本文關(guān)鍵詞：外向延遲整流型鉀離子通道Kv2.1參與甲基苯丙胺引起海馬神經(jīng)元損傷及機(jī)制研究　出處：《南京醫(yī)科大學(xué)》2015年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 甲基苯丙胺 海馬神經(jīng)元 Kv2.1 細(xì)胞凋亡 甲基苯丙胺 MAPK Kv2.1 細(xì)胞凋亡 甲基苯丙胺 sigma-1受體 Kv2.1 p38 MAPK 細(xì)胞凋亡

【摘要】：第一部分 Kv2.1在甲基苯丙胺引起海馬神經(jīng)元損傷中的作用甲基苯丙胺(Meth)是一類非法的精神興奮劑,其濫用已成為全球性的公共衛(wèi)生難題。Meth可引起中樞神經(jīng)系統(tǒng)退行性改變,其中,細(xì)胞凋亡引起的神經(jīng)元丟失是重要機(jī)制之一。外向延遲整流型鉀通道亞型Kv2.1激活或高表達(dá)可引起鉀離子持續(xù)外流,細(xì)胞皺縮,最終誘導(dǎo)凋亡。因此,本實(shí)驗(yàn)以鉀離子通道亞型Kv2.1為研究靶點(diǎn),探討其在Meth作用下,與海馬神經(jīng)元損傷間的關(guān)系。實(shí)驗(yàn)室前期結(jié)果表明Meth可引起外向鉀電流的增加,在此基礎(chǔ)上,通過western blot的方法我們發(fā)現(xiàn),Meth引起Kv2.1蛋白表達(dá)量呈時(shí)間及劑量依賴性的增多,并伴隨細(xì)胞凋亡水平升高。而利用Kv2.1特異性抑制劑GxTX-1E與海馬神經(jīng)元共孵后,發(fā)現(xiàn)GxTX-1E能有效抑制Meth引起的細(xì)胞凋亡。借助于慢病毒感染海馬神經(jīng)元的方法,使Kv2.1高表達(dá),結(jié)果顯示,Kv2.1轉(zhuǎn)染組較正常細(xì)胞組或空載體組,凋亡水平明顯升高(cleavedcaspase3表達(dá)升高,bcl-2減少),進(jìn)一步提示Kv2.1可能是Meth促凋亡作用中的重要靶點(diǎn)。第二部分MAPK參與甲基苯丙胺引起的海馬神經(jīng)元損傷絲裂原活化蛋白激酶(Mitogen-activated protein kinase,MAPK)參與調(diào)節(jié)細(xì)胞活化、分化、增殖、生存及細(xì)胞因子產(chǎn)生等多種細(xì)胞活動,亦可介導(dǎo)細(xì)胞凋亡過程。Meth入胞后,MAPK通路廣泛激活,參與不同的生理、病理調(diào)節(jié)過程。本實(shí)驗(yàn)探討了 MAPK通路參與Meth引起海馬神經(jīng)元損傷中的作用。Western blot結(jié)果顯示ERK1/2、JNK、p38MAPK通路均可被Meth磷酸化激活,而分別加入三者特異性的抑制劑預(yù)孵后,僅p38MAPK抑制劑(SB203580)可顯著下調(diào)細(xì)胞凋亡水平(JNK通路抑制后可在一定程度下降低cleaved-caspase 3表達(dá)),同時(shí)p38抑制劑及JNK抑制劑(SP600125)均能降低Meth誘導(dǎo)的Kv2.1的表達(dá)。以上結(jié)果提示Meth可能通過磷酸化激活p38 MAPK通路對Kv2.1鉀通道進(jìn)行調(diào)控從而介導(dǎo)海馬神經(jīng)元的損傷。第三部分Sigma-1受體在甲基苯丙胺引起的海馬神經(jīng)元損傷中的作用Sigma-1受體廣泛存在于中樞神經(jīng)系統(tǒng)中,被視為多種神經(jīng)退行性病變的潛在治療靶點(diǎn)。其作為特異性精神類藥物結(jié)合蛋白,是Meth在胞內(nèi)的重要受體之一,其突出特點(diǎn)還在于,該受體能夠調(diào)節(jié)多種離子通道功能。鑒于sigma-1受體的配體能可逆性的抑制延遲整流型鉀通道,同時(shí)結(jié)合鉀通道主要亞型Kv2.1可能作為Meth作用下細(xì)胞早凋的重要靶點(diǎn)這一結(jié)果,本部分內(nèi)容利用western blot的方法,通過細(xì)胞膜蛋白提取技術(shù),探討在Meth引起的細(xì)胞凋亡過程中,sigma-1受體激動劑與抑制劑對Kv2.1表達(dá)與分布的影響及相關(guān)信號轉(zhuǎn)導(dǎo)通路。本部分實(shí)驗(yàn)中,我們發(fā)現(xiàn)sigma-1受體表達(dá)隨Meth濃度的增大而升高。sigma-1受體激動劑PRE-084能抑制Meth誘導(dǎo)的細(xì)胞凋亡作用,而sigma-1受體抑制劑BD-1063對細(xì)胞凋亡未有明顯影響。同時(shí),sigma-1受體的激動劑PRE-084可下調(diào)Kv2.1蛋白的表達(dá),抑制通道蛋白的膜遷移,并降低p38MAPK通路的磷酸化水平,該現(xiàn)象可為sigma-1受體激動劑抑制細(xì)胞凋亡的作用提供部分解釋。
[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.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R749.64

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