miRNA-137在癫痫中的表达和功能研究

发布时间：2018-01-19 21:21

本文关键词： miRNA-137 癫痫 agomir antagomir mIPSCs　出处：《重庆医科大学》2016年博士论文　论文类型：学位论文

【摘要】：第一部分:miRNA-137在颞叶癫痫患者及模型动物脑组织中的表达研究目的:研究miRNA-137在颞叶癫痫患者术后脑组织中的表达以及在小鼠匹罗卡品癫痫模型和戊四氮慢性点燃癫痫模型脑组织中的表达特点。方法:1.从课题组已经建立的难治性颞叶癫痫患者术后脑组织库中随机抽取18例颞叶脑组织标本作为癫痫组,抽取12例年龄性别匹配的颅脑外伤患者术后颞叶脑组织标本作为对照组。2.选择成年雄性C57BL/6小鼠,由腹腔给予匹罗卡品或者戊四氮构建癫痫慢性期动物模型,选择自发性发作(或完全点燃)的小鼠作为癫痫组,未自发性发作(或未点燃)的小鼠为对照组。3.用荧光定量PCR技术检测miRNA-137在颞叶癫痫患者及癫痫模型小鼠脑组织标本中的表达变化。结果:1.miRNA-137在颞叶癫痫患者脑组织中的表达水平较对照组明显降低(P0.05);2.在匹罗卡品癫痫慢性期小鼠模型中,miRNA-137在有自发性发作的小鼠的海马和皮质中的表达水平均较无自发性发作的小鼠降低(p0.05);3.在戊四氮慢性点燃癫痫小鼠模型中,mirna-137在完全点燃小鼠的海马和皮质中的表达水平均较未点燃的小鼠降低(p0.05)。结论:mirna-137在颞叶癫痫患者和癫痫模型小鼠脑组织中的表达水平均降低,提示mirna-137可能与癫痫的发生发展过程具有密切关系。第二部分mirna-137体内干预对癫痫动物模型行为学的影响目的:利用mirna-137特异性激动剂agomir和抑制剂antagomir对小鼠进行海马区微量注射干预,研究mirna-137在脑内表达水平的变化对癫痫模型小鼠行为学的影响。方法:1.选择成年雄性c57bl/6小鼠,随机分为五组,分别为对照组(control组),agomirnc组,agomir组,antagomirnc组和antagomir组。各组分别给予生理盐水,agomirscramblednc0.2nmol,agomir0.2nmol,antagomirscramblednc0.8nmol以及antagomir0.8nmol海马区双侧立体定位微量注射。2.采用荧光定量pcr技术和激光共聚焦技术检测agomir及antagomir海马立体定位注射后的干预效率。3.腹腔注射匹罗卡品(320mg/kg)建立匹罗卡品癫痫模型,观察各组小鼠首次自发性发作的潜伏期和发作的次数;每日给予腹腔注射阈下剂量(35mg/kg)的戊四氮建立戊四氮慢性点燃癫痫模型,观察各组小鼠完全点燃所需的时间和痫性发作的级别。结果:1.mirna-137agomir海马立体定位注射干预后,mirna-137的表达水平升高。与control组相比,其在干预后3天,1周,2周及4周组的表达均升高(p0.05);antagomir海马注射干预后,海马区mirna-137的表达水平降低。与control组相比,其在干预后3天,1周,2周及4周组的表达均显著降低(p0.05)。激光共聚焦结果显示,agomir及antagomir干预后,海马区可见带有绿色荧光的干预剂表达;2.在匹罗卡品癫痫模型中,agomir组的首次自发性发作的潜伏期较control组和agomirnc组延长,自发性发作的次数较control组和agomirnc组降低;与control组和antagomirnc组相比,首次自发性发作的潜伏期在antagomir组明显缩短,自发性发作的次数较control组和antagomirnc组增加(p0.05);3.在戊四氮慢性点燃模型中,与control组和agomirnc组相比,agomir组完全点燃所需要的时间明显延长(p0.05)。agomir组痫性发作级别在第5-12天时较control组降低,其差异具有统计学意义(p0.05);antagomir组完全点燃所需的时间较control组和antagomirnc组缩短,而其痫性发作级别在第4-9天时较control组升高,这些差异同样具有统计学意义(p0.05)。结论:1、mirna-137agomir干预可特异性增加海马mirna-137的表达水平;mirna-137antagomir干预能够特异性抑制海马mirna-137的表达。2、mirna-137体内干预可以引起癫痫模型小鼠发作潜伏期和严重程度的改变。第三部分mirna-137对小鼠海马神经元兴奋性的影响目的:利用mirna-137特异性激动剂agomir和抑制剂antagomir对小鼠进行海马区立体定位微量注射干预,探讨mirna-137在脑内表达水平的变化对小鼠海马脑片椎体神经元兴奋性的影响。方法:1.选择健康雄性c57bl/6小鼠,随机分为五组:对照组(control组),agomirnc组,agomir组,antagomirnc组和antagomir组,并通过海马区立体定位微量注射的方式给与相应的干预。2.用全细胞膜片钳技术记录各组小鼠海马脑片ca3区椎体神经元在无镁脑脊液灌流下所诱发的动作电位(ap),微小兴奋性突触后电流(mepscs),微小抑制性突触后电流(mipscs)以及配对脉冲比率(ppr)的变化情况。结果:1.agomir组海马区椎体神经元ap的放电频率较control组和agomirnc组均降低,而antagomir组的ap放电频率较control组和antagomirnc组均升高(p0.05);2.与control组和agomir NC组相比,海马椎体神经元m IPSCs的放电频率在agomir组升高,而antagomir组mIPSCs放电的频率较control组和antagomir NC组均降低(P0.05),它们的差异具有统计学意义。另一方面,mIPSCs放电的幅值在各组之间并无明显统计学差异(P0.05);3.agomir组和antagomir组海马椎体神经元EPSCs的放电频率和幅值均较对照组无明显差异(P0.05);4.agomir组海马区椎体神经元的PPR值较control组明显降低(P0.05)。结论:1、miRNA-137体内干预可对小鼠海马脑片椎体神经元的兴奋性产生负性调控效应。2、miRNA-137体内干预可通过影响突触前抑制性神经递质的释放,来调控小鼠海马脑片椎体神经元抑制性突触后电流放电频率。
[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.

【学位授予单位】：重庆医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R742.1

【相似文献】