【作者】 郭林；

【导师】 冯美云；

【作者基本信息】 北京体育大学 ， 运动人体科学， 2005， 博士

【摘要】 随着对细胞因子研究的不断深入和分子生物学技术的迅速发展,更多的细胞因子的生物学功能为人们所认识,有关大负荷训练与细胞因子关系的研究越来越受到重视,许多研究证明细胞因子与运动训练具有密切的关系,细胞因子在大负荷训练导致组织结构和功能变化上起着重要的作用。缺血再灌注损伤是一个十分复杂的而又普遍存在的过程,肾脏是一个高灌注的器官,对缺血和缺血再灌注损伤非常敏感,运动及其恢复期肾血流量的两个时相的改变导致了肾缺血再灌注的整个过程。近年来研究发现细胞因子在缺血再灌注急性损伤发挥着重要的作用,在缺血再灌注过程中细胞因子的表达异常活跃,正常情况下抑炎因子和促炎因子之间的动态平衡状态被打破。目前这种过度的细胞因子表达被认为是引起包括炎症、细胞凋亡等一系列再灌注损伤的基础。近年的研究表明IL-1、IL-6、IL-18和TNF-α这组功能密切相关的炎症细胞因子,在急性肾缺血再灌注损伤中发挥重要作用。肾缺血-再灌注损伤所导致的IL-6、IL-1β和TNF-α等可以引起肾脏固有细胞的增殖,刺激其表达粘附分子并生成过多的细胞外基质而影响肾脏细胞外基质代谢,导致ECM合成与分解代谢的改变,而会直接影响到组织细胞结构和功能的变化。所以研究ECM在肾缺血再灌注损伤的代谢规律及炎症细胞因子对ECM积聚合成和降解的影响与机制,对阐明运动导致的肾组织微细结构变化和肾缺血再灌注损伤的分子机制具有极其重要的意义。目前有关运动对细胞因子影响的研究主要集中在运动导致血液和尿液中细胞因子水平变化,运动性缺血再灌注肾损伤肾脏细胞因子及相关基因的表达还未见有直接的研究报道。运动对ECM影响的研究主要在与运动直接相关的组织如骨、骨骼肌、肌腱及心脏等,有关运动对肾脏ECM影响的研究及与细胞因子相互关系还未有报道。因此研究大负荷训练状态对运动性缺血再灌注肾损伤细胞因子和相关基因的表达以及肾脏细胞外基质代谢变化的影响,对在分子及基因水平上探讨大负荷训练导致的肾组织微细结构变化机制、导致运动性缺血再灌注肾损伤发生机理、以及对于对抗与预防过度训练的发生及防止对机体运动能力和健康水平的不利影响具有积极意义。本实验通过研究大负荷训练导致缺血再灌注肾脏损害血清和肾组织炎性细胞因子蛋白表达及基因表达的变化和作用,为进一步探讨大负荷训练导致的肾组织微细结构变化机制、导致运动性缺血再灌注肾损伤发生机理提供分子生物学依据。研究肾脏细胞外基质代谢在大负荷训练导致缺血再灌注肾脏损害时的变化与作用,为在分子水平上进一步探讨大负荷训练导致运动性缺血再灌注肾损伤发生机理及大负荷训练导致的肾组织微细结构变化机制提供依据和新的研究目标。通过研究运动性缺血再灌注肾脏损害血清和肾组织炎性细胞因子蛋白表达及基因表达与肾脏细胞外基质的变化,阐述运动性缺血再灌注肾脏损害细胞因子与肾脏细胞外基质的相互关系和作用,进一步阐明大负荷训练导致运动性缺血再灌注肾损伤发生和肾组织微细结构变化机理的复杂性和网络性。1.大负荷训练导致大鼠肾功能的下降、肾组织超微结构的破坏、组织病理学改变、血清IL-1β和肾组织IL-1β明显升高及肾组织的IL-1βmRNA表达上调,且IL-1β表达均与BUN、Cr成正相关。证明IL-1β在大负荷训练导致大鼠肾缺血再灌注损伤可能起一定的作用。运动训练导致大鼠肾缺血再灌注损伤时,血清IL-1β水平可作为反映运动性肾缺血再灌注损伤严重程度的一个指标。2.大负荷训练组大鼠血清TNF-α和肾组织TNF-α明显高于对照组与一般训练组大鼠,肾组织的TNF-αmRNA也明显升高,经相关分析发现TNF-α表达均与BUN,Cr成正相关。证明大负荷训练导致大鼠TNF-α表达升高与肾缺血再灌注损伤有密切的关系,TNF-α在大负荷训练导致大鼠肾缺血再灌注损伤可能起一定的作用。大负荷训练导致大鼠肾组织TNF-α表达升高,不但在TNF-α蛋白表达水平上的上调,而且也体现在TNF-α基因表达水平上的上调。运动训练导致大鼠肾缺血再灌注损伤时,血清IL-1β水平可作为反映运动性肾缺血再灌注损伤严重程度的一个指标。3.大负荷训练导致大鼠肾功能的下降、肾组织超微结构的破坏、组织病理学改变、血清IL-6和肾组织IL-6明显升高及肾组织的IL-6mRNA表达上调,且IL-6表达均与BUN、Cr成正相关。证明IL-6在大负荷训练导致大鼠肾缺血再灌注损伤可能起一定的作用。运动训练导致大鼠肾缺血再灌注损伤时,血清IL-6水平可作为反映运动性肾缺血再灌注损伤严重程度的一个指标。4.大负荷训练导致大鼠肾功能的下降、肾组织超微结构的破坏、组织病理学改变、血清IL-18和肾组织IL-18明显升高及肾组织的IL-18mRNA表达上调,且IL-18表达均与BUN、Cr成正相关。证明IL-18在大负荷训练导致大鼠肾缺血再灌注损伤可能起一定的作用。运动训练导致大鼠肾缺血再灌注损伤时,血清IL-18水平可作为反映运动性肾缺血再灌注损伤严重程度的一个指标。5.大负荷训练导致大鼠肾小球ECM代谢发生异常,ECM沉积加重,且经相关分析发现ECM沉积均与BUN、Cr成正相关。证明大负荷训练导致大鼠肾小球ECM沉积加重与肾缺血再灌注损伤有密切的关系,ECM沉积在大负荷训练导致大鼠肾缺血再灌注损伤时可能起一定的作用。6.大负荷训练导致大鼠肾缺血再灌注损伤肾小球ECM沉积加重与细胞因子高度表达有密切的关系。大负荷训练致使大鼠免疫反应激活了肾脏炎症效应细胞,激活的效应细胞增殖、释放炎症介质及细胞外基质致肾脏损害,炎症介质、细胞外基质又反作用于效应细胞。如此,炎症细胞、炎症介质及细胞外基质间的“交互应答”作用,导致并加重了缺血再灌注肾脏损害的发生和不断进展。7.中等强度运动的大鼠肾功能、肾组织超微结构正常,无组织病理学改变。与对照组相比,大鼠血清和肾组IL-1β、TNF-α、IL-6、IL-18蛋白表达及肾组织的IL-1βmRNA、TNF-αmRNA、IL-6mRNA、IL-18mRNA表达无显著性差异。大鼠肾小球ECM代谢正常,无ECM沉积加重。大负荷训练导致大鼠肾血清和肾组织IL-1β、TNF-α、IL-6、IL-18蛋白表达及肾组织的IL-1βmRNA、TNF-αmRNA、IL-6mRNA、IL-18mRNA表达上调与肾缺血再灌注损伤有密切的关系,在大负荷训练导致大鼠肾缺血再灌注损伤可能起一定的作用。大负荷训练导致大鼠肾小球ECM代谢发生异常,ECM沉积加重,肾小球ECM沉积加重与肾缺血再灌注损伤高度相关。大负荷训练导致大鼠肾缺血再灌注损伤肾小球ECM沉积加重与细胞因子高度表达有密切的关系。大负荷训练导致大鼠肾血清和肾组织IL-1β、TNF-α、IL-6、IL-18蛋白表达及肾组织的IL-1βmRNA、TNF-αmRNA、IL-6mRNA、IL-18mRNA表达上调与肾小球ECM代谢发生异常和ECM沉积加重在导致肾脏组织微细结构发生变化上起者重要分子生物学作用。更多还原

【Abstract】 Ischemia and reperfusion injury is an ubiquitous and complicated process. Renal is a high perfusion organ and is very sensitive to ischemia and reperfusion injury. The redistribution of blood among organs in exercise results in the increase of blood volume in muscle and decrease in renal, which is called the sports renal ischemia. The renal blood volume recoveries after exercise forms the reperfusion following the sports renal ischemia. Therefore, the change of blood volume during the two period of exercise and recovery induces the whole period of renal ischemia and reperfusion.Cytokines plays a very important role in ischemia and reperfusion injury found in recent years studies. The Cytokines is abnormal active and the dynamic balance of inflammation inhibitor and inflammation promoter in normal state is broken up during ischemia and reperfusion process. At present, the excess expression of cytokines reckoned as the foundation of inflammation and apoptosis of cells. The group of inflammation inducing cytokines including IL-1、IL-6、TNF-αand IL-18 is closely related in function and also plays a very important role in ischemia and reperfusion injury. IL-1、IL-6 and TNF-αresulted from ischemia and reperfusion could induce in the proliferation of the renal inherent cells, stimulate the expression of adhesive molecules and produce more Extracellular matrix, as a results the Extracellular matrix metabolism is changed and the structure and function of tissue and cells is influenced directly. As the progress of studying in cellular metabolism and function, it is found that Extracellular matrix build up the upport materials of tissues and cells and the fill-up tissues of cell interstice as well as plays a very important role to the proliferation、differentiation、metabolism、movement、signal conduction and so on. The quality and quantity of Extracellular matrix changes will result in the environment alteration of cells and influence cells function and organ figure directly. Therefore, the studying in the metabolism of function and organ reperfusion injury resulted from exercise.But at present, the studying in the effects of exercise on cytokines mainly focus on the level of cytokines in blood and urine. There is no direct report on the expression of renal cytokines and related gene by now. The research of the influence of exercise on Extracellular matrix metabolism is carried out mainly in the tissues of bone、skeletal muscle、tendor and heart which is directly related with exercise. The studying of effects of exercise on renal Extracellular matrix metabolism has no reports till now. Therefore, the studying in the effects of load training and exercise on the expression of cytokines and related gene as well as renal Extracellular matrix metabolism changes, will has significant meaning for the studying in the pathogenesis of load training and sports ischemia and reperfusion renal injury onset at the level of cell、molecule and gene, counteraction and prevention of overload training onset as well as avoiding harmful effects on sports capacity and health.The experiment studied the changes and effects of inflammatory cytokines protein and gene expression in serum and renal tissues during ischemia and reperfusion renal injury resulted from load training and provided molecule biological basis for the further studying in the pathogenesis of load training and sports ischemia and reperfusion renal injury onset at the level of cell、molecule and gene. The experiment studied the changes and effects of ECM during ischemia and reperfusion renal injury resulted from load training and provided basis and new studying targets for the further studying in the pathogenesis of load training and sports ischemia and reperfusion renal injury onset at the level of cell and molecule. Furthermore, with the studying the cytokines protein and gene expression in serum and renal tissues and the changes of ECM during ischemia and reperfusion renal injury, the research expounded the relation and effects of cytokines and ECM during sports ischemia and reperfusion renal injury and had further illumination of the complication and network character of the pathogenesis of load training and sports ischemia and reperfusion renal injury onset.1.Load training resulting in the decrease of renal function of rats、the damage to the microstructure of renal tissues、changes of tissues pathology and significant increase of IL-1βin serum and renal tissues as well as significant increase of IL-1βmRNA in renal tissues. The expression of IL-1βhad positive correlation with the increase of BUN and SCr. This indicated that IL-1βmight play some role in ischemia and reperfusion renal injury resulted from overload training. The level of IL-1βin serum were a suitable indexes that could evaluate the degree of sports ischemia and reperfusion renal injury.2.The level of TNF-αin serum and renal tissues of the rats in load training group was significantly higher than it in control and middle exercise intensity group. The TNF-αmRNA in renal tissues increased significantly too in load training group and TNF-αexpression had a positive correlation with BUN and Cr. It proved that the increase of TNF-αexpression resulted from overload training had closely relation with mia and reperfusion renal injury. The TNF-αexpression increased resulted from load training expression had a positive correlation with BUN and Cr. It proved that the increase of TNF-αexpression resulted from overload training had closely relation with mia and reperfusion renal injury. The TNF-αexpression increased resulted from load training not only at the level of TNF-αprotein but also at the level of TNF-αgene. The level of IL-1βin serum can be used as indexes of the degree of sports ischemia and reperfusion renal injury.3.Load training resulting in the decrease of renal function of rats、the damage to the microstructure of renal tissues、changes of tissues pathology and significant increase of IL-6 in serum and renal tissues as well as significant increase of IL- IL-6mRNA in renal tissues. The expression of IL-6 was correlated positively with the increase of BUN and SCr. This indicated that IL-6 might play some role in ischemia and reperfusion renal injury resulted from load training. The level of IL-6 in serum were a suitable indexes that could evaluate the degree of sports ischemia and reperfusion renal injury.4.Load training resulting in the decrease of renal function of rats、the damage to the microstructure of renal tissues、changes of tissues pathology and significant increase of IL-18 in serum and renal tissues as well as significant increase of IL-18mRNA in renal tissues. The expression of IL-18 had positive correlation with the increase of BUN and SCr. This indicated that IL-18 might play some role in ischemia and reperfusion renal injury resulted from load training. The level of IL-18 in serum were a suitable indexes that could evaluate the degree of sports ischemia and reperfusion renal injury.5.Load training resulted in the abnormal metabolism of ECM in glomerulus and ECM deposition enhancement and ECM deposition was correlated positively with BUN and SCr. It demonstrated that the ECM deposition enhancement resulted from load training had closely relation with ischemia and reperfusion renal injury. ECM deposition might play some role in ischemia and reperfusion renal injury resulted from overload training.6.ECM deposition enhancement in glomerulus with the ischemia and reperfusion renal injury resulted from load training was closely correlated with the high expression of cytokine. load training induced Immunoreaction and activated renal inflammatory cells. The activated renal inflammatory cells proliferated and released inflammatory mediator and ECM and as a result it damaged kidney. Inflammatory mediator and ECM had a counteraction to inflammatory cells. Therefore, the effects of the“cross talk”among inflammatory cells、inflammatory mediator and ECM induced and enhanced generation and evolution of ischemia and reperfusion renal injury.7.The renal function and microstructure of renal tissues of rats in the middle exercise intensity group were normal and had no changes of tissues pathology. The expression level of IL-1β、IL-6、TNF-αand IL-18 in serum and renal tissues and the expression level of IL-1βmRNA、IL-6mRNA、TNF-αmRNA and IL-18mRNA in renal tissues in the middle exercise intensity group had no significantly difference compared with it in control group. The ECM metabolism of the rats in the middle exercise intensity group were normal and there was no ECM deposition enhancement in this group.更多还原