本文關(guān)鍵詞： miRNA-137 癲癇 agomir antagomir mIPSCs　出處：《重慶醫(yī)科大學(xué)》2016年博士論文　論文類型：學(xué)位論文

【摘要】：第一部分:miRNA-137在顳葉癲癇患者及模型動物腦組織中的表達(dá)研究目的:研究miRNA-137在顳葉癲癇患者術(shù)后腦組織中的表達(dá)以及在小鼠匹羅卡品癲癇模型和戊四氮慢性點燃癲癇模型腦組織中的表達(dá)特點。方法:1.從課題組已經(jīng)建立的難治性顳葉癲癇患者術(shù)后腦組織庫中隨機(jī)抽取18例顳葉腦組織標(biāo)本作為癲癇組,抽取12例年齡性別匹配的顱腦外傷患者術(shù)后顳葉腦組織標(biāo)本作為對照組。2.選擇成年雄性C57BL/6小鼠,由腹腔給予匹羅卡品或者戊四氮構(gòu)建癲癇慢性期動物模型,選擇自發(fā)性發(fā)作(或完全點燃)的小鼠作為癲癇組,未自發(fā)性發(fā)作(或未點燃)的小鼠為對照組。3.用熒光定量PCR技術(shù)檢測miRNA-137在顳葉癲癇患者及癲癇模型小鼠腦組織標(biāo)本中的表達(dá)變化。結(jié)果:1.miRNA-137在顳葉癲癇患者腦組織中的表達(dá)水平較對照組明顯降低(P0.05);2.在匹羅卡品癲癇慢性期小鼠模型中,miRNA-137在有自發(fā)性發(fā)作的小鼠的海馬和皮質(zhì)中的表達(dá)水平均較無自發(fā)性發(fā)作的小鼠降低(p0.05);3.在戊四氮慢性點燃癲癇小鼠模型中,mirna-137在完全點燃小鼠的海馬和皮質(zhì)中的表達(dá)水平均較未點燃的小鼠降低(p0.05)。結(jié)論:mirna-137在顳葉癲癇患者和癲癇模型小鼠腦組織中的表達(dá)水平均降低,提示mirna-137可能與癲癇的發(fā)生發(fā)展過程具有密切關(guān)系。第二部分mirna-137體內(nèi)干預(yù)對癲癇動物模型行為學(xué)的影響目的:利用mirna-137特異性激動劑agomir和抑制劑antagomir對小鼠進(jìn)行海馬區(qū)微量注射干預(yù),研究mirna-137在腦內(nèi)表達(dá)水平的變化對癲癇模型小鼠行為學(xué)的影響。方法:1.選擇成年雄性c57bl/6小鼠,隨機(jī)分為五組,分別為對照組(control組),agomirnc組,agomir組,antagomirnc組和antagomir組。各組分別給予生理鹽水,agomirscramblednc0.2nmol,agomir0.2nmol,antagomirscramblednc0.8nmol以及antagomir0.8nmol海馬區(qū)雙側(cè)立體定位微量注射。2.采用熒光定量pcr技術(shù)和激光共聚焦技術(shù)檢測agomir及antagomir海馬立體定位注射后的干預(yù)效率。3.腹腔注射匹羅卡品(320mg/kg)建立匹羅卡品癲癇模型,觀察各組小鼠首次自發(fā)性發(fā)作的潛伏期和發(fā)作的次數(shù);每日給予腹腔注射閾下劑量(35mg/kg)的戊四氮建立戊四氮慢性點燃癲癇模型,觀察各組小鼠完全點燃所需的時間和癇性發(fā)作的級別。結(jié)果:1.mirna-137agomir海馬立體定位注射干預(yù)后,mirna-137的表達(dá)水平升高。與control組相比,其在干預(yù)后3天,1周,2周及4周組的表達(dá)均升高(p0.05);antagomir海馬注射干預(yù)后,海馬區(qū)mirna-137的表達(dá)水平降低。與control組相比,其在干預(yù)后3天,1周,2周及4周組的表達(dá)均顯著降低(p0.05)。激光共聚焦結(jié)果顯示,agomir及antagomir干預(yù)后,海馬區(qū)可見帶有綠色熒光的干預(yù)劑表達(dá);2.在匹羅卡品癲癇模型中,agomir組的首次自發(fā)性發(fā)作的潛伏期較control組和agomirnc組延長,自發(fā)性發(fā)作的次數(shù)較control組和agomirnc組降低;與control組和antagomirnc組相比,首次自發(fā)性發(fā)作的潛伏期在antagomir組明顯縮短,自發(fā)性發(fā)作的次數(shù)較control組和antagomirnc組增加(p0.05);3.在戊四氮慢性點燃模型中,與control組和agomirnc組相比,agomir組完全點燃所需要的時間明顯延長(p0.05)。agomir組癇性發(fā)作級別在第5-12天時較control組降低,其差異具有統(tǒng)計學(xué)意義(p0.05);antagomir組完全點燃所需的時間較control組和antagomirnc組縮短,而其癇性發(fā)作級別在第4-9天時較control組升高,這些差異同樣具有統(tǒng)計學(xué)意義(p0.05)。結(jié)論:1、mirna-137agomir干預(yù)可特異性增加海馬mirna-137的表達(dá)水平;mirna-137antagomir干預(yù)能夠特異性抑制海馬mirna-137的表達(dá)。2、mirna-137體內(nèi)干預(yù)可以引起癲癇模型小鼠發(fā)作潛伏期和嚴(yán)重程度的改變。第三部分mirna-137對小鼠海馬神經(jīng)元興奮性的影響目的:利用mirna-137特異性激動劑agomir和抑制劑antagomir對小鼠進(jìn)行海馬區(qū)立體定位微量注射干預(yù),探討mirna-137在腦內(nèi)表達(dá)水平的變化對小鼠海馬腦片椎體神經(jīng)元興奮性的影響。方法:1.選擇健康雄性c57bl/6小鼠,隨機(jī)分為五組:對照組(control組),agomirnc組,agomir組,antagomirnc組和antagomir組,并通過海馬區(qū)立體定位微量注射的方式給與相應(yīng)的干預(yù)。2.用全細(xì)胞膜片鉗技術(shù)記錄各組小鼠海馬腦片ca3區(qū)椎體神經(jīng)元在無鎂腦脊液灌流下所誘發(fā)的動作電位(ap),微小興奮性突觸后電流(mepscs),微小抑制性突觸后電流(mipscs)以及配對脈沖比率(ppr)的變化情況。結(jié)果:1.agomir組海馬區(qū)椎體神經(jīng)元ap的放電頻率較control組和agomirnc組均降低,而antagomir組的ap放電頻率較control組和antagomirnc組均升高(p0.05);2.與control組和agomir NC組相比,海馬椎體神經(jīng)元m IPSCs的放電頻率在agomir組升高,而antagomir組mIPSCs放電的頻率較control組和antagomir NC組均降低(P0.05),它們的差異具有統(tǒng)計學(xué)意義。另一方面,mIPSCs放電的幅值在各組之間并無明顯統(tǒng)計學(xué)差異(P0.05);3.agomir組和antagomir組海馬椎體神經(jīng)元EPSCs的放電頻率和幅值均較對照組無明顯差異(P0.05);4.agomir組海馬區(qū)椎體神經(jīng)元的PPR值較control組明顯降低(P0.05)。結(jié)論:1、miRNA-137體內(nèi)干預(yù)可對小鼠海馬腦片椎體神經(jīng)元的興奮性產(chǎn)生負(fù)性調(diào)控效應(yīng)。2、miRNA-137體內(nèi)干預(yù)可通過影響突觸前抑制性神經(jīng)遞質(zhì)的釋放,來調(diào)控小鼠海馬腦片椎體神經(jīng)元抑制性突觸后電流放電頻率。
[Abstract]:The first part: Objective To study the expression of miRNA-137 in temporal lobe epilepsy patients and animal models of brain tissue: the expression of miRNA-137 in brain tissue of patients with temporal lobe epilepsy in mice and in pilocarpine induced epilepsy model and e four n chronic kindling expression characteristics of brain tissue in the epilepsy model. Methods: 1. from the research group has been established intractable temporal lobe epilepsy in patients with brain tissue Library in a random sample of 18 patients with temporal lobe brain tissue specimens as the epilepsy group, selected 12 cases of craniocerebral trauma patients with age and gender matched after temporal lobe brain tissue samples as control group.2. adult male C57BL/6 mice, pilocarpine or e four nitrogen construction of chronic epilepsy animal the model given by intraperitoneal, selection of spontaneous seizures (or fully lit) mice as the epilepsy group, no spontaneous seizures (or unlit) mice as the control group by.3. fluorescence quantitative PCR detection technology M Expression of iRNA-137 in brain tissue of patients with temporal lobe epilepsy and epileptic mice specimens. Results: the expression level of 1.miRNA-137 in brain tissue of patients with temporal lobe epilepsy was lower than that in the control group (P0.05); 2. in the chronic phase of pilocarpine induced epilepsy model in mice, the expression level of miRNA-137 in the hippocampus of mice and spontaneous seizures in the cortex were lower than that of non spontaneous seizures in mice (P0.05); 3. in e four n chronic kindling model in mice, reduce the mirna-137 expression level in fully kindled mice in hippocampus and cortex were unignited mice (P0.05). Conclusion: the expression level of mirna-137 in patients with temporal lobe epilepsy and epilepsy model in the brain of mice were reduced, suggesting that mirna-137 may be the occurrence and development of epilepsy and has a close relationship. The second part of the mirna-137 in vivo intervention effects on the behavior of the epilepsy animal model to study: The use of mirna-137 specific activator agomir and inhibitor antagomir in the hippocampus of mice by microinjection of intervention, effect of the change of the expression of mirna-137 in brain of epileptic behavior model mice. Methods: 1. adult male c57bl/6 mice were randomly divided into five groups, including control group (control group), agomirnc group. Agomir group, antagomirnc group and antagomir group. Each group were given normal saline, agomirscramblednc0.2nmol, agomir0.2nmol, antagomirscramblednc0.8nmol and antagomir0.8nmol in hippocampus of bilateral stereotactic microinjection of.2. by fluorescence quantitative PCR and confocal laser technology to detect the hippocampal agomir and antagomir stereotactic injection intervention after intraperitoneal injection of pilocarpine.3. efficiency (320mg/kg) of pilocarpine induced epilepsy the model mice were observed for the first time, spontaneous seizure latency and the number of attacks; Daily intraperitoneal injection of subthreshold dose (35mg/kg) of e four e four nitrogen nitrogen to establish chronic kindling model mice were observed, and the time required for the fully kindled seizures level. Results: 1.mirna-137agomir hippocampal stereotaxic injection intervention, increased the expression level of mirna-137. Compared with the control group, after intervention. 3 days, 1 weeks, 2 weeks and 4 weeks expression group were significantly increased (P0.05); antagomir after injection of intervention, the expression level of mirna-137 in hippocampus decreased. Compared with control group, the intervention after 3 days, 1 weeks, 2 weeks and 4 weeks group was significantly decreased (P0.05) confocal laser. Results showed that agomir and antagomir intervention, hippocampus with green fluorescent agent intervention expression; 2. in the pilocarpine model of epilepsy, agomir group for the first time the latency to spontaneous seizures compared with control group and agomirnc group prolonged, the number of spontaneous seizures Compared with control group and agomirnc group decreased; compared with control group and antagomirnc group for the first time, spontaneous seizures was significantly shortened in group antagomir, the number of spontaneous seizures increased compared with control group and antagomirnc group (P0.05); 3. in e four n chronic kindling model, compared with control group and agomirnc group, agomir group fully lit the time required was significantly prolonged (P0.05) group.Agomir seizure level lower than that of control group in the first 5-12 days, the difference was statistically significant (P0.05); group antagomir was required for ignition time than control group and antagomirnc group were shortened, and its seizure level was higher than that in control group at day 4-9. When these differences were also statistically significant (P0.05). Conclusion: 1. Mirna-137agomir intervention can specifically increase the expression level of mirna-137 in hippocampus; mirna-137antagomir intervention can specifically inhibit hippocampal miRNA-1 The expression of.2 37, mirna-137 model mice in vivo intervention can cause epilepsy seizure latency and severity of the change. The third part: the effect of mirna-137 on the excitability of hippocampal neurons in mice Objective: using mirna-137 specific activator agomir and inhibitor antagomir in hippocampus of stereotactic microinjection intervention on mice, to investigate the change of the expression of mirna-137 in brain effect on the excitability of pyramidal neurons in mouse hippocampal slices. Methods: 1. healthy male c57bl/6 mice were randomly divided into five groups: control group (control group), agomirnc group, agomir group, antagomirnc group and antagomir group, and the hippocampus by stereotaxic microinjection of ways to give the corresponding intervention.2. using whole cell the patch clamp technique to record vertebral mice hippocampus CA3 neurons in the magnesium free action potential induced by cerebrospinal fluid perfusion (AP), miniature excitatory Postsynaptic currents (mEPSCs), minimal inhibitory postsynaptic currents (mIPSCs) and paired pulse ratio (PPR) were observed. Results: the discharge frequency of the 1.agomir ertebra group of hippocampal neurons in AP than in control group and agomirnc group were decreased, and the discharge frequency of AP in antagomir group compared with control group and antagomirnc group were higher (P0.05; 2.) compared with control group and agomir NC group, the discharge frequency of m in hippocampal pyramidal neurons of IPSCs increased in agomir group, antagomir group and mIPSCs discharge frequency compared with control group and antagomir NC group were lower (P0.05), with significant difference between them. On the other hand, the amplitude of mIPSCs discharge in each group. There was no statistically significant difference (P0.05); the discharge frequency and the amplitude of 3.agomir group and antagomir group in hippocampal pyramidal neurons of EPSCs were lower than those in the control group had no significant difference (P0.05); 4.agomir group in hippocampus neurons of the sea vertebral PPR value is Co The ntrol group was significantly lower (P0.05). Conclusion: 1. MiRNA-137 in the intervention can produce a negative regulatory effect of.2 on excitatory pyramidal neurons in mouse hippocampal slices, miRNA-137 in vivo intervention can affect presynaptic inhibition of neurotransmitter release in mouse hippocampal slices to control vertebral body neuron inhibitory postsynaptic current discharge frequency.

【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】：2016
【分類號】：R742.1

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