本文選題：G蛋白偶聯(lián)受體40 + 癲癇發(fā)作　； 參考：《重慶醫(yī)科大學(xué)》2017年博士論文

【摘要】：第一部分GPR40在癲癇患者及動物模型腦組織中的表達研究目的:檢測GPR40在癲癇患者術(shù)后腦組織及癲癇小鼠模型腦組織中的表達情況。方法:1.從課楲組已建立的包含了300多例難治性癲癇患者術(shù)后腦組織和100多例腦外傷減壓術(shù)切除的正常顳葉腦組織的腦庫中,隨機選取癲癇腦組織標本20例和正常腦組織10例。2.成年雄性C57BL/6鼠,海人酸海馬內(nèi)注射誘導(dǎo)顳葉癲癇(TLE)動物模型,根據(jù)是否有反復(fù)自發(fā)性發(fā)作(SRS),分為SRS組(n=5)和non-SRS組(n=5),獲取海馬和皮質(zhì)。3.免疫熒光檢測GPR40的表達部位,免疫印跡檢測GPR40的表達水平。結(jié)果:1.免疫熒光顯示GPR40主要分布在海馬CA1和CA3區(qū)的錐體細胞層,在癲癇病人組織和動物模型腦組織中,GPR40都不在星狀膠質(zhì)細胞中表達,而在神經(jīng)元上表達,且與PSD95共表達。2.在癲癇患者術(shù)后腦組織中,GPR40的表達水平較對照組腦組織顯著升高。3.在癲癇小鼠模型的皮質(zhì)和海馬中,有反復(fù)自發(fā)性發(fā)作小鼠的GPR40表達水平較無反復(fù)自發(fā)性發(fā)作小鼠顯著升高。結(jié)論:GPR40與興奮性神經(jīng)元共表達,癲癇患者和癲癇動物模型腦組織中GPR40表達均顯著升高,提示GPR40有可能參與了癲癇的發(fā)生發(fā)展。第二部分GPR40對兩種癲癇動物模型癲癇發(fā)作的影響目的:為進一步探討GPR40在癲癇發(fā)生發(fā)展中的作用,我們在癲癇發(fā)生發(fā)展過程中,應(yīng)用GPR40選擇性激動劑GW9508和選擇性抑制劑GW1100,了解GPR40在海人酸模型和戊四氮慢性點燃模型中對癲癇發(fā)作的影響。方法:1.海人酸模型:成年雄性C57BL/6小鼠海馬內(nèi)注射海人酸(200ng)誘導(dǎo)癲癇持續(xù)狀態(tài),3天后經(jīng)側(cè)腦室置管每天給予GW9508、GW1100和DMSO溶劑(每組6只),連續(xù)7天。監(jiān)測動物的癲癇發(fā)作情況,30天后進行海馬內(nèi)局部場電位記錄。2.PTZ慢性點燃模型:腹腔注射閾下劑量PTZ(35mg/kg)點燃成年雄性C57BL/6小鼠,點燃前給予GW9508、GW1100和DMSO溶劑(每組12只),每隔48 h點燃1次,共15次,觀察點燃過程中的發(fā)作分級。結(jié)果:1.海人酸模型中,行為學(xué)觀察發(fā)現(xiàn),與對照組相比,GW9508顯著降低了癲癇發(fā)作頻率,GW1100增加了癲癇發(fā)作頻率。局部場電位記錄發(fā)現(xiàn),與對照組相比,GW9508降低了30分鐘內(nèi)癲癇樣放電(SLEs)的次數(shù)和總時間,GW1100增加了SLEs的次數(shù)和總時間。2.PTZ慢性點燃模型中,與對照組相比,GW9508降低了各時間點的平均發(fā)作分級,提高了點燃過程的生存率;GW1100顯示了相反的結(jié)果。結(jié)論:通過兩種動物模型證實,在癲癇發(fā)生發(fā)展過程中,干預(yù)GPR40可影響癲癇發(fā)作,激動劑使癲癇發(fā)作活性減輕,抑制劑使癲癇發(fā)作活性增加。第三部分GPR40對突觸傳遞的影響目的:利用全細胞膜片鉗技術(shù),探討GPR40對突觸傳遞的影響。方法:制備小鼠腦片,利用全細胞膜片鉗技術(shù)記錄海馬CA1區(qū)錐體神經(jīng)元的神經(jīng)電生理,包括動作電位(AP),微小興奮性突觸后電流(m EPSC)、記錄微小抑制性突觸后電流(m IPSC),AMPA/NMDA比率以及成對脈沖比例(PPR)。結(jié)果:與基線水平相比,GW9508降低了動作電位的頻率,GW1100增加了動作電位的頻率;GW9508降低了m EPSC的頻率和幅值,GW1100增加了m EPSC的頻率和幅值;GPR40干預(yù)對m IPSC的頻率和幅值的影響無顯著差異。AMPA/NMDA比值結(jié)果發(fā)現(xiàn),GW9508降低了NMDA受體介導(dǎo)電流的幅度,GW1100增加了NMDA受體介導(dǎo)電流的幅度;GPR40干預(yù)對AMPA受體介導(dǎo)的電流幅度的影響無顯著差異。PPR在各組間無顯著差異。結(jié)論:GPR40影響NMDA受體介導(dǎo)的興奮性突觸后傳遞。第四部分GPR40在癲癇發(fā)作中的作用機制目的:通過了解GPR40對NMDA受體的調(diào)節(jié)作用,以探討GPR40調(diào)節(jié)癲癇發(fā)作的機制。方法:1.在動物實驗后的腦組織中,免疫印跡法檢測NMDA受體NR2A和NR2B的總蛋白和膜蛋白的表達變化。2.免疫熒光檢測GPR40與NR2A和NR2B的共定位,免疫共沉淀檢測GPR40與NR2A和NR2B相互作用,并采用定量免疫共沉淀檢測GPR40對這種相互作用的影響。結(jié)果:1.在海人酸和PTZ實驗后的腦組織中,免疫印跡結(jié)果顯示,GW9508組NR2A和NR2B的膜蛋白/總蛋白比值顯著減低,GW1100組膜蛋白/總蛋白比值明顯升高;NR2A和NR2B的總蛋白表達量無顯著變化。2.免疫熒光顯示GPR40與NR2A和NR2B共定位;免疫共沉淀結(jié)果顯示GPR40與NR2A和NR2B相互作用;定量免疫共沉淀結(jié)果顯示,與對照相比,GW9508使這種結(jié)合減弱,GW1100使這種結(jié)合增強。結(jié)論:GPR40調(diào)節(jié)NR2A和NR2B的神經(jīng)元細胞膜上的表達;這種改變可能是由于影響了GPR40與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.

【學(xué)位授予單位】：重慶醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R742.1

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