UCP2同向调控ATP、ROS作用的初步研究
发布时间：2018-10-09 14:40
【摘要】： 目的：线粒体是细胞能量代谢的中心,同时也是细胞内活性氧(reactive oxygen species, ROS)生成的主要部位。解偶联蛋白2(uncoupling protein 2, UCP2)是1997年发现的线粒体内膜上的质子转运蛋白,广泛分布于人类及啮齿类动物多种组织器官中。UCP2具有很高的质子转运活性,能使质子直接进入线粒体基质而不参与ATP的合成,使氧化磷酸化解偶联,ATP合成减少；同时由于线粒体内膜两侧质子梯度降低和呼吸链电子漏减少,活性氧产生减少。 UCP2自被发现以来,对其抗自由基作用和调节能量代谢作用研究较多,但极少数研究将二者结合起来。线粒体产生ROS的过程与氧化磷酸化过程一样是线粒体固有的、必然的和同等重要的功能。根据UCP2的解偶联效应,我们提出UCP2同向调控ATP与ROS理论假设：UCP2解偶联效应提高,ATP水平降低,同时因线粒体内膜两侧质子梯度降低,膜电位、电子漏降低,ROS的生成也降低;反之,UCP2解偶联效应降低,ATP水平升高,ROS水平也升高。然而,因解偶联效应改变引发的ATP、ROS同向变化,在细胞功能和抗损伤的作用分析中似有矛盾和不解之处：若UCP2低表达、解偶联效应降低,ATP增加但ROS也增加,可能导致细胞损伤；若UCP2高表达、解偶联效应增强,ROS减少但ATP也降低,可能导致细胞功能降低,对损伤的耐受能力也会下降。UCP2到底扮演什么角色?究竟是UCP2高表达还是低表达对细胞正常功能的维持有利?高水平ATP和低水平ROS在细胞抗损伤中哪一个可能有更为重要的作用?本研究同时关注UCP2解偶联效应引起的ATP、ROS水平变化,为UCP2生理意义的探明提供了重要的实验依据。 方法：将正常肝细胞株Chang Liver作为实验对象,用含10%胎牛血清的RPMI-1640培养液,37℃、5%CO2恒温孵育箱培养,隔天换液一次,0.25%胰酶EDTA消化传代。常规培养2-3代后,待细胞状态良好时开始进行实验。应用京尼平(genipin)作为UCP2特异性抑制剂。将细胞分为四组,分别为对照组、京尼平25μM组、京尼平50μM组、京尼平100μM组,对照组正常换液,给药组分别给与不同浓度的京尼平作用40min后,用荧光分光光度法检测细胞线粒体膜电位和细胞内总ROS的含量；用荧光虫素酶生物发光法测定ATP含量;通过免疫细胞化学的方法检测张氏肝细胞内UCP2的表达。 结果： 1.通过免疫细胞化学方法检测,可见张氏肝细胞内阳性染色,表明张氏肝细胞内有UCP2表达。京尼平作用后,UCP2表达有减少的趋势。 2.给与不同浓度京尼平作用40min后,京尼平处理组与对照组相比,肝细胞线粒体膜电位水平显著升高(P0.001),且具有剂量依赖性。 3.给与不同浓度京尼平作用40min后,京尼平处理组与对照组相比,肝细胞内ROS水平显著增加(P0.001),且具有剂量依赖性。 4.京尼平作用40min后,京尼平处理组与对照组相比,肝细胞内ATP水平显著增加(P0.05)。 结论： 1.正常张氏肝细胞内可见UCP2阳性表达,京尼平作用后,UCP2表达有减少的趋势。 2.京尼平能够增加张氏肝细胞的线粒体膜电位,且有剂量依赖性。 3.京尼平能够使张氏肝细胞的ATP水平产生增加。
[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，
本文编号：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，
本文编号：2259712
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