体外神经元网络4-AP痫性模型建立及能量代谢特征研究

发布时间：2019-05-27 20:19

【摘要】：第一部分体外培养海马神经元网络痫性电活动模型的建立目的在体外培养原代大鼠胚胎海马神经元网络中,应用4-AP(50μM)分别进行致痫,通过与经典的低镁海马神经元痫样放电(SLEs)模型进行比较,建立4-AP的致痫神经网络模型。观察两种模型上神经元痫样放电的情况,为进一步探讨癫痫的能量代谢特征提供神经网络模型。方法取孕第18-19天的胚胎SD大鼠,分离海马神经元培养,在倒置相差显微镜下观察神经元的生长情况。采用Map2免疫荧光细胞染色鉴定神经元。培养至第8天采用全细胞膜片钳技术记录神经元在无镁培养液、4-AP处理不同后30main内的放电情况。结果神经元在种植24h后大部分贴壁,突起逐渐增多,在神经元间逐渐互相连接形成网络。培养第8～10d时细胞最为丰满。第8天的神经元经无镁培养液或4-AP处理后马上进行膜片钳检测,发现两组培养细胞均出现高频与高幅度痫样放电。在5-10min时间段达到高峰后逐渐下降,在每个时间段的放电频率及平均振幅均远远高于正常对照组(p0.001)。而低镁及4-AP两种致痫模型的比较,平均振幅及放电频率在30min内差异无统计学意义。三组间的平均振幅及放电频率在各时间变化均有显著性差异。结论体外培养的胚胎海马神经元经4-AP处理后,可诱发出与低镁处理一样的高频与高幅度痫样放电,可视为一种体外痫性神经网络模型,作为体外研究痫性网络的工具。第二部分体外培养胚胎海马神经元网络的能量代谢特征及与痫性标志变化间的关系目的在体外培养的大鼠胚胎海马神经元细胞上,建立低镁及4-AP诱发痫性神经网络模型,检测30min内线粒体膜电位的变化,及细胞能量代谢标志分子ATP, ADP浓度,及网络致痫标志分子细胞外谷氨酸(Glu)及γ氨基丁酸(GABA)的含量。观察致痫神经网络线粒体膜电位水平、与能量代谢及网络致痫状态的关系。方法分别用低镁培养液及4-AP对培养的海马神经元处理Omin,5min,1Omin,15min,20min,25min,30min后,利用荧光分光光度法检测胚胎海马神经元网络线粒体膜电位,用比色法检测海马神经元内ATP、 ADP含量,用ELISA法检测海马神经网络中Glu及GABA浓度。结果大鼠胚胎海马神经元网络诱导SLEs后呈,线粒体膜电位、ADP及谷氨酸短期内升高,之后逐渐降低；而ATP及GABA则呈现相反变化。表明神经元网络诱导SLEs的事件可视为神经元网络的高电位爆发过程,并伴有高代谢特征。但相对而言,痫性标志分子的变化较电位变化迟滞。结论大鼠胚胎海马神经元网络诱导SLEs后呈现规律性时相变化。在此过程中,细胞线粒体膜电位与痫性活动标志分子Glu呈现先升后降,而ATP与GABA呈现先降后升的变化趋势。这表明神经元网络诱导SLEs的事件可视为神经元网络高电位爆发过程,并伴有高代谢特征。但相对而言,痫性标志分子的变化较电位变化迟滞。图21幅,表16个,参考文献42篇
[Abstract]:The first part is the establishment of epileptic electrical activity model of hippocampal neuron network cultured in vitro. Objective to induce eclampsia by 4-AP (50 渭 M) in primary rat embryonic hippocampal neuron network in vitro. Compared with the classical epileptic discharge (SLEs) model of low magnesium hippocampal neurons, the epileptic neural network model of 4-AP was established. To observe the epileptic discharge of neurons in the two models, and to provide a neural network model for further study of the characteristics of energy metabolism in seizures. Methods the hippocampal neurons were isolated from embryonic SD rats on the 18th and 19th day of gestation, and the growth of neurons was observed under inverted phase contrast microscope. The neurons were identified by Map2 immunofluorescence cell staining. On the 8th day, the discharge of neurons in magnesium-free medium and 30main treated with 4-AP was recorded by whole-cell patch clamp technique. Results after 24 hours of implantation, most of the neurons adhered to the wall, the processes increased gradually, and the neurons gradually connected to each other to form a network. The cells were the most plump on the 8th to 10th day of culture. On the 8th day, the neurons were treated with magnesium-free medium or 4-AP as soon as they were treated with patch clamp. It was found that the cells in both groups showed high frequency and high amplitude epileptic discharge. After reaching the peak in 5-10min time period, the discharge frequency and average amplitude were much higher than those in the normal control group (p0. 001), and the discharge frequency and average amplitude in each time group were much higher than those in the normal control group (p0. 001). However, there was no significant difference in average amplitude and discharge frequency between low magnesium and 4-AP epileptic models in 30min. There were significant differences in average amplitude and discharge frequency among the three groups at each time. Conclusion the embryonic hippocampal neurons cultured in vitro can induce the same high frequency and high amplitude epileptic discharge as low magnesium treatment after 4-AP treatment, which can be regarded as a epileptic neural network model in vitro and used as a tool to study epileptic network in vitro. The second part is the energy metabolism characteristics of embryonic hippocampal neuron network cultured in vitro and its relationship with the changes of epileptic markers. Objective to investigate the relationship between energy metabolism and epileptic markers in rat embryonic hippocampal neuron cells cultured in vitro. The neural network model of epileptic induced by low magnesium and 4-AP was established. The changes of mitochondrial membrane potential and the concentration of ATP, ADP, a marker of cell energy metabolism, were detected in 30min. And the contents of extracellular glutamic acid (Glu) and gamma aminobutyric acid (GABA) in eclampsia induced by the network. To observe the relationship between the level of mitochondrial membrane potential and energy metabolism and the state of eclampsia induced by epileptic neural network. Methods the cultured hippocampal neurons were treated with Omin,5min,1Omin,15min,20min,25min,30min in low magnesium medium and 4-AP, respectively. the mitochondrial membrane potential of embryonic hippocampal neuron network was measured by fluorescence spectrophotometry. The content of ATP, ADP in hippocampal neurons was measured by colorimetric method, and the concentrations of Glu and GABA in hippocampal neural network were detected by ELISA method. Results after SLEs induced by rat embryonic hippocampal neuron network, the mitochondrial membrane potential, ADP and glutamic acid increased in a short period of time, and then decreased gradually, while ATP and GABA showed opposite changes. It is suggested that the event of SLEs induced by neural network can be regarded as the process of high potential burst of neural network, accompanied by high metabolic characteristics. But relatively speaking, the change of epileptic marker molecules is slower than the change of potential. Conclusion SLEs induced by embryonic hippocampal neuron network in rats shows regular temporal changes. In this process, the mitochondrial membrane potential and epileptic activity marker Glu increased at first and then decreased, while ATP and GABA decreased at first and then increased. This suggests that the event of SLEs induced by neural network can be regarded as a process of high potential burst of neural network, accompanied by high metabolic characteristics. But relatively speaking, the change of epileptic marker molecules is slower than the change of potential. Figure 21, table 16, references 42
【学位授予单位】：中南大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R742.1;R-332

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