N-甲基-D-天冬氨酸受体在癫痫轴突损害中的作用及其分子机制研究

发布时间：2018-03-14 00:46

本文选题：癫痫　切入点：轴突损害　出处：《重庆医科大学》2017年博士论文　论文类型：学位论文

【摘要】：第一部分癫痫患者及PTZ点燃癫痫大鼠模型脑组织中的轴突损害目的:探讨癫痫患者脑组织和戊四氮(PTZ)点燃癫痫大鼠模型脑组织中轴突损害的情况。方法:收集难治性癫痫患者手术脑组织标本,建立PTZ点燃癫痫大鼠模型并分离收集其脑白质,用Western blot检测难治性癫痫患者脑组织和PTZ腹腔注射1天、1周、2周、3周和4周的大鼠脑白质中高分子量神经细丝(NF-H)、淀粉样前体蛋白(APP)、总Tau蛋白、磷酸化Tau蛋白S396(p-Tau S396)和磷酸化Tau蛋白T231(p-Tau T231),并计算Tau蛋白磷酸化水平。结果:相比对照组,难治性癫痫患者脑组织中NF-H水平明显降低,APP水平升高,总Tau蛋白减少,Tau蛋白磷酸化水平也升高。PTZ点燃癫痫大鼠脑白质中也存在NF-H减少、APP水平升高、总Tau蛋白减少和Tau蛋白磷酸化水平升高,并且其程度随PTZ注射时间延长而逐渐加重。结论:难治性癫痫患者和PTZ点燃癫痫大鼠脑组织中均存在以NF-H减少、APP积聚和总Tau蛋白减少为表现的轴突损害,并同时伴有Tau蛋白磷酸化水平升高。第二部分NMDA受体、GSK-3β和Cdk5在癫痫轴突损害中的作用目的:探索NMDA受体、GSK-3β和Cdk5是否参与了癫痫中轴突损害的形成。方法:对大鼠每日腹腔注射PTZ共21天并成功建立点燃癫痫模型后,给予每日腹腔注射非选择性NMDA受体拮抗剂美金刚、NR2B亚单位选择性NMDA受体拮抗剂艾芬地尔、GSK-3β拮抗剂氯化锂、Cdk5拮抗剂Roscovitine或生理盐水共7天,并用免疫组织化学术检测大鼠胼胝体中NF-H和APP水平,用western blot检测大鼠脑白质中NF-H、APP、Tau、p-Tau S396、和p-Tau T231的水平,并计算Tau蛋白磷酸化水平。结果:使用美金刚和艾芬地尔阻滞NDMA受体可以减轻PTZ点燃大鼠脑白质中NF-H减少和APP积聚的程度,并减轻PTZ点燃导致的总Tau蛋白减少和Tau蛋白磷酸化水平升高。用氯化锂和Roscovitine分别抑制GSK-3β和Cdk5也可以减轻PTZ点燃导致的脑白质中NF-H减少和APP积聚,并减轻PTZ点燃导致的Tau蛋白磷酸化水平升高。此外,氯化锂可逆转PTZ点燃导致的总Tau蛋白减少,而Roscovitine则并不影响总Tau蛋白的水平。结论:抑制NMDA受体、GSK-3β和Cdk5均可以改善PTZ点燃癫痫大鼠模型中的轴突损害。提示NMDA受体、GSK-3β和Cdk5兴奋参与了癫痫轴突损害的形成。第三部分NMDA受体参与癫痫轴突损害的分子机制目的:研究NMDA受体是否可能通过兴奋GSK-3β和Cdk5进而介导癫痫的轴突损害。方法:对大鼠每日腹腔注射PTZ 21天并成功建立点燃癫痫模型后,再行每日腹腔注射非选择性NMDA受体拮抗剂美金刚、NR2B亚单位选择性NMDA受体拮抗剂艾芬地尔、GSK-3β拮抗剂氯化锂、Cdk5拮抗剂Roscovitine或生理盐水共7天,用western blot检测大鼠脑白质中总GSK-3β、磷酸化GSK-3βY216(p-GSK-3βY216)、磷酸化GSK-3βS9(p-GSK-3βS9)和Cdk5的蛋白水平,用多聚酶链式反应(PCR)检测大鼠脑白质中GSK-3βm RNA和Cdk5 m RNA的水平。结果:在PTZ点燃大鼠中使用美金刚和艾芬地尔阻滞NDMA受体可以降低Cdk5的m RNA表达和蛋白水平,并且可以升高p-GSK-3βS9、降低p-GSK-3βY216从而抑制GSK-3β的活性,但与美金刚不同的是,艾芬地尔并不影响GSK-3β的m RNA表达和蛋白水平。结论:NMDA受体,尤其是含NR2B亚单位的NMDA受体,可通过兴奋Cdk5和GSK-3β介导癫痫的轴突损害。
[Abstract]:The first part of epilepsy patients and PTZ light axon damage of brain tissues in a rat model of epilepsy Objective: To investigate the brain tissue of patients with epilepsy and four with nitrogen (PTZ) light axonal loss in brain tissue in a rat model of epilepsy. Methods: collecting the brain tissue of patients with refractory epilepsy surgical specimens, the establishment of PTZ kindling rat model of epilepsy the collection and separation of the white matter of the brain, using Western blot to detect brain tissue of patients with refractory epilepsy and PTZ intraperitoneal injection for 1 days, 1 weeks, 2 weeks, the rats of cerebral white matter in high molecular neurofilament 3 and 4 weeks (NF-H), amyloid precursor protein (APP), total Tau protein phosphorylation of Tau protein, S396 (p-Tau S396) and phosphorylated Tau protein T231 (p-Tau T231), and calculate the phosphorylation of Tau protein. Results: compared with control group, NF-H level of brain tissue of patients with refractory epilepsy were significantly decreased, APP increased the level of total Tau protein decreased, Tau protein phosphorylation level also increased.P TZ NF-H also reduced light epileptic rat brain white matter, elevated levels of APP and elevated total Tau protein decreased and Tau protein phosphorylation, and the degree with PTZ injection time gradually increased. Conclusion: the patients with intractable epilepsy and PTZ kindled seizures in the rat brain have to reduce NF-H reduce the accumulation of APP, Tau and total protein of axonal damage, and accompanied by phosphorylation of Tau protein increased. The second part of the NMDA receptor, GSK-3 beta and Cdk5 in axonal damage in epilepsy Objective: To explore the NMDA receptor, GSK-3 beta and Cdk5 is involved in axonal damage in epilepsy. Methods: rats were intraperitoneally injected with PTZ for 21 days and successfully establish a kindling model of epilepsy after given daily intraperitoneal injection of non selective NMDA receptor antagonist memantine, NR2B subunit of NMDA receptor selective antagonist ifenprodil, GSK-3 beta antagonists of lithium chloride, Cdk5 Antagonist Roscovitine or saline for 7 days, and the level of NF-H and APP in rat corpus callosum were detected with immunohistochemistry, Western blot detection of rat brain white matter in NF-H, APP, Tau, p-Tau, S396, p-Tau and T231 levels, and calculate the phosphorylation of Tau protein. Results: the use of memantine and ifenprodil blocking NDMA receptor can reduce PTZ light NF-H cerebral white matter in rats and reduce the degree of accumulation of APP, and reduce PTZ lit caused total Tau protein decreased and Tau protein phosphorylation levels. Brain white matter with lithium chloride and Roscovitine inhibition of GSK-3 beta and Cdk5 can also reduce the PTZ light LED NF-H decrease and APP accumulation, and reduce the level of Tau phosphorylation of PTZ light induced increase. In addition, lithium chloride can lead to the reversal of PTZ kindling total Tau protein decreased, while Roscovitine does not affect the level of total Tau protein. Conclusion: inhibition of NMDA receptor, GSK-3 beta and Cdk5 can improve PTZ kindling axonal injury in rat models of epilepsy. The results indicate that NMDA receptor, GSK-3 beta and Cdk5 in epilepsy excited axonal injury. The purpose of the third part of the NMDA receptor is involved in the molecular mechanism of epilepsy axonal injury: NMDA receptor on the possibility by exciting the GSK-3 beta and Cdk5 mediated axonal damage and epilepsy. Methods: the rats were intraperitoneally injected with PTZ 21 days and successfully establish a kindling model of epilepsy, and daily intraperitoneal injection of non selective NMDA receptor antagonist memantine, NR2B subunit selective NMDA receptor antagonist iseefair to Seoul, GSK-3 beta antagonists of lithium chloride, Cdk5 antagonist Roscovitine or saline 7 day, with total GSK-3 Western blot detection of rat brain white matter in beta, the phosphorylation of GSK-3 Y216 beta (p-GSK-3 beta Y216), phosphorylated GSK-3 S9 beta (p-GSK-3 beta S9) protein and Cdk5, polymerase chain reaction (PCR). Measurement of cerebral white matter in rats with GSK-3 m RNA and Cdk5 m beta RNA level. Results: in PTZ light memantine and ifenprodil blocking NDMA receptor using rat can decrease m RNA expression and protein level of Cdk5, and increased p-GSK-3 beta S9, Y216 inhibits GSK-3 reduce p-GSK-3 beta beta activity. But with memantine is different, ifenprodil did not affect m RNA expression and protein level of GSK-3 beta. Conclusion: the NMDA receptor, especially NR2B subunit containing NMDA receptors, can guide axonal loss in epilepsy by exciting Cdk5 and GSK-3 beta.

【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】：R742.9

【相似文献】