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孕期HMGB1干预对子鼠脑发育和神经行为的影响

发布时间:2018-05-27 21:25

  本文选题:难治性癫痫 + 皮质发育障碍 ; 参考:《第三军医大学》2017年硕士论文


【摘要】:研究背景:脑的发育是一个复杂而序贯的过程:包括神经前体细胞(Neural Precursor Cells,NPCs)的增殖、迁移、分化,神经突触的形成,神经递质的合成以及最终整合为具有完整功能的神经网络。在胚胎发育期,由于各种内在和外部刺激因素导致NPCs的异常增殖、迁移和分化则形成皮质发育障碍(Malformation of cortical dysplasia,MCD),并表现为脑组织结构和功能的异常。目前的研究发现,MCD内存在具有起搏性质的结构异常神经元,同时伴有抑制性神经递质减少、兴奋性神经递质增加及胶质细胞活化增生,这些因素可导致神经元兴奋性增高并产生自发性放电,最终引起临床癫痫发作。已有多个研究显示,在难治性癫痫手术标本中,MCD发生率在25%-53%,在3岁以前的婴幼儿难治性癫痫,MCD的发生率高达80%。因此,MCD成为难治性癫痫的重要病因,研究其发生及形成机制成为目前癫痫研究的重要内容。目前,MCD动物模型多采用冰冻损伤、致畸剂(如BCNU、MAM等)、辐射照射等方式模拟外源性损伤、化学致畸和放射照射干扰NPCs发育而诱导MCD的形成,这些方式能够部分地模拟外源性致畸因素诱导MCD的过程,并被广泛应用于目前的MCD研究中。与外源性损伤诱导MCD不同的是,在胚胎期还存在内源性的MCD诱导方式,包括遗传改变、缺血、缺氧、感染、炎症和内环境改变等,这些因素导致的内源性脑损伤及继发的炎症反应(内源性炎症反应)是胚胎期MCD形成重要原因。大量的研究表明,高迁移率族蛋白B1(High Mobility Group Box 1,HMGB1)是各种内源性损伤产生的共同结果和重要的内源性炎症反应诱导剂,作为固有免疫的主要配体,能启动Toll样受体(Toll Like Receptors,TLRs)和晚期糖基化终末产物受体(Receptor For Advanced Glycation Endproducts,RAGE)介导的免疫反应。HMGB1是一种具有高度保守性的非组蛋白染色体结合蛋白,主要分布于细胞核,能与DNA特异性结合,生理情况下参与基因转录和修复,除自然分泌外,在细胞损伤、死亡情况下大量释放到细胞外,具有很强的致炎活性,能启动并放大炎症反应。已有的研究显示,胚胎期损伤和内源性炎症能干扰NPCs的增殖、迁移及分化过程,而HMGB1启动的TLRs、RAGE受体激活是其中重要的起始信号。因此,本研究拟在脑发育的关键时期(即胚胎早期),通过注射HMGB1模拟内源性在体损伤,激活系列炎症反应,观察其对脑发育和神经行为学的影响,为建立内源性MCD动物模型和MCD发生研究提供新的思路。目的:通过孕期腹腔注射HMGB1模拟内源性损伤,明确HMGB1诱导子鼠脑发育和神经行为异常的最佳时间及最佳剂量;研究HMGB1干预对子鼠脑发育的病理学改变;研究HMGB1干预对子鼠的癫痫易感性和电生理改变;研究HMGB1干预对子鼠情绪、认知等神经行为改变。方法:1.分别对妊娠7天、妊娠14天(分别以E7、E14表示)的SD大鼠予以腹腔注射HMGB1,剂量分别为1μg/2ml PBS、5μg/2ml PBS、10μg/2ml PBS,对照组予以腹腔注射2ml PBS。2.观察子鼠的一般行为学表现;子鼠于第90天龄予BrdU(100mg/kg)连续腹腔注射3天后取材,采用琉瑾-吉姆萨染色(尼氏染色)、免疫组化(Neu N)观察脑组织病理学改变;DCX-Brd U、NeuN-Brd U免疫荧光双标技术检测海马神经发生情况;子鼠于第90天龄用匹鲁卡品(270mg/kg)诱发癫痫发作,并予以脑电图记录,以研究子鼠的癫痫易感性和电生理改变。3.子鼠于第90天龄进行Open Field Test实验、新旧物体识别实验和Morris水迷宫实验(定位航行实验和空间探索实验),研究HMGB1干预对子鼠抑郁焦虑、学习记忆等情绪、神经行为的影响。结果:一、孕期HMGB1腹腔注射影响子鼠脑发育的研究1.对照组、E7各组及E14-1μg组子鼠一般行为学无异常表现;E14-5μg组、E14-10μg组子鼠表现出活动量少、易激惹、兴奋躁动、“洗脸样”动作频繁等一般行为学改变。2.形态学显示对照组、E7各组子鼠双侧大脑饱满对称、皮质表面光滑,脑组织体积、重量无明显差异。E14-1μg组出现皮层表面呈颗粒状改变;E14-5μg组出现脑组织畸形,双侧大脑萎缩、皮层呈颗粒状改变;E14-10μg组脑组织畸形更明显,双侧大脑半球变小、四叠体过度外露伴上下丘过度增生、小脑巨大畸形、枕叶部分缺失变形。E14各组子鼠脑组织体积减小、重量减轻,随着剂量增加,脑组织体积越小、重量越轻。3.尼氏染色、免疫组化(Neu N标记)显示对照组、E7各组子鼠脑组织皮层层状结构清楚、细胞排列有序,海马形态正常;E14-1μg组子鼠表现为皮层层状结构紊乱,海马齿状回颗粒细胞排列稀疏;E14-5μg组、E14-10μg组子鼠均显示皮层变薄、层状结构消失、细胞排列紊乱、伴异形神经元出现,海马齿状回颗粒细胞及神经元丢失、宽度变窄;其中E14-10μg组皮层和海马均出现团块状增殖结节。4.免疫荧光双标技术(DCX-Brd U,Neu N-BrdU)显示对照组、E7各组子鼠海马齿状回神经发生无明显差异;E14-1μg组、E14-5μg组、E14-10μg组子鼠齿状回新生神经元减少,且剂量越高,其增殖、分化能力越低。5.匹鲁卡品诱发癫痫检测癫痫易感性,显示对照组、E7各组、E14-1μg组子鼠癫痫发作的起始时间及症状无明显差异,均无自发性癫痫;E14-5μg组、E14-10μg组子鼠癫痫发作的起始时间短、症状重,且E14-10μg组出现癫痫持续状态,癫痫点燃后出现自发性癫痫和痫性放电。二、孕期HMGB1腹腔注射对子鼠神经行为的影响1.OFT实验显示对照组子鼠活跃,中央活动及周边活动路程多;E7各组、E14-1μg组子鼠中央活动路程减少,周边活动增加;E14-5μg组和E14-10μg组子鼠更喜好于周边活动,表现为周边活动的路程及时间明显增多,其差异有统计学意义(P0.05)。2.新旧物体识别实验显示E7各组子鼠物体分辨指数(Discriminationhidex,DI)与对照组无明显差异;E14各组子鼠DI值均低于对照组,其中E14-10μg组DI值最低且统计分析其差异有意义(P=0.0280.05)。3.Morris水迷宫定位航行实验显示对照组、E7各组子鼠逃避潜伏期无明显差异;E14各组子鼠逃避潜伏期均长于对照组,且随着剂量的增加,逃避潜伏期逐渐延长,其差异有统计学意义(P0.05)。空间探索实验显示对照组、E7各组子鼠穿越第三平台的次数均无明显差异;E14各组子鼠穿越第三平台次数均少于对照组,且随着剂量增加而减少,其差异有统计学意义(P0.05)。结论:1.HMGB1高剂量孕期腹腔注射后可诱导子鼠脑皮层结构紊乱,齿状回神经发生减少,出现异形细胞和增殖性结节,且伴有一般行为学异常,子鼠癫痫易感性增高,显示HMGB1孕期注射能够模拟MCD的基本病理学和电生理特征,且E14-10μg是HMGB1诱导MCD动物模型的较佳时间和剂量。2.HMGB1高剂量孕期腹腔注射后子鼠学习、记忆等认知功能下降,且表现出焦虑抑郁倾向。3.E14予以高剂量HMGB1腹腔注射能诱导出MCD的基本病理改变,并伴有学习、记忆能力下降,表现焦虑抑郁倾向,是模拟内源性损伤机制诱导MCD的较佳模型。
[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.
【学位授予单位】:第三军医大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:R742.1

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