EphB3在癫痫发作和形成中的作用及其可能机制研究
发布时间：2018-08-18 08:23
【摘要】：癫痫是一种慢性反复发作的严重的神经系统常见疾病之一。尽管目前各种各样的抗癫痫药物在临床上使用,但由于其发病机理尚不清楚,仍有20-30%的癫痫患者症状得不到有效控制。因此,对癫痫发病机制的深入研究,有益于指导癫痫的预防和治疗。越来越多的研究表明异常的神经网络在癫痫发病中起着重要作用。属于酪氨酸家族成员的EphB/ephrinB系统,是一类神经轴突导向因子,他们相互作用在中枢神经系统内神经元轴突导向、特异性突触连接、神经元树突结构等方面具有非常重要作用。本课题主要研究EphB/ephrinB各亚型在临床难治性颞叶癫痫患者和癫痫大鼠模型中的表达,以求找出可能与癫痫形成有关的亚型;并在无镁诱导的离体海马脑片癫痫模型和两种经典的慢性癫痫动物模型上,应用功能性重组融合蛋白和慢病毒介导的基因转染技术进行干预,通过电生理学、分子生物学、动物行为学以及形态学检测,探讨其在癫痫发作和形成过程中的作用和可能机制,为癫痫发病机制提供新思路和新途径,以及为临床上寻找抗癫痫治疗新靶点和新的抗癫痫药物研发提供实验依据。第一部分EphB/ephrinB在难治性颞叶癫痫患者及大鼠癫痫模型中的表达研究目的:检测EphB/ephrinB各亚型在临床难治性颞叶癫痫患者和氯化锂-匹罗卡品诱导的癫痫大鼠脑组织中的表达,以求找出可能与癫痫形成有关的EphB/ephrinB亚型。方法:1.从第三军医大学附属第二医院(新桥医院)神经外科脑标本库中随机收集20例难治性颞叶癫痫患者和20例严重颅脑外伤需要去骨瓣减压手术治疗的对照患者手术后颞叶皮层标本。2.SD成年雄性大鼠随机分成正常对照组和癫痫模型组(每组10只),模型组给与氯化锂-匹罗卡品腹腔注射诱导癫痫模型。3.用RT-qPCR检测EphB/ephrinB各亚型在临床标本和动物模型上的表达,筛选出mRNA表达一致的亚型,然后用Westernblot和免疫组织化学染色验证其蛋白表达,接着进一步用Westernblot检测其功能性磷酸化蛋白水平,并用免疫荧光双标对其细胞表达进行定位。结果:1.在癫痫患者和癫痫大鼠脑组织中EphB3和ephrinB3mRNA表达一致性升高(p0.05),而EphBl/2/4/6和ephrinl/2mRNA表达趋势不一致。2.Westernblot和免疫组织化学染色证实EphB3和ephrinB3蛋白表达在癫痫标本中一致性升高(p0.05)。3.功能性p-EphB3蛋白表达在癫痫标本中升高(p0.05),而p-ephrinB蛋白表达在癫痫标本中没有改变(p0.05)。4.免疫荧光双标提示EphB3在癫痫标本中主要表达于神经元胞质和胞膜。结论:EphB3和p-EphB3在难治性颞叶癫痫患者和氯化锂-匹罗卡品诱导的癫痫大鼠脑组织中表达升高,提示EphB3可能与癫痫的形成有关。第二部分EphB3功能改变对大鼠海马脑片神经元兴奋性的影响目的:为了探讨EphB3对癫痫发作的影响,我们在无镁诱导的离体海马脑片癫痫模型上,用全细胞膜片钳检测EphB3功能改变对海马神经元兴奋性的影响。方法:健康雄性SD大鼠麻醉后直接断头取脑制作海马脑片,在正常人工脑脊液灌流基础上,换成无镁人工脑脊液诱导离体脑片癫痫模型,然后将EphB3活性调节剂功能性重组融合蛋白(抑制剂EphB3-Fc、激活剂ephrinB3-Fc)和对照Fc加入灌流液中进行干预,用全细胞膜片钳检测各干预前后神经元动作电位频率。结果:无镁人工脑脊液可明显升高神经元动作电位频率(p0.05);EphB3-Fc(10nm和100nm)可降低无镁诱导的神经元动作电位频率(p0.05);ephrinB3-Fc(10nm和100nm)可升高无镁诱导的神经元动作电位频率(p0.05);而Fc(10nm和100nm)对无镁诱导的神经元动作电位频率没有影响(p0.05)。融合蛋白(Fc、EphB3-Fc和ephrinB3-Fc10nm)对正常人工脑脊液环境下的神经元动作电位频率没有影响(p0.05)。结论:无镁人工脑脊液可诱导离体海马脑片癫痫模型。抑制EphB3活性可降低无镁诱导的神经元兴奋性,激活EphB3活性可升高无镁诱导的神经元兴奋性,提示EphB3功能改变可影响癫痫的发作。第三部分EphB3功能改变对癫痫大鼠行为学及下游相关RhoGEF基因表达的影响目的:为了进一步明确EphB3对癫痫发作和形成的影响,我们在两种经典慢性癫痫动物模型上,观察EphB3功能改变对大鼠行为学、海马齿状回区苔藓纤维出芽、内分子层树突棘密度和EphB3下游相关RhoGEF基因表达的影响。方法:健康雄性SD大鼠侧脑室置管后恢复一周,随机分成单纯功能性重组融合蛋白干预、氯化锂-匹罗卡品诱导癫痫模型和戊四氮点燃癫痫模型三个大组。单纯功能性重组融合蛋白干预组大鼠又随机分成ephrinB3-Fc组、EphB3-Fc组、对照Fc组和正常对照(PBS)组(每组5只),分别用ephrinB3-Fc、EphB3-Fc、Fc和PBS通过侧脑室置管注射连续干预一周,于干预后第7、14、30和60天麻醉后断头取脑,用Westernblot检测各组海马组织中EphB3和p-EphB3蛋白表达。癫痫模型组大鼠又随机分成4个亚组,即模型对照(PBS)组、模型+对照Fc组、模型+ephrinB3-Fc组和模型+EphB3-Fc组(每组10只),通过侧脑室置管分别注射等量无菌PBS和融合蛋白(Fc、ephrinB3-Fc和EphB3-Fc)连续干预一周后造模。氯化锂-匹罗卡品模型观察大鼠急性发作程度、急性潜伏期和慢性期自发性发作次数,戊四氮模型组观察大鼠每天发作程度和完全被点燃的时间。应用Timm染色检测癫痫大鼠海马齿状回苔藓纤维出芽情况,高尔基染色检测癫痫大鼠海马齿状回内分子层区树突棘密度,RT-qPCR和Westernblot检测癫痫大鼠海马组织中EphB3下游相关RhoGEF基因(Kalirin、Intersectin1和Intersectin2)表达情况。结果:1.EphB3-Fc可长时间(7-60d)降低大鼠海马组织p-EphB3蛋白表达(p0.05),ephrinB3-F可长时间(7-60d)增加大鼠海马组织p-EphB3蛋白表达(p0.05),而Fc对大鼠海马组织p-EphB3蛋白表达没有影响(p0.05)。2.EphB3-Fc可降低氯化锂-匹罗卡品癫痫大鼠的急性发作级别、延长急性潜伏期和减少慢性期自发性发作次数(p0.05),ephrinB3-Fc则可加重氯化锂-匹罗卡品癫痫大鼠的急性发作级别、缩短急性潜伏期和增加慢性期自发性发作次数(p0.05)。同时EphB3-Fc可降低戊四氮癫痫大鼠的发作级别(11-33d)和延长发作潜伏期(p0.05),ephrinB3-Fc则可加重戊四氮癫痫大鼠的发作级别(9-29d)和缩短发作潜伏期(p0.05),Fc对氯化锂-匹罗卡品和戊四氮癫痫大鼠的上述行为学没有影响(p0.05)。3.EphB3-Fc可减轻癫痫大鼠海马齿状回区苔藓纤维出芽程度和降低内分子层树突棘密度,ephrinB3-Fc则加重癫痫大鼠海马齿状回区苔藓纤维出芽程度和增加内分子层树突棘密(p0.05),Fc对癫痫大鼠海马齿状回区苔藓纤维出芽程度和内分子层树突棘密没有影响(p0.05)。4.功能性重组融合蛋白对癫痫大鼠海马组织Intersectin1和Intersectin2mRNA表达没有影响(p0.05)。EphB3-Fc可降低癫痫大鼠海马组织中KalirinmRNA和蛋白表达,ephrinB3-Fc则增加癫痫大鼠海马组织KalirinmRNA和蛋白表达(p0.05),而Fc对癫痫大鼠海马组织KalirinmRNA和蛋白表达没有影响(p0.05)。结论:1.功能性重组融合蛋白(ephrinB3-Fc和EphB3-Fc)可长时间(7-60d)影响EphB3功能。2.EphB3功能改变可影响癫痫大鼠急性发作程度、潜伏期长短以及慢性期自发性发作频率。3.EphB3功能改变可调节癫痫大鼠海马区苔藓纤维出芽和内分子层树突棘密度。4.Kalirin可能为EphB3潜在的下游作用靶点。第四部分EphB3通过Kalirin参与癫痫的发作与形成目的:为了探索EphB3在癫痫发作和形成中的可能机制,我们在两种经典慢性癫痫动物模型上,用慢病毒介导Kalirin-shRNA沉默Kalirin基因后,观察激活EphB3功能对大鼠行为学、海马齿状回区苔藓纤维出芽、内分子层树突棘密度的影响,以明确EphB3是否是通过Kalirin参与癫痫的发作与形成。方法:用Westernblot检测难治性颞叶癫痫患者和癫痫大鼠脑组织中Kalirin蛋白表达。健康雄性SD大鼠随机分成Control、LV-GFP和LV-Kalirin-shRNA三组,分别通过侧脑室置管注射等量生理盐水、LV-GFP和LV-Kalirin-shRNA。一部分LV-Kalirin-shRNA干预组大鼠分别于注射后第3、7、14、30和60天(7天组大鼠10只,其余每组5只)麻醉后断头取脑,荧光显微镜下观察大鼠海马以及皮层绿色荧光表达,Westernblot检测海马Kalirin蛋白表达。另一部分大鼠(30只)于侧脑室置管注射生理盐水和慢病毒后一周,用氯化锂-匹罗卡品和戊四氮制备癫痫模型(每组大鼠5只)。观察各组大鼠行为学改变,Westernblot检测癫痫大鼠海马组织中EphB3和p-EphB3蛋白表达情况。剩余部分大鼠(30只)于侧脑室置管注射生理盐水和慢病毒一周后,继续通过侧脑室置管注射ephrinB3-Fc连续干预一周,然后用氯化锂-匹罗卡品和戊四氮制备癫痫模型(每组大鼠5只)。观察各组大鼠行为学改变、海马齿状回苔藓纤维出芽和内分子层区树突棘密度情况。结果:1.在难治性颞叶癫痫患者和癫痫大鼠脑组织中Kalirin蛋白表达升高(p0.05)。2.荧光显微镜下观察到慢病毒绿色荧光广泛分布于大鼠大脑皮层和海马区。LV-Kalirin-shRNA从注射后第7天开始有效抑制Kalirin蛋白表达,并持续至注射后的第60天(p0.05)。3.LV-Kalirin-shRNA降低氯化锂-匹罗卡品癫痫大鼠的急性发作级别、延长急性潜伏期和减少慢性期自发性发作次数(p0.05),同时也降低戊四氮癫痫大鼠的发作级别(9-35d)和延长发作潜伏期(p0.05)。LV-Kalirin-shRNA对癫痫大鼠海马组织EphB3和p-EphB3蛋白表达没有影响(p0.05)。4.沉默内源性的Kalirin能降低ephrinB3-Fc所引起的氯化锂-匹罗卡品癫痫大鼠的急性发作级别、急性潜伏期、慢性期自发性发作次数以及戊四氮癫痫大鼠的发作级别(11-29d)和发作潜伏期(p0.05)。5.沉默内源性的Kalirin能减轻和降低ephrinB3-Fc所引起的癫痫大鼠海马齿状回区苔藓纤维出芽程度和内分子层树突棘密度(p0.05)。结论:1.Kalirin在难治性TLE患者和癫痫大鼠脑组织中表达升高。2.沉默内源性Kalirin可减轻癫痫大鼠发作程度、延长潜伏期和减少慢性期大鼠自发性发作频率。3.沉默内源性Kalirin可逆转EphB3激活所引起的癫痫大鼠行为学和海马神经环路形态学改变。4.EphB3通过对RhoGEFKalirin的调节影响海马神经环路重建参与癫痫的发作和形成。
[Abstract]:Epilepsy is one of the most common chronic recurrent neurological diseases. Although various antiepileptic drugs are used in clinic, there are still 20-30% epileptic patients whose symptoms can not be effectively controlled because of the unclear pathogenesis. Therefore, in-depth study on the pathogenesis of epilepsy is helpful to guide epilepsy. Prevention and treatment. More and more studies have shown that abnormal neural networks play an important role in epilepsy. EphB/ephrinB system, a member of tyrosine family, is a class of neuronal axon-directing factors that interact with neuronal axon-directing, specific synaptic connections, and neuronal dendritic structures in the central nervous system. Masks play an important role in the development of intractable temporal lobe epilepsy and epilepsy in rats. EphB/ephrinB subtypes were used to identify potential epilepsy-related subtypes, and in magnesium-free hippocampal slice epilepsy model and two classical chronic epilepsy animal models. Sexual recombinant fusion protein and lentivirus-mediated gene transfection were used to intervene in the process of epileptic seizure and its possible mechanism by electrophysiology, molecular biology, animal behavior and morphology detection, which provided new ideas and new ways for epileptic pathogenesis and clinical anti-epileptic treatment. Objective: To detect the expression of EphB/ephrinB subtypes in the brain of patients with refractory temporal lobe epilepsy and epileptic rats induced by lithium-pilocarpine. Methods: 1. Twenty patients with refractory temporal lobe epilepsy and 20 control patients with severe craniocerebral trauma requiring decompressive craniotomy were randomly collected from the neurosurgical brain specimen bank of the Second Affiliated Hospital of the Third Military Medical University (Xinqiao Hospital) and compared with the control group. 2. SD Male rats were randomly divided into normal control group and epilepsy model group (10 rats in each group). The model group was given lithium chloride-pilocarpine intraperitoneally to induce epilepsy model. 3. The expression of EphB/ephrinB subtypes in clinical specimens and animal models was detected by RT-qPCR, and the mRNA subtypes with the same expression were screened out. The expression of ephB3 and ephrinB3 mRNA in the brain tissues of epileptic patients and epileptic rats was consistent with that of epileptic rats (p0.05), but the expression of EphBl/2/4/6 and ephrinl/2 mRNA tended to increase. Western blot and immunohistochemical staining confirmed that EphB3 and ephrinB3 protein expression in epileptic specimens increased consistently (p0.05). 3. Functional p-EphB3 protein expression in epileptic specimens increased (p0.05), while p-ephrinB protein expression in epileptic specimens did not change (p0.05). 4. Immunofluorescence double labeling suggested that EphB3 was present in epileptic specimens. EphB3 and p-EphB3 were mainly expressed in the cytoplasm and membrane of neurons. Conclusion: EphB3 and p-EphB3 were elevated in the brain tissues of refractory temporal lobe epilepsy patients and lithium-pilocarpine-induced epilepsy rats, suggesting that EphB3 may be related to the formation of epilepsy. To investigate the effect of EphB3 on seizures, we used whole-cell patch clamp technique to detect the effect of EphB3 on the excitability of hippocampal neurons in a magnesium-free hippocampal slice epilepsy model. The epilepsy model was induced by artificial cerebrospinal fluid (ACF), and then the functional recombinant fusion protein (inhibitor EphB3-Fc, activator ephrinB3-Fc) and the control Fc were added into the perfusion fluid for intervention. The action potential frequencies of neurons before and after intervention were detected by whole-cell patch clamp. Neuronal action potential frequency (p0.05); EphB3-Fc (10 nm and 100 nm) decreased Mg-free neuronal action potential frequency (p0.05); ephrinB3-Fc (10 nm and 100 nm) increased Mg-free neuronal action potential frequency (p0.05); Fc (10 nm and 100 nm) had no effect on Mg-free neuronal action potential frequency (p0.05). Fusion protein (Fc) EphB3-Fc and ephrinB3-Fc10nm had no effect on neuronal action potential frequencies in normal artificial cerebrospinal fluid environment (p0.05). Conclusion: Magnesium-free artificial cerebrospinal fluid can induce epilepsy model in isolated hippocampal slices. It is suggested that the changes of EphB3 function may influence the onset of epilepsy. Part III The effect of EphB3 function on the behavior and downstream rhoGEF gene expression in epileptic rats Objective: To further clarify the effect of EphB3 on seizures and formation, we observed the behavior of epileptic rats in two classical chronic epileptic animal models. Methods: Healthy male SD rats were randomly divided into three groups: single functional recombinant fusion protein intervention, lithium chloride-pilocarpine induced epilepsy model and pentylenetetrazol kindled epilepsy model. Rats in the simple functional recombinant fusion protein intervention group were randomly divided into ephrinB3-Fc group, EphB3-Fc group, control Fc group and normal control (PBS) group (5 rats in each group). EphrinB3-Fc, EphB3-Fc, Fc and PBS were injected into the lateral ventricle for one week. Brains were cut off at the 7th, 14th, 30th and 60th days after the intervention, and brain was taken out by Western blot. EphB3 and p-EphB3 protein expression were detected in the hippocampus of each group. Epileptic model rats were randomly divided into four subgroups: model control group (PBS), model + control group (Fc), model + ephrinB3-Fc and model + EphB3-Fc (10 rats in each group). The same amount of aseptic PBS and fusion protein (Fc, ephrinB3-Fc and EphB3-Fc) were injected into the lateral ventricle of rats respectively. Lithium chloride-pilocarpine model was used to observe the degree of acute attack, the number of spontaneous attacks in acute latency and chronic phase, and pentylenetetrazol model group was used to observe the degree of attack and the time of complete kindling. The dendritic spine density in the molecular layer of the dentate gyrus of the hippocampus of epileptic rats was measured. The expression of RhoGEF gene (Kalirin, Intersectin 1 and Intersectin2) downstream of EphB3 in the hippocampus of epileptic rats was detected by RT-qPCR and Western blot. Results: 1. EphB3-Fc could decrease the expression of p-EphB3 protein (p0.05) for a long time (7-60 days) in the hippocampus of epileptic rats. Time (7-60 days) increased the expression of p-EphB3 protein in rat hippocampus (p0.05), but Fc had no effect on the expression of p-EphB3 protein in rat hippocampus (p0.05). 2. EphB3-Fc could decrease the acute seizure grade, prolong the acute latency and reduce the number of spontaneous seizures in chronic phase (p0.05). EphrinB3-Fc could aggravate chlorine content in rat hippocampus. EphB3-Fc could decrease the onset grade (11-33 days) and prolong the onset latency (p0.05). EphrinB3-Fc could aggravate the onset grade (9-29 days) and shorten the onset of pentylenetetrazol epilepsy in rats. During the incubation period (p0.05), Fc had no effect on the above behavior of lithium chloride-pilocarpine and pentylenetetrazol epileptic rats (p0.05). 3. EphB3-Fc could reduce the degree of mossy fiber sprouting in hippocampal dentate gyrus and the density of dendritic spines in inner molecular layer of epileptic rats, while ephrinB3-Fc aggravated the degree and increase of mossy fiber sprouting in hippocampal dentate gyrus of epileptic rats. Fc had no effect on the degree of mossy fiber sprouting and dendritic dendritic dendrite density in hippocampal dentate gyrus of epileptic rats (p0.05). 4. Functional recombinant fusion protein had no effect on the expression of Intersectin1 and Intersectin2 mRNA in hippocampus of epileptic rats (p0.05). EphB3-Fc could decrease the expression of K in hippocampus of epileptic rats. Alarin mRNA and protein expression, ephrin B3-Fc increased Kalirin mRNA and protein expression in hippocampus of epileptic rats (p0.05), but Fc had no effect on Kalirin mRNA and protein expression in hippocampus of epileptic rats (p0.05). Conclusion: 1. Functional recombinant fusion proteins (ephrin B3-Fc and EphB3-Fc) can affect the function of EphB3 for a long time (7-60 days). EphB3 can regulate mossy fiber budding and dendritic spine density in hippocampus of epileptic rats. 4. Kalirin may be a potential downstream target of EphB3. Part IV EphB3 participates in epileptic attack and shape through Kalirin. AIM: To explore the possible mechanism of EphB3 in epileptic seizures and epilepsy formation, we observed the effects of activating EphB3 on rat behavior, mossy fiber budding and dendritic spine density in hippocampal dentate gyrus after Kalirin-shRNA silencing in two classical chronic epileptic animal models. Methods: The expression of Kalirin protein in brain tissues of refractory temporal lobe epilepsy patients and epileptic rats was detected by Western blot. Healthy male SD rats were randomly divided into three groups: control, LV-GFP and LV-Kalirin-shRNA, which were injected with normal saline, LV-GFP and LV-Kalir through lateral ventricle catheterization respectively. In-shRNA. Some LV-Kalirin-shRNA-treated rats were anesthetized at 3,7,14,30 and 60 days after injection (10 rats in 7-day group and 5 rats in each group). The expression of green fluorescence in hippocampus and cortex was observed under fluorescence microscope. The expression of Kalirin protein in hippocampus was detected by Western blot. Another part of rats (30 rats) were placed cannula in lateral ventricle. Epilepsy models were established with lithium chloride-pilocarpine and pentylenetetrazol (5 rats in each group) one week after injection of normal saline and lentiviruses. Behavioral changes were observed in each group. Expression of EphB3 and p-EphB3 protein in hippocampus of epileptic rats was detected by Western blot. The remaining 30 rats (30 rats) were injected with normal saline and lentiviruses in lateral ventricles. After one week, the epileptic model was established by injecting ephrinB3-Fc into the lateral ventricle for one week, then lithium chloride-pilocarpine and pentylenetetrazol were used to prepare the epileptic model (5 rats in each group). Behavioral changes, mossy fiber sprouting in the dentate gyrus and dendritic spine density in the inner molecular layer of the hippocampus were observed. LV-Kalirin-shRNA effectively inhibited the expression of Kalirin protein from the 7th day after injection and lasted until the 60th day after injection (p0.05). 3. LV-Kalirin-shRNA reduced chlorination. LV-Kalirin-shRNA had no effect on the expression of EphB3 and p-EphB3 protein in hippocampus of epileptic rats (p0.05). The silencing of endogenous Kalirin can reduce the acute epileptic grade, acute latency, the number of spontaneous seizures in chronic phase, the onset grade (11-29 days) and the onset latency (p0.05) of epileptic rats induced by ephrinB3-Fc. The silencing of endogenous Kalirin can alleviate and reduce the induction of ephrinB3-Fc. The degree of mossy fiber sprouting and dendritic spine density of inner molecular layer in dentate gyrus of hippocampus in epileptic rats (p0.05). Conclusion: 1. Kalirin in refractory TLE
【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
本文编号：2188920
[Abstract]:Epilepsy is one of the most common chronic recurrent neurological diseases. Although various antiepileptic drugs are used in clinic, there are still 20-30% epileptic patients whose symptoms can not be effectively controlled because of the unclear pathogenesis. Therefore, in-depth study on the pathogenesis of epilepsy is helpful to guide epilepsy. Prevention and treatment. More and more studies have shown that abnormal neural networks play an important role in epilepsy. EphB/ephrinB system, a member of tyrosine family, is a class of neuronal axon-directing factors that interact with neuronal axon-directing, specific synaptic connections, and neuronal dendritic structures in the central nervous system. Masks play an important role in the development of intractable temporal lobe epilepsy and epilepsy in rats. EphB/ephrinB subtypes were used to identify potential epilepsy-related subtypes, and in magnesium-free hippocampal slice epilepsy model and two classical chronic epilepsy animal models. Sexual recombinant fusion protein and lentivirus-mediated gene transfection were used to intervene in the process of epileptic seizure and its possible mechanism by electrophysiology, molecular biology, animal behavior and morphology detection, which provided new ideas and new ways for epileptic pathogenesis and clinical anti-epileptic treatment. Objective: To detect the expression of EphB/ephrinB subtypes in the brain of patients with refractory temporal lobe epilepsy and epileptic rats induced by lithium-pilocarpine. Methods: 1. Twenty patients with refractory temporal lobe epilepsy and 20 control patients with severe craniocerebral trauma requiring decompressive craniotomy were randomly collected from the neurosurgical brain specimen bank of the Second Affiliated Hospital of the Third Military Medical University (Xinqiao Hospital) and compared with the control group. 2. SD Male rats were randomly divided into normal control group and epilepsy model group (10 rats in each group). The model group was given lithium chloride-pilocarpine intraperitoneally to induce epilepsy model. 3. The expression of EphB/ephrinB subtypes in clinical specimens and animal models was detected by RT-qPCR, and the mRNA subtypes with the same expression were screened out. The expression of ephB3 and ephrinB3 mRNA in the brain tissues of epileptic patients and epileptic rats was consistent with that of epileptic rats (p0.05), but the expression of EphBl/2/4/6 and ephrinl/2 mRNA tended to increase. Western blot and immunohistochemical staining confirmed that EphB3 and ephrinB3 protein expression in epileptic specimens increased consistently (p0.05). 3. Functional p-EphB3 protein expression in epileptic specimens increased (p0.05), while p-ephrinB protein expression in epileptic specimens did not change (p0.05). 4. Immunofluorescence double labeling suggested that EphB3 was present in epileptic specimens. EphB3 and p-EphB3 were mainly expressed in the cytoplasm and membrane of neurons. Conclusion: EphB3 and p-EphB3 were elevated in the brain tissues of refractory temporal lobe epilepsy patients and lithium-pilocarpine-induced epilepsy rats, suggesting that EphB3 may be related to the formation of epilepsy. To investigate the effect of EphB3 on seizures, we used whole-cell patch clamp technique to detect the effect of EphB3 on the excitability of hippocampal neurons in a magnesium-free hippocampal slice epilepsy model. The epilepsy model was induced by artificial cerebrospinal fluid (ACF), and then the functional recombinant fusion protein (inhibitor EphB3-Fc, activator ephrinB3-Fc) and the control Fc were added into the perfusion fluid for intervention. The action potential frequencies of neurons before and after intervention were detected by whole-cell patch clamp. Neuronal action potential frequency (p0.05); EphB3-Fc (10 nm and 100 nm) decreased Mg-free neuronal action potential frequency (p0.05); ephrinB3-Fc (10 nm and 100 nm) increased Mg-free neuronal action potential frequency (p0.05); Fc (10 nm and 100 nm) had no effect on Mg-free neuronal action potential frequency (p0.05). Fusion protein (Fc) EphB3-Fc and ephrinB3-Fc10nm had no effect on neuronal action potential frequencies in normal artificial cerebrospinal fluid environment (p0.05). Conclusion: Magnesium-free artificial cerebrospinal fluid can induce epilepsy model in isolated hippocampal slices. It is suggested that the changes of EphB3 function may influence the onset of epilepsy. Part III The effect of EphB3 function on the behavior and downstream rhoGEF gene expression in epileptic rats Objective: To further clarify the effect of EphB3 on seizures and formation, we observed the behavior of epileptic rats in two classical chronic epileptic animal models. Methods: Healthy male SD rats were randomly divided into three groups: single functional recombinant fusion protein intervention, lithium chloride-pilocarpine induced epilepsy model and pentylenetetrazol kindled epilepsy model. Rats in the simple functional recombinant fusion protein intervention group were randomly divided into ephrinB3-Fc group, EphB3-Fc group, control Fc group and normal control (PBS) group (5 rats in each group). EphrinB3-Fc, EphB3-Fc, Fc and PBS were injected into the lateral ventricle for one week. Brains were cut off at the 7th, 14th, 30th and 60th days after the intervention, and brain was taken out by Western blot. EphB3 and p-EphB3 protein expression were detected in the hippocampus of each group. Epileptic model rats were randomly divided into four subgroups: model control group (PBS), model + control group (Fc), model + ephrinB3-Fc and model + EphB3-Fc (10 rats in each group). The same amount of aseptic PBS and fusion protein (Fc, ephrinB3-Fc and EphB3-Fc) were injected into the lateral ventricle of rats respectively. Lithium chloride-pilocarpine model was used to observe the degree of acute attack, the number of spontaneous attacks in acute latency and chronic phase, and pentylenetetrazol model group was used to observe the degree of attack and the time of complete kindling. The dendritic spine density in the molecular layer of the dentate gyrus of the hippocampus of epileptic rats was measured. The expression of RhoGEF gene (Kalirin, Intersectin 1 and Intersectin2) downstream of EphB3 in the hippocampus of epileptic rats was detected by RT-qPCR and Western blot. Results: 1. EphB3-Fc could decrease the expression of p-EphB3 protein (p0.05) for a long time (7-60 days) in the hippocampus of epileptic rats. Time (7-60 days) increased the expression of p-EphB3 protein in rat hippocampus (p0.05), but Fc had no effect on the expression of p-EphB3 protein in rat hippocampus (p0.05). 2. EphB3-Fc could decrease the acute seizure grade, prolong the acute latency and reduce the number of spontaneous seizures in chronic phase (p0.05). EphrinB3-Fc could aggravate chlorine content in rat hippocampus. EphB3-Fc could decrease the onset grade (11-33 days) and prolong the onset latency (p0.05). EphrinB3-Fc could aggravate the onset grade (9-29 days) and shorten the onset of pentylenetetrazol epilepsy in rats. During the incubation period (p0.05), Fc had no effect on the above behavior of lithium chloride-pilocarpine and pentylenetetrazol epileptic rats (p0.05). 3. EphB3-Fc could reduce the degree of mossy fiber sprouting in hippocampal dentate gyrus and the density of dendritic spines in inner molecular layer of epileptic rats, while ephrinB3-Fc aggravated the degree and increase of mossy fiber sprouting in hippocampal dentate gyrus of epileptic rats. Fc had no effect on the degree of mossy fiber sprouting and dendritic dendritic dendrite density in hippocampal dentate gyrus of epileptic rats (p0.05). 4. Functional recombinant fusion protein had no effect on the expression of Intersectin1 and Intersectin2 mRNA in hippocampus of epileptic rats (p0.05). EphB3-Fc could decrease the expression of K in hippocampus of epileptic rats. Alarin mRNA and protein expression, ephrin B3-Fc increased Kalirin mRNA and protein expression in hippocampus of epileptic rats (p0.05), but Fc had no effect on Kalirin mRNA and protein expression in hippocampus of epileptic rats (p0.05). Conclusion: 1. Functional recombinant fusion proteins (ephrin B3-Fc and EphB3-Fc) can affect the function of EphB3 for a long time (7-60 days). EphB3 can regulate mossy fiber budding and dendritic spine density in hippocampus of epileptic rats. 4. Kalirin may be a potential downstream target of EphB3. Part IV EphB3 participates in epileptic attack and shape through Kalirin. AIM: To explore the possible mechanism of EphB3 in epileptic seizures and epilepsy formation, we observed the effects of activating EphB3 on rat behavior, mossy fiber budding and dendritic spine density in hippocampal dentate gyrus after Kalirin-shRNA silencing in two classical chronic epileptic animal models. Methods: The expression of Kalirin protein in brain tissues of refractory temporal lobe epilepsy patients and epileptic rats was detected by Western blot. Healthy male SD rats were randomly divided into three groups: control, LV-GFP and LV-Kalirin-shRNA, which were injected with normal saline, LV-GFP and LV-Kalir through lateral ventricle catheterization respectively. In-shRNA. Some LV-Kalirin-shRNA-treated rats were anesthetized at 3,7,14,30 and 60 days after injection (10 rats in 7-day group and 5 rats in each group). The expression of green fluorescence in hippocampus and cortex was observed under fluorescence microscope. The expression of Kalirin protein in hippocampus was detected by Western blot. Another part of rats (30 rats) were placed cannula in lateral ventricle. Epilepsy models were established with lithium chloride-pilocarpine and pentylenetetrazol (5 rats in each group) one week after injection of normal saline and lentiviruses. Behavioral changes were observed in each group. Expression of EphB3 and p-EphB3 protein in hippocampus of epileptic rats was detected by Western blot. The remaining 30 rats (30 rats) were injected with normal saline and lentiviruses in lateral ventricles. After one week, the epileptic model was established by injecting ephrinB3-Fc into the lateral ventricle for one week, then lithium chloride-pilocarpine and pentylenetetrazol were used to prepare the epileptic model (5 rats in each group). Behavioral changes, mossy fiber sprouting in the dentate gyrus and dendritic spine density in the inner molecular layer of the hippocampus were observed. LV-Kalirin-shRNA effectively inhibited the expression of Kalirin protein from the 7th day after injection and lasted until the 60th day after injection (p0.05). 3. LV-Kalirin-shRNA reduced chlorination. LV-Kalirin-shRNA had no effect on the expression of EphB3 and p-EphB3 protein in hippocampus of epileptic rats (p0.05). The silencing of endogenous Kalirin can reduce the acute epileptic grade, acute latency, the number of spontaneous seizures in chronic phase, the onset grade (11-29 days) and the onset latency (p0.05) of epileptic rats induced by ephrinB3-Fc. The silencing of endogenous Kalirin can alleviate and reduce the induction of ephrinB3-Fc. The degree of mossy fiber sprouting and dendritic spine density of inner molecular layer in dentate gyrus of hippocampus in epileptic rats (p0.05). Conclusion: 1. Kalirin in refractory TLE
【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
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