì)分析其差異有意義(P=0.0280.05)。3.Morris水迷宮定位航行實(shí)驗(yàn)顯示對(duì)照組、E7各組子鼠逃避潛伏期無(wú)明顯差異;E14各組子鼠逃避潛伏期均長(zhǎng)于對(duì)照組,且隨著劑量的增加,逃避潛伏期逐漸延長(zhǎng),其差異有統(tǒng)計(jì)學(xué)意義(P0.05)�？臻g探索實(shí)驗(yàn)顯示對(duì)照組、E7各組子鼠穿越第三平臺(tái)的次數(shù)均無(wú)明顯差異;E14各組子鼠穿越第三平臺(tái)次數(shù)均少于對(duì)照組,且隨著劑量增加而減少,其差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論:1.HMGB1高劑量孕期腹腔注射后可誘導(dǎo)子鼠腦皮層結(jié)構(gòu)紊亂,齒狀回神經(jīng)發(fā)生減少,出現(xiàn)異形細(xì)胞和增殖性結(jié)節(jié),且伴有一般行為學(xué)異常,子鼠癲癇易感性增高,顯示HMGB1孕期注射能夠模擬MCD的基本病理學(xué)和電生理特征,且E14-10μg是HMGB1誘導(dǎo)MCD動(dòng)物模型的較佳時(shí)間和劑量。2.HMGB1高劑量孕期腹腔注射后子鼠學(xué)習(xí)、記憶等認(rèn)知功能下降,且表現(xiàn)出焦慮抑郁傾向。3.E14予以高劑量HMGB1腹腔注射能誘導(dǎo)出MCD的基本病理改變,并伴有學(xué)習(xí)、記憶能力下降,表現(xiàn)焦慮抑郁傾向,是模擬內(nèi)源性損傷機(jī)制誘導(dǎo)MCD的較佳模型。
[Abstract]:Research background: brain development is a complex and sequential process: proliferation, migration, differentiation, synapse formation, synthesis of neurotransmitters, synthesis of neurotransmitters, and final integration into a complete functional neural network, including the Neural Precursor Cells (NPCs). In embryonic development, N is caused by various internal and external stimuli. The abnormal proliferation, migration and differentiation of PCs forms cortical dysplasia (Malformation of cortical dysplasia, MCD), and shows abnormal structure and function of the brain. Current studies have found that MCD is in abnormal neurons with pacing properties, accompanied by reduced neurotransmitters, increased excitatory neurotransmitters and glue. A number of studies have shown that the incidence of MCD is in 25%-53%, and the incidence of MCD in infants with refractory epilepsy before 3 years of age is up to 80%., and MCD becomes a refractory epilepsy. The important cause of epilepsy and the study of its formation and formation mechanism have become an important content of the current study of epilepsy. At present, MCD animal models mostly use freezing injury, teratogenic agents (such as BCNU, MAM, etc.), radiation exposure and other methods to simulate exogenous damage. Chemical teratogenicity and radiation exposure interference with the formation of MCD, which can induce the formation of MCD, which can be partially modeled. The process of inducing exogenous teratogenic factors to induce MCD and is widely used in current MCD studies. Unlike exogenous injury induced MCD, there are endogenous MCD induction modes in the embryonic stage, including genetic changes, ischemia, hypoxia, infection, inflammation and internal environment changes, and the endogenous brain damage and secondary inflammation caused by these factors. The disease response (endogenous inflammatory response) is an important reason for the formation of MCD in the embryonic stage. A large number of studies have shown that the high mobility group protein B1 (High Mobility Group Box 1, HMGB1) is a common result of various endogenous injuries and an important endogenous inflammatory response inducer. As the main ligand of the fixed immune system, the Toll like receptor (Toll Like) can be started. Receptors, TLRs) and late glycosylation end product receptor (Receptor For Advanced Glycation Endproducts, RAGE) mediated immune response.HMGB1 is a highly conserved non histone chromosome binding protein, mainly distributed in the nucleus, can bind to DNA specificity, and participate in gene transcription and repair in physiological conditions, except for nature. Secretory, a large number of release to cells in cell injury and death, has strong inflammatory activity and can initiate and amplify the inflammatory response. Previous studies have shown that embryonic injury and endogenous inflammation can interfere with the proliferation, migration and differentiation of NPCs, while HMGB1 activated TLRs, RAGE receptor activation is an important starting signal. This study is aimed at the critical period of brain development (early embryo). Through the injection of HMGB1 to simulate endogenous body damage and activate a series of inflammatory reactions, observe its effects on brain development and neurobehavioral studies, and provide new ideas for the establishment of endogenous MCD animal models and the development of MCD. Objective: to intraperitoneal intraperitoneal injection of HMGB1 to simulate endogenous sources by intraperitoneal injection during pregnancy. The best time and optimal dose of HMGB1 induced brain development and neurobehavioral abnormalities in the rats were determined, and the pathological changes of the HMGB1 intervention on the brain development of the rat were studied. The susceptibility and electrophysiological changes of the HMGB1 intervention on the epileptic rats were studied. The changes of the HMGB1 intervention on the emotion and cognition of the rats were studied. Methods: 1. to 7 of pregnancy, respectively. Day, 14 days of pregnancy (E7, E14, respectively) of SD rats were injected HMGB1 intraperitoneally, the dose was 1 g/2ml PBS, 5 mu g/2ml PBS, 10 micron PBS, and the control group was given celiac injection 2ml PBS.2. to observe the general behavior of the rats. The rats were selected for 3 days after ninetieth days of intraperitoneal injection, using ryukon GIM staining. (Nissl's staining), immunohistochemistry (Neu N) was used to observe the pathological changes of brain tissue; DCX-Brd U, NeuN-Brd U immunofluorescence double labeling technique was used to detect the occurrence of hippocampal neurogenesis; the rats were induced by pilocarpine (270mg/kg) at ninetieth days of age, and the electroencephalogram was recorded to study the epilepsy susceptibility and electrophysiological changes of the rat in ninetieth of the rats. Open Field Test experiment, new and old object recognition experiment and Morris water maze experiment (positioning navigation experiment and space exploration experiment) were used to study the effects of HMGB1 intervention on depression and anxiety, learning and memory, and neurobehavioral effects of HMGB1 intervention. Results: first, the study of 1. control groups, E7 groups and E14-1 in pregnancy HMGB1 intraperitoneal injection of projective rounded rats There was no abnormal behavior in the rats of the G group, and the rats in the group of E14-5 mu g and the group E14-10 mu g showed less activity, irritability, excitement and agitation, and the frequent behavior of "wash face" changed the.2. morphology to display the control group. The bilateral brains of the rats in each group of E7 were full symmetry, the skin surface was smooth, the volume of brain tissue and weight had no obvious difference.E14-1 There was a granular change on the surface of the cortical surface in the G group, and the brain tissue malformation, bilateral cerebral atrophy and granular change in the group of E14-5 mu g; the cerebral abnormality in the group of E14-10 mu g was more obvious, the bilateral cerebral hemisphere became smaller, the overexposure of the teas with the upper hypothalamus, the giant malformation of the cerebellum, and the partial absence of the occipital lobe in the brain tissue of each group of.E14. The volume was reduced and the weight was reduced. The smaller the brain tissue was, the smaller the volume of the brain tissue, the lighter the.3. Nissl staining, the Neu N marker showed in the control group. The cortical layer structure of the brain tissue of each group of E7 rats was clear, the cells arranged orderly, the hippocampus was normal, and the E14-1 Mug G group showed the layer structure disorder of the cortex and the granular cells of the dentate gyrus in the hippocampus. In group E14-5 mu g, the rats in group E14-10 mu g showed that the cortex became thinner, the layer structure disappeared, the cell arrangement was disorganized, the appearance of the heteromorphic neurons, the loss of granular cells and neurons in the dentate gyrus of the hippocampus, and the narrowing of the width of neurons, and the.4. immunofluorescence double labeling technique (DCX-Brd U, Neu N-BrdU) in the cortex and hippocampus of the E14-10 Mug G group. In the control group, there was no significant difference in the dentate gyrus of the E7 rats in each group, and in the E14-1 mu g group, the E14-5 mu g group and the E14-10 mu g group, the dentate neurons of the dentate gyrus were reduced, and the higher the dose, the proliferation, the lower the differentiation ability, the epilepsy induced seizure susceptibility of.5. pilocarpine, which showed that the epileptic seizures of the control group, E7 group and E14-1 u g group were the onset of epileptic seizures. There was no significant difference in initial time and symptoms, and no spontaneous epilepsy, in group E14-5 mu g, the onset time of epileptic seizures in group E14-10 mu g was short, and the symptoms were heavy, and the status epilepticus occurred in group E14-10 mu g, spontaneous epilepsy and epileptic discharge appeared after the kindling of epilepsy. Two, the effect of intraperitoneal injection of HMGB1 on the neurobehavioral effects of HMGB1 in pregnancy showed that the effect of 1.OFT in 1.OFT experiment showed control The group of rats were active, the central activity and the peripheral activity were much more, and the central activity of the group of E7 group, E14-1 mu g group decreased and the peripheral activity increased, and the E14-5 mu g group and the E14-10 mu g group preferred the peripheral activity, which showed a significant increase in the distance and time of the peripheral activities. The difference was statistically significant (P0.05).2. new and old object recognition experiment showed. The object resolution index (Discriminationhidex, DI) of each group of E7 rats was not significantly different from that of the control group; the DI value of the rats in each group of E14 was lower than the control group, and the DI of the E14-10 mu g group was the lowest and the statistical analysis of the difference was significant (P=0.0280.05).3.Morris water maze navigation experiment showed the control group, E7 groups had no obvious difference in escape latency; E14 The escape latency of each group was longer than that of the control group, and with the increase of the dose, the escape latency extended gradually, and the difference was statistically significant (P0.05). The space exploration experiment showed that there was no significant difference between the control group and the control group, and the number of third groups in each group of E14 in each group of E14 was less than that of the control group. The difference was statistically significant (P0.05). Conclusion: after intraperitoneal injection of high dose of 1.HMGB1, intraperitoneal injection could induce the derangement of cerebral cortex structure, decrease of dentate gyrus, heteromorphic cells and proliferative nodules, with general behavioral abnormalities, and increased susceptibility to epileptic rats, which showed that HMGB1 injection could simulate MCD during pregnancy. The basic pathological and electrophysiological characteristics, and E14-10 mu g is the better time and the dose of HMGB1 to induce the MCD animal model, and the dose of.2.HMGB1 in the high dose.2.HMGB1 rats after intraperitoneal injection, the cognitive function decline, and the expression of anxiety and depression.3.E14 with high dose HMGB1 intraperitoneal injection can induce the basic pathological changes of MCD, accompanied by the study. Learning and memory decline, showing anxiety and depression tendency, is a better model to simulate MCD induced by endogenous injury mechanism.
【學(xué)位授予單位】：第三軍醫(yī)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R742.1

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