外向延迟整流型钾离子通道Kv2.1参与甲基苯丙胺引起海马神经元损伤及机制研究

发布时间：2018-01-01 14:17

本文关键词：外向延迟整流型钾离子通道Kv2.1参与甲基苯丙胺引起海马神经元损伤及机制研究　出处：《南京医科大学》2015年硕士论文　论文类型：学位论文

【摘要】：第一部分 Kv2.1在甲基苯丙胺引起海马神经元损伤中的作用甲基苯丙胺(Meth)是一类非法的精神兴奋剂,其滥用已成为全球性的公共卫生难题。Meth可引起中枢神经系统退行性改变,其中,细胞凋亡引起的神经元丢失是重要机制之一。外向延迟整流型钾通道亚型Kv2.1激活或高表达可引起钾离子持续外流,细胞皱缩,最终诱导凋亡。因此,本实验以钾离子通道亚型Kv2.1为研究靶点,探讨其在Meth作用下,与海马神经元损伤间的关系。实验室前期结果表明Meth可引起外向钾电流的增加,在此基础上,通过western blot的方法我们发现,Meth引起Kv2.1蛋白表达量呈时间及剂量依赖性的增多,并伴随细胞凋亡水平升高。而利用Kv2.1特异性抑制剂GxTX-1E与海马神经元共孵后,发现GxTX-1E能有效抑制Meth引起的细胞凋亡。借助于慢病毒感染海马神经元的方法,使Kv2.1高表达,结果显示,Kv2.1转染组较正常细胞组或空载体组,凋亡水平明显升高(cleavedcaspase3表达升高,bcl-2减少),进一步提示Kv2.1可能是Meth促凋亡作用中的重要靶点。第二部分MAPK参与甲基苯丙胺引起的海马神经元损伤丝裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)参与调节细胞活化、分化、增殖、生存及细胞因子产生等多种细胞活动,亦可介导细胞凋亡过程。Meth入胞后,MAPK通路广泛激活,参与不同的生理、病理调节过程。本实验探讨了 MAPK通路参与Meth引起海马神经元损伤中的作用。Western blot结果显示ERK1/2、JNK、p38MAPK通路均可被Meth磷酸化激活,而分别加入三者特异性的抑制剂预孵后,仅p38MAPK抑制剂(SB203580)可显著下调细胞凋亡水平(JNK通路抑制后可在一定程度下降低cleaved-caspase 3表达),同时p38抑制剂及JNK抑制剂(SP600125)均能降低Meth诱导的Kv2.1的表达。以上结果提示Meth可能通过磷酸化激活p38 MAPK通路对Kv2.1钾通道进行调控从而介导海马神经元的损伤。第三部分Sigma-1受体在甲基苯丙胺引起的海马神经元损伤中的作用Sigma-1受体广泛存在于中枢神经系统中,被视为多种神经退行性病变的潜在治疗靶点。其作为特异性精神类药物结合蛋白,是Meth在胞内的重要受体之一,其突出特点还在于,该受体能够调节多种离子通道功能。鉴于sigma-1受体的配体能可逆性的抑制延迟整流型钾通道,同时结合钾通道主要亚型Kv2.1可能作为Meth作用下细胞早凋的重要靶点这一结果,本部分内容利用western blot的方法,通过细胞膜蛋白提取技术,探讨在Meth引起的细胞凋亡过程中,sigma-1受体激动剂与抑制剂对Kv2.1表达与分布的影响及相关信号转导通路。本部分实验中,我们发现sigma-1受体表达随Meth浓度的增大而升高。sigma-1受体激动剂PRE-084能抑制Meth诱导的细胞凋亡作用,而sigma-1受体抑制剂BD-1063对细胞凋亡未有明显影响。同时,sigma-1受体的激动剂PRE-084可下调Kv2.1蛋白的表达,抑制通道蛋白的膜迁移,并降低p38MAPK通路的磷酸化水平,该现象可为sigma-1受体激动剂抑制细胞凋亡的作用提供部分解释。
[Abstract]:The first part of Kv2.1 induced injury of hippocampal neurons in methamphetamine methamphetamine (Meth) is a kind of illegal stimulant abuse has become a public health problem of global.Meth can cause central nervous system degenerative changes, the apoptosis induced by neuronal loss is one of the important mechanisms. Outward delayed rectifier potassium channel subtypes Kv2.1 activation or overexpression can induce potassium ion flow, cell shrinkage, and ultimately induce apoptosis. Therefore, the potassium ion channel subtype Kv2.1 as the research target, to explore its relationship with the action of Meth, injury of hippocampal neurons. The previous results show that Meth can cause the increase of the outward current in on the basis of blot by western method we found that Meth induced the expression of Kv2.1 protein increased in a dose and time dependent, accompanied by apoptosis in water Flat elevated. The use of Kv2.1 specific inhibitor GxTX-1E and hippocampal neurons were cultured, found that apoptosis of GxTX-1E can effectively inhibit Meth induced by virus infection. Methods the hippocampal neurons, the expression of Kv2.1. The results show that Kv2.1 transfection group than normal cell group or empty vector group, the apoptosis levels were significantly increased (cleavedcaspase3 increased expression of Bcl-2 decrease), further suggesting that Kv2.1 may be an important target for Meth induced apoptosis in MAPK. The second part involved in the neuronal damage induced by methamphetamine mitogen activated protein kinase (Mitogen-activated protein, kinase, MAPK) in the regulation of cell activation, differentiation, proliferation, survival and production of cytokines may be mediated cellular activities. The process of.Meth induced apoptosis in the cell, the MAPK pathway is widely involved in different physiological activation, regulation of pathology. The experiment studied MAPK The road is involved in Meth induced injury of hippocampal neurons of.Western blot results in ERK1/2, JNK, p38MAPK pathway can be phosphorylated by Meth activation inhibitor, pretreatment and were added to three specific, only p38MAPK inhibitor (SB203580) could significantly decrease the apoptosis level (inhibition of JNK pathway can decrease the expression of cleaved-caspase 3 in at the same time, under a certain degree) p38 inhibitor and JNK inhibitor (SP600125) could reduce the expression of Meth induced by Kv2.1. These results suggest that Meth may activate p38 by phosphorylation of MAPK pathway on the regulation of Kv2.1 potassium channel which mediates hippocampal neuron damage. Hippocampal neurons damage caused by the third part of the Sigma-1 receptor in the role of methamphetamine Sigma-1 receptors are widely distributed in the central nervous system, is regarded as a potential target for the treatment of various neurodegenerative diseases. As a specific kind of spirit Drug binding protein, is one of the most important receptor in the cytosolic Meth, its outstanding characteristic is that the receptor can regulate a variety of ion channel function. Whereas sigma-1 receptor ligands can inhibit the reversibility of the delayed rectifier potassium channel, combined with the main potassium channel subtype Kv2.1 may serve as an important target of Meth cells and withered early the results of this part, using the method of Western blot, through the cell membrane protein extraction technology, study on Meth induced apoptosis pathway, sigma-1 receptor agonists and inhibitors on Kv2.1 expression and distribution effect and related signal transduction. This part of the experiment, we found that sigma-1 receptor expression with cell apoptosis the increase of the concentration of Meth increased.Sigma-1 receptor agonist PRE-084 can inhibit Meth induced, and sigma-1 receptor inhibitor BD-1063 on apoptosis was not affected. At the same time, sIgM PRE-084, an agonist of A-1 receptor, can down regulate the expression of Kv2.1 protein, inhibit the membrane migration of channel proteins, and decrease the phosphorylation level of p38MAPK pathway. This phenomenon can provide some explanation for the role of sigma-1 receptor agonists in inhibiting cell apoptosis.

【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R749.64

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