颞叶癫痫发生过程中海马神经元核膜通透性改变的实验研究

发布时间：2018-03-12 13:34

本文选题：氯化锂-匹罗卡品癫痫动物模型　切入点：颞叶癫痫　出处：《中南大学》2014年硕士论文　论文类型：学位论文

【摘要】：目的：探讨颞叶癫痫发生过程中海马神经元核膜通透性改变状况。方法：将6-8周龄健康雄性SD大鼠114只,随机分为对照组(n=57只)和实验组(n=57只),每大组再分为SE后2h、7d和60d三个亚组。应用氯化锂-匹罗卡品腹腔注射的方法制备癫痫模型,分别进行：1.观察所有致痫大鼠行为学和脑电图变化；2.用尼氏染色法观察大鼠海马的病理学改变(分别从对照组和实验组随机取6只)；3.余大鼠采用蔗糖密度梯度离心技术,分离纯化大鼠海马组织神经元细胞核,并观察分离纯化的效果；4.对提取的神经元细胞核用免疫荧光法,检测核膜通透性变化、细胞核凋亡情况,并验证核膜通透性改变发生部位与核孔的关系。结果： 1.氯化锂-匹罗卡品大鼠癫痫模型：癫痫造模成功率为70.8%,总死亡率为18.6%。 2.致痫大鼠自发性痫性发作：致痫大鼠慢性期均发生了自发性痫性发作。大鼠在SE后8-22天(14.9±6.4天)开始出现自发性痫性发作。 3.脑电图变化：对照组以慢波为主,致痫大鼠急性期脑电图出现多种形式的痫样放电,典型波形有尖波、单棘波、多相棘波等发作性节律波；静止期大鼠脑电波亦以慢波为主,未见明显的痫样波；慢性期大鼠自发性痫性发作的脑电图可见以尖波为主的痫样波,且波幅较正常组高。 4.SE后海马CA1区、CA3区和门区病理学改变：模型组大鼠门区神经元显著减少,CA1、CA3区锥体细胞排列紊乱及神经元丢失；部分模型大鼠齿状回颗粒细胞减少、细胞排列紊乱,或颗粒细胞层弥散化。 5.蔗糖密度梯度离心结果：大鼠海马组织经蔗糖0.32M、2.2M、2.4M、2.6M和2.8M浓度梯度离心后,2.6-2.8M蔗糖层所含细胞核主要是神经元细胞核,还有极少量星形胶质细胞核。 6.大鼠海马神经元细胞核免疫荧光结果 (1)核膜通透性检测结果：从SE后2h、7d和60d组分离到海马神经元细胞核中,均包括数量不等的淡绿色阳性细胞核(渗漏细胞核),且显著多于对照组(P0.01)；且渗漏细胞核的数量以2h和60d组最多,2h组尤甚(P0.01)。 (2)凋亡细胞核检测结果：从SE后2h、7d和60d组分离到海马神经元细胞核,均包括数量不等的凋亡细胞核。与对照组相比,2h和60d组凋亡细胞核最多,7d组较少(P0.01)。 (3)核膜通透性改变发生部位与核孔的关系：将SE后2h、7d和60d组大鼠海马组织中分离纯化的神经元细胞核分两份：一份未加核孔阻塞剂麦胚凝集素(WGA),可见不同数量的发生核膜渗漏的阳性细胞核。另一份预先加入WGA,各组细胞核均未发现明显核膜渗漏。结论： 1.SE将导致海马神经元核孔损伤,增加其核膜通透性。 2.核膜通透性增加的存活海马神经元可能参与了颞叶癫痫形成。
[Abstract]:Objective: to investigate the changes of nuclear membrane permeability of hippocampal neurons during temporal lobe epilepsy. Methods: 114 healthy male Sprague-Dawley rats aged 6-8 weeks were randomly divided into control group (n = 57) and experimental group (n = 57). Each group was subdivided into three subgroups: 2 h after SE for 7 d and 60 d. The epileptic model was established by intraperitoneal injection of lithium-pilocarpine. Observe the changes of behavior and electroencephalogram in all epileptic rats 2.The pathological changes of hippocampus of rats were observed by Nissl staining method (6 rats were randomly selected from control group and experimental group respectively. The remaining rats were centrifuged by sucrose density gradient centrifugation. Isolation and purification of neuronal nuclei in rat hippocampal tissue and observation of the effect of isolation and purification 4.The changes of nuclear membrane permeability and nuclear apoptosis were detected by immunofluorescence assay. The relationship between the location of nuclear membrane permeability change and the nuclear pore was verified. Results:. 1. The epileptic model of rats with lithium chloride and pilocarpine: the success rate of epileptic model was 70.8 and the total mortality was 18.6. 2. Spontaneous seizures in epileptic rats: spontaneous epileptic seizures occurred in chronic phase of epileptic rats, and spontaneous seizures began to occur in rats 8-22 days after SE (14.9 卤6.4 days). 3. EEG changes: the control group was dominated by slow waves, and various forms of epileptiform discharges were observed in the electroencephalogram of epileptic rats in the acute phase. The typical waves included sharp wave, single spike wave, polyphase spike wave and so on. There was no obvious epileptiform wave and the amplitude of epileptiform wave was higher than that of normal group. 4. Pathological changes of hippocampal CA1, CA3 and portal area: in model group, the neuronal disarrangement and neuronal loss in the hippocampal CA1 region were significantly decreased, while in some model rats, the number of granular cells in dentate gyrus decreased and the cell arrangement was disordered. Or granular cell layer dispersion. 5. The results of sucrose density gradient centrifugation: after sucrose concentration gradient centrifugation, the nuclei in sucrose layer of rat hippocampus were mainly neuronal nuclei and a few astrocytes. 6. Results of nuclear immunofluorescence of hippocampal neurons in rats. 1) the results of nuclear membrane permeability test: the nuclei of hippocampal neurons were isolated from the groups of 2 h, 7 d and 60 d after SE. The number of leakage nuclei was significantly higher than that of control group (P 0.01), and the number of leakage nuclei was especially high in 2 h and 60 d groups, especially in 2 h and 60 d groups, especially in 2 h and 60 d groups, especially in 2 h and 60 d groups, especially in 2 h and 60 d groups, especially in 2 h and 60 d groups. (2) the results of apoptotic nucleus detection: hippocampal neuronal nuclei were isolated from 7 d and 60 d groups after SE, including different numbers of apoptotic nuclei. Compared with the control group, the number of apoptotic nuclei in the 2 h and 60 d groups was less than that in the 7 d group. (3) the relationship between the location of nuclear membrane permeability change and the nuclear pores: the neurons isolated and purified from hippocampal tissues of rats in 2 h and 60 d groups after SE were divided into two parts: one without wheat germ agglutinin agglutinin (WGAN), with different numbers. The positive nuclei with nuclear membrane leakage and the other with WGA were not found in each group. Conclusion:. 1. SE will cause the injury of hippocampal neurons' nuclear foramen and increase the permeability of nuclear membrane. 2. Surviving hippocampal neurons with increased nuclear membrane permeability may be involved in the formation of temporal lobe epilepsy.
【学位授予单位】：中南大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R742.1

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