GPR40在癫痫发作中的作用和机制研究

发布时间：2018-04-12 21:11

本文选题：G蛋白偶联受体40 + 癫痫发作　；参考：《重庆医科大学》2017年博士论文

【摘要】：第一部分GPR40在癫痫患者及动物模型脑组织中的表达研究目的:检测GPR40在癫痫患者术后脑组织及癫痫小鼠模型脑组织中的表达情况。方法:1.从课楲组已建立的包含了300多例难治性癫痫患者术后脑组织和100多例脑外伤减压术切除的正常颞叶脑组织的脑库中,随机选取癫痫脑组织标本20例和正常脑组织10例。2.成年雄性C57BL/6鼠,海人酸海马内注射诱导颞叶癫痫(TLE)动物模型,根据是否有反复自发性发作(SRS),分为SRS组(n=5)和non-SRS组(n=5),获取海马和皮质。3.免疫荧光检测GPR40的表达部位,免疫印迹检测GPR40的表达水平。结果:1.免疫荧光显示GPR40主要分布在海马CA1和CA3区的锥体细胞层,在癫痫病人组织和动物模型脑组织中,GPR40都不在星状胶质细胞中表达,而在神经元上表达,且与PSD95共表达。2.在癫痫患者术后脑组织中,GPR40的表达水平较对照组脑组织显著升高。3.在癫痫小鼠模型的皮质和海马中,有反复自发性发作小鼠的GPR40表达水平较无反复自发性发作小鼠显著升高。结论:GPR40与兴奋性神经元共表达,癫痫患者和癫痫动物模型脑组织中GPR40表达均显著升高,提示GPR40有可能参与了癫痫的发生发展。第二部分GPR40对两种癫痫动物模型癫痫发作的影响目的:为进一步探讨GPR40在癫痫发生发展中的作用,我们在癫痫发生发展过程中,应用GPR40选择性激动剂GW9508和选择性抑制剂GW1100,了解GPR40在海人酸模型和戊四氮慢性点燃模型中对癫痫发作的影响。方法:1.海人酸模型:成年雄性C57BL/6小鼠海马内注射海人酸(200ng)诱导癫痫持续状态,3天后经侧脑室置管每天给予GW9508、GW1100和DMSO溶剂(每组6只),连续7天。监测动物的癫痫发作情况,30天后进行海马内局部场电位记录。2.PTZ慢性点燃模型:腹腔注射阈下剂量PTZ(35mg/kg)点燃成年雄性C57BL/6小鼠,点燃前给予GW9508、GW1100和DMSO溶剂(每组12只),每隔48 h点燃1次,共15次,观察点燃过程中的发作分级。结果:1.海人酸模型中,行为学观察发现,与对照组相比,GW9508显著降低了癫痫发作频率,GW1100增加了癫痫发作频率。局部场电位记录发现,与对照组相比,GW9508降低了30分钟内癫痫样放电(SLEs)的次数和总时间,GW1100增加了SLEs的次数和总时间。2.PTZ慢性点燃模型中,与对照组相比,GW9508降低了各时间点的平均发作分级,提高了点燃过程的生存率;GW1100显示了相反的结果。结论:通过两种动物模型证实,在癫痫发生发展过程中,干预GPR40可影响癫痫发作,激动剂使癫痫发作活性减轻,抑制剂使癫痫发作活性增加。第三部分GPR40对突触传递的影响目的:利用全细胞膜片钳技术,探讨GPR40对突触传递的影响。方法:制备小鼠脑片,利用全细胞膜片钳技术记录海马CA1区锥体神经元的神经电生理,包括动作电位(AP),微小兴奋性突触后电流(m EPSC)、记录微小抑制性突触后电流(m IPSC),AMPA/NMDA比率以及成对脉冲比例(PPR)。结果:与基线水平相比,GW9508降低了动作电位的频率,GW1100增加了动作电位的频率;GW9508降低了m EPSC的频率和幅值,GW1100增加了m EPSC的频率和幅值;GPR40干预对m IPSC的频率和幅值的影响无显著差异。AMPA/NMDA比值结果发现,GW9508降低了NMDA受体介导电流的幅度,GW1100增加了NMDA受体介导电流的幅度;GPR40干预对AMPA受体介导的电流幅度的影响无显著差异。PPR在各组间无显著差异。结论:GPR40影响NMDA受体介导的兴奋性突触后传递。第四部分GPR40在癫痫发作中的作用机制目的:通过了解GPR40对NMDA受体的调节作用,以探讨GPR40调节癫痫发作的机制。方法:1.在动物实验后的脑组织中,免疫印迹法检测NMDA受体NR2A和NR2B的总蛋白和膜蛋白的表达变化。2.免疫荧光检测GPR40与NR2A和NR2B的共定位,免疫共沉淀检测GPR40与NR2A和NR2B相互作用,并采用定量免疫共沉淀检测GPR40对这种相互作用的影响。结果:1.在海人酸和PTZ实验后的脑组织中,免疫印迹结果显示,GW9508组NR2A和NR2B的膜蛋白/总蛋白比值显著减低,GW1100组膜蛋白/总蛋白比值明显升高;NR2A和NR2B的总蛋白表达量无显著变化。2.免疫荧光显示GPR40与NR2A和NR2B共定位;免疫共沉淀结果显示GPR40与NR2A和NR2B相互作用;定量免疫共沉淀结果显示,与对照相比,GW9508使这种结合减弱,GW1100使这种结合增强。结论:GPR40调节NR2A和NR2B的神经元细胞膜上的表达;这种改变可能是由于影响了GPR40与NR2A和NR2B的相互作用。
[Abstract]:Objective to study the expression of GPR40 in the first part of the brain tissue of epileptic patients and animal model: To investigate the expression of GPR40 in brain tissue of brain tissue in patients with epilepsy and epilepsy in mice. Methods: 1. from the class Wei group has been established including more than 300 cases of intractable epilepsy patients after temporal normal brain tissue and more than 100 cases of cerebral trauma resection of lobe Tsinhua, randomly selected 20 cases of epileptic brain tissues and normal brain tissues in 10 cases of adult male.2. C57BL/6 rats induced by injection of kainic acid in hippocampus of temporal lobe epilepsy (TLE) animal model, according to whether there is a recurrent spontaneous seizure (SRS), divided into SRS group (n=5) and non-SRS group (n=5), expression of.3. in hippocampus and cortex for immunofluorescence detection of GPR40, the expression level of GPR40 was detected by Western blot. Results: 1. immunofluorescence showed the pyramidal cell layer of GPR40 mainly distributed in the CA1 and CA3 of hippocampus in epileptic. The brain tissue of epileptic patients and in animal models of GPR40 are expressed in astrocytes, while the expression in neurons, and PSD95 expression of.2. in brain tissue of patients with epilepsy, the expression level of GPR40 in brain tissue was significantly increased compared with the control group.3. in the epilepsy mouse model of cortex and hippocampus, a recurrent spontaneous seizures in mice GPR40 expression was no recurrent spontaneous seizures in mice increased significantly. Conclusion: GPR40 and excitatory neurons co expression significantly increased the expression of GPR40 in patients with epilepsy and epilepsy animal model of brain tissue, suggesting that GPR40 may be involved in the occurrence and development of epilepsy. The second part GPR40 of two kinds of animal models of epilepsy epilepsy attack. Objective: to further explore the GPR40 in the occurrence and development of epilepsy, we in the process of the occurrence and development of epilepsy, a selective GPR40 agonist GW9508 and the selective inhibition Agent GW1100, GPR40 in kainic acid kindling model and e four nitrogen model of epilepsy. Methods: 1. adult hippocampus kainic acid model: male C57BL/6 mice injected with kainic acid (200ng) induced status epilepticus, 3 days after lateral ventricle puncture and DMSO GW1100 GW9508 every day. The solvent (n = 6), for 7 consecutive days. The monitoring of animal seizures, 30 days after hippocampal local field potentials recorded.2.PTZ kindling model: intraperitoneal injection of subthreshold dose of PTZ (35mg/kg) lit C57BL/6 male mice were given GW9508 and GW1100 before ignition, DMSO solvent (n = 12), every 48 h light 1 times, a total of 15 times, observe the grading process light attack. Results: 1. kainic acid model, behavioral observation found that, compared with the control group, GW9508 significantly decreased the frequency of seizures, GW1100 increased the frequency of seizures. The local field potential recording Found that, compared with the control group, GW9508 decreased 30 minutes of epileptiform discharges (SLEs) and the number of total time, GW1100 increased the number of SLEs and the total time of.2.PTZ kindling model, compared with the control group, GW9508 reduced the average seizure classification at each time point, improve the survival rate of the ignition process; GW1100 showed opposite results. Conclusion: the two kinds of animal models confirm that in the process of the occurrence and development of epilepsy, the intervention of GPR40 can affect the seizure, seizure activity reduced agonist, inhibitors of seizure activity increased. In the third part, the effect of GPR40 on synaptic transmission Objective: Using whole cell patch clamp technique to investigate the effect of GPR40 on the synaptic transmission. Methods: the preparation of mouse brain slices, using electrophysiological whole cell patch clamp recording of hippocampal CA1 pyramidal neurons, including action potential (AP), miniature excitatory postsynaptic currents ( M EPSC), recorded miniature inhibitory postsynaptic current (m IPSC), AMPA/NMDA ratio and paired pulse ratio (PPR). Results: compared with the baseline, GW9508 decreased the frequency of action potential, GW1100 increased the frequency of action potential; GW9508 reduced the frequency and amplitude of the m of EPSC, GW1100 increased the frequency and the amplitude of M EPSC; effect of GPR40 intervention on m frequency and amplitude of the IPSC had no significant difference in the ratio of.AMPA/NMDA, GW9508 reduced NMDA receptor mediated current amplitude, GW1100 increased NMDA receptor-mediated current amplitude; GPR40 effect of dry pretreatment on the current amplitude of AMPA receptor mediated no significant no significant difference the difference in.PPR between the two groups. Conclusion: the effect of GPR40 NMDA receptor mediated excitatory postsynaptic transmission. To the fourth part of the GPR40 mechanism in epilepsy: through the regulating effect of GPR40 on NMDA receptor, on GPR40 The mechanism of epilepsy. Methods: section 1. in animal experiments in brain tissue after CO localization, total protein and membrane protein by Western blot detection of NMDA receptor NR2A and NR2B expression of.2. by immunofluorescence detection of GPR40 and NR2A and NR2B, CO immunoprecipitation assay of GPR40 and NR2A and NR2B interaction, and the use of quantitative effect of immune co precipitation of GPR40 on this interaction. Results: 1. in kainic acid and PTZ in brain tissue after experiment, Western blotting showed that membrane protein / GW9508 group NR2A and NR2B total protein ratio was significantly decreased in group GW1100 membrane protein / total protein ratio increased significantly; the total protein of NR2A and NR2B the expression of.2. had no significant change of GPR40 and NR2A and immunofluorescence showed the co localization of NR2B; the results showed that GPR40 and NR2A interact with NR2B immunoprecipitation; quantitative immunoprecipitation results showed that, compared with the control, the combination of GW9508 decreased, GW1 100, enhance the binding. Conclusion: GPR40 regulates the expression of NR2A and NR2B on the cell membrane of neurons. This change may be due to the interaction between GPR40 and NR2A and NR2B.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R742.1

【相似文献】