UCP2同向調(diào)控ATP、ROS作用的初步研究
發(fā)布時(shí)間：2018-10-09 14:40
【摘要】： 目的：線粒體是細(xì)胞能量代謝的中心,同時(shí)也是細(xì)胞內(nèi)活性氧(reactive oxygen species, ROS)生成的主要部位。解偶聯(lián)蛋白2(uncoupling protein 2, UCP2)是1997年發(fā)現(xiàn)的線粒體內(nèi)膜上的質(zhì)子轉(zhuǎn)運(yùn)蛋白,廣泛分布于人類及嚙齒類動(dòng)物多種組織器官中。UCP2具有很高的質(zhì)子轉(zhuǎn)運(yùn)活性,能使質(zhì)子直接進(jìn)入線粒體基質(zhì)而不參與ATP的合成,使氧化磷酸化解偶聯(lián),ATP合成減少；同時(shí)由于線粒體內(nèi)膜兩側(cè)質(zhì)子梯度降低和呼吸鏈電子漏減少,活性氧產(chǎn)生減少。 UCP2自被發(fā)現(xiàn)以來,對(duì)其抗自由基作用和調(diào)節(jié)能量代謝作用研究較多,但極少數(shù)研究將二者結(jié)合起來。線粒體產(chǎn)生ROS的過程與氧化磷酸化過程一樣是線粒體固有的、必然的和同等重要的功能。根據(jù)UCP2的解偶聯(lián)效應(yīng),我們提出UCP2同向調(diào)控ATP與ROS理論假設(shè)：UCP2解偶聯(lián)效應(yīng)提高,ATP水平降低,同時(shí)因線粒體內(nèi)膜兩側(cè)質(zhì)子梯度降低,膜電位、電子漏降低,ROS的生成也降低;反之,UCP2解偶聯(lián)效應(yīng)降低,ATP水平升高,ROS水平也升高。然而,因解偶聯(lián)效應(yīng)改變引發(fā)的ATP、ROS同向變化,在細(xì)胞功能和抗損傷的作用分析中似有矛盾和不解之處：若UCP2低表達(dá)、解偶聯(lián)效應(yīng)降低,ATP增加但ROS也增加,可能導(dǎo)致細(xì)胞損傷；若UCP2高表達(dá)、解偶聯(lián)效應(yīng)增強(qiáng),ROS減少但ATP也降低,可能導(dǎo)致細(xì)胞功能降低,對(duì)損傷的耐受能力也會(huì)下降。UCP2到底扮演什么角色?究竟是UCP2高表達(dá)還是低表達(dá)對(duì)細(xì)胞正常功能的維持有利?高水平ATP和低水平ROS在細(xì)胞抗損傷中哪一個(gè)可能有更為重要的作用?本研究同時(shí)關(guān)注UCP2解偶聯(lián)效應(yīng)引起的ATP、ROS水平變化,為UCP2生理意義的探明提供了重要的實(shí)驗(yàn)依據(jù)。 方法：將正常肝細(xì)胞株Chang Liver作為實(shí)驗(yàn)對(duì)象,用含10%胎牛血清的RPMI-1640培養(yǎng)液,37℃、5%CO2恒溫孵育箱培養(yǎng),隔天換液一次,0.25%胰酶EDTA消化傳代。常規(guī)培養(yǎng)2-3代后,待細(xì)胞狀態(tài)良好時(shí)開始進(jìn)行實(shí)驗(yàn)。應(yīng)用京尼平(genipin)作為UCP2特異性抑制劑。將細(xì)胞分為四組,分別為對(duì)照組、京尼平25μM組、京尼平50μM組、京尼平100μM組,對(duì)照組正常換液,給藥組分別給與不同濃度的京尼平作用40min后,用熒光分光光度法檢測(cè)細(xì)胞線粒體膜電位和細(xì)胞內(nèi)總ROS的含量；用熒光蟲素酶生物發(fā)光法測(cè)定ATP含量;通過免疫細(xì)胞化學(xué)的方法檢測(cè)張氏肝細(xì)胞內(nèi)UCP2的表達(dá)。 結(jié)果： 1.通過免疫細(xì)胞化學(xué)方法檢測(cè),可見張氏肝細(xì)胞內(nèi)陽性染色,表明張氏肝細(xì)胞內(nèi)有UCP2表達(dá)。京尼平作用后,UCP2表達(dá)有減少的趨勢(shì)。 2.給與不同濃度京尼平作用40min后,京尼平處理組與對(duì)照組相比,肝細(xì)胞線粒體膜電位水平顯著升高(P0.001),且具有劑量依賴性。 3.給與不同濃度京尼平作用40min后,京尼平處理組與對(duì)照組相比,肝細(xì)胞內(nèi)ROS水平顯著增加(P0.001),且具有劑量依賴性。 4.京尼平作用40min后,京尼平處理組與對(duì)照組相比,肝細(xì)胞內(nèi)ATP水平顯著增加(P0.05)。 結(jié)論： 1.正常張氏肝細(xì)胞內(nèi)可見UCP2陽性表達(dá),京尼平作用后,UCP2表達(dá)有減少的趨勢(shì)。 2.京尼平能夠增加張氏肝細(xì)胞的線粒體膜電位,且有劑量依賴性。 3.京尼平能夠使張氏肝細(xì)胞的ATP水平產(chǎn)生增加。
[Abstract]:Objective: The mitochondria are the centers of energy metabolism in cells, and also the main parts of active oxygen species (ROS). uncoupling protein 2 (UCP2) is a proton transport protein found in the mitochondrial membrane found in 1997, widely distributed in human and various tissues and organs. UCP2 has high proton transport activity, which can lead protons to directly enter the mitochondria matrix without participating in the synthesis of ATP, so that the oxidative phosphorylation is coupled and ATP synthesis is reduced; meanwhile, due to the reduction of proton gradient on both sides of the mitochondrial membrane and the reduction of the electron leakage of the respiratory chain, the active oxygen generation is reduced. UCP2 has more research on its anti-free radical effect and regulation of energy metabolism since its discovery, but very few studies will In combination, the process of producing ROS in mitochondria is the same as the oxidative phosphorylation process, which is inherent in mitochondria, inevitable and equal According to the decoupling effect of UCP2, we propose that UCP2 can increase the coupling effect of UCP2 and decrease ATP level, but also decrease the proton gradient on both sides of mitochondria, decrease the electron leakage and decrease the generation of ROS; otherwise, UCP2 is decoupled. Reduction in effect, increased ATP level, ROS water However, due to the change of ATP and ROS induced by the change of uncoupling effect, there seems to be a contradiction and incomprehension in the function analysis of cell function and anti-injury: if UCP2 is low in expression, the uncoupling effect decreases, ATP increases but ROS also increases, which may lead to cell damage; if UCP 2 High expression, enhanced uncoupling effect, reduced ROS but decreased ATP, which could lead to decreased cell function and resistance to damage It's also going down. UCP2 's playing What role? Is the UCP2 high expression or low expression on the normal function of the cell Maintenance? High levels of ATP and low levels of ROS may be more heavy in cell damage It is important to study the changes of ATP and ROS caused by UCP2 uncoupling effect. Methods: Chang Liver, a normal liver cell strain, was used as an experimental object, and cultured at 37 鈩，
本文編號(hào)：2259712
[Abstract]:Objective: The mitochondria are the centers of energy metabolism in cells, and also the main parts of active oxygen species (ROS). uncoupling protein 2 (UCP2) is a proton transport protein found in the mitochondrial membrane found in 1997, widely distributed in human and various tissues and organs. UCP2 has high proton transport activity, which can lead protons to directly enter the mitochondria matrix without participating in the synthesis of ATP, so that the oxidative phosphorylation is coupled and ATP synthesis is reduced; meanwhile, due to the reduction of proton gradient on both sides of the mitochondrial membrane and the reduction of the electron leakage of the respiratory chain, the active oxygen generation is reduced. UCP2 has more research on its anti-free radical effect and regulation of energy metabolism since its discovery, but very few studies will In combination, the process of producing ROS in mitochondria is the same as the oxidative phosphorylation process, which is inherent in mitochondria, inevitable and equal According to the decoupling effect of UCP2, we propose that UCP2 can increase the coupling effect of UCP2 and decrease ATP level, but also decrease the proton gradient on both sides of mitochondria, decrease the electron leakage and decrease the generation of ROS; otherwise, UCP2 is decoupled. Reduction in effect, increased ATP level, ROS water However, due to the change of ATP and ROS induced by the change of uncoupling effect, there seems to be a contradiction and incomprehension in the function analysis of cell function and anti-injury: if UCP2 is low in expression, the uncoupling effect decreases, ATP increases but ROS also increases, which may lead to cell damage; if UCP 2 High expression, enhanced uncoupling effect, reduced ROS but decreased ATP, which could lead to decreased cell function and resistance to damage It's also going down. UCP2 's playing What role? Is the UCP2 high expression or low expression on the normal function of the cell Maintenance? High levels of ATP and low levels of ROS may be more heavy in cell damage It is important to study the changes of ATP and ROS caused by UCP2 uncoupling effect. Methods: Chang Liver, a normal liver cell strain, was used as an experimental object, and cultured at 37 鈩，
本文編號(hào)：2259712
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