缺血后肝损伤标书.doc

一研究意义及国内外研究现状 缺血一再灌注(ischemia and reperfusion，I-R)常见于外科手术中的器官血流阻断、器官移植、休克复苏等过程。目前，临床上针对IR损伤的防治策略的研究除了药物保护和基因治疗外，对机械性或应激性干预尤为关注，如缺血预处理(ischemic preconditioning，IpreC)、控制性再灌注以及新近提出的缺血后处理(ischemic postconditioning，lpost)1,2。Iprec是目前研究的最为广泛的保护性策略，应用IpreC需要确定器官何时将发生缺血，这在临床多数情况下常常难以预测，并且给尚未缺血的器官采用短暂的缺血处理，常使人在心理上难以接受。 IpostC与IpreC不同，IpostC在缺血器官的再灌注起始应用，大多能被人接受，并且其操作的简易性使临床医生易于掌握和实施，尤其在条件一般的广大的中小医疗单位中易于推广。IpostC作为一种新的抗I-R损伤策略，愈来愈受到广泛的关注。现有IpostC研究主要集中在心脏方面，其减轻心肌I-R损伤的机制是多方面的。由于Ipostc在再灌注一开始实施，其对I-R器官的保护作用机制存在两个方面，一是可能削弱了已经发生的或即将发生的损伤机制，再则可能引发了机体的内源性保护机制。与Ipostc减少ROS的生成、减轻线粒体钙超载和抑制线粒体通透性转换孔(mitochondrial permeability transition pore，MPTP)开放、激活促生存激酶PI3K/Akt和MEKl/2-ERKl2信号途径。促生存激酶PI3K/Akt和ERK 12通路在心脏I-R时被激活，并通过各种抗凋亡机制减少再灌注诱导的心肌细胞死亡。Iprec和再灌注起始应用许多生长因子和药物如腺苷受体促进剂、缓激肽、胰岛素、吗啡等均可募集和上调PI3 K/Akt和或ERKl/2通路再灌注损伤补救激酶(Reperfusion Injury Salvage Kinase，RISK)信号通路而对I-R心脏发挥抗损伤作用。研究表明Ipostc能够上调RISK通路发挥主动性保护效应12, 20-22, 25-28。激活后的RISK通路可使caspases 3和caspases 9失话并抑制细胞凋亡，从而启动心脏保护机制33。 钙蛋白酶(calpain) 是一种需要Ca2+激活的胞质蛋白酶，可作用于许多底物，包括多种细胞骨架蛋白、膜受体以及参与信号转导和代谢的酶69。在生理条件下，活化的calpain通过对底物有限的水解作用(而非完全的破坏)，参与许多与细胞重建有关的生理过程，如增殖、分化和迁移。calpain参与了许多疾病的病理发展过程，如A1zheimer病、多发性硬化症、肌营养不良、创伤性脊髓脑损伤以及多种器官的缺血性损伤包括肝脏。在这些疾病中，calpain活性常常增加，抑制其活性具有器官保护作用。calpain引起组织损伤至少通过机制不同的两条途径：多数情况下如肌营养不良，calpain调节失衡导致胞内calpain的过度活化进而引起细胞损伤；另一种是正在死亡的细胞可释放calpain进入周围环境，被胞外高浓度的Ca2+活化，进而攻击临近健康细胞，引起组织损伤的进一步加重69。目前研究证实，calpain参与包括肝脏在内的多种器官的I-R损伤，应用calpain抑制剂能够减轻I-R损伤。大鼠肝缺血或肝移植后应用calpain抑制剂Cbz-Leu-Leu-Tyr-CHN2能够减轻缺血、保存再灌注损伤和增加存活率70, 71。Iprec可抑制calpain活性，减轻心脏IR损伤72，但IpreC对I-R肝损伤的保护作用则与calpain活性无关76。在细胞死亡发生过程中，calpain和caspase发挥重要的作用，两者可单独或相互作用促进细胞的死亡73。心脏I-R时，活化的Calpain分解Bid为tBid，通过线粒体途径促进细胞的死亡74, 75。IpostC能否影响Calpain活性，减少I-R损伤目前尚未见报道。揭示calpain 在肝IpostC中的变化可能为探索抗I-R损伤策略提供新的治疗靶点。 肝细胞死亡(坏死和戚凋亡)是I-R肝损伤的重要特征。长久以来，坏死和凋亡被认为是截然不同的两个过程。然而，近年来研究表明，两者的发生经过共同的“necrapoptosis”44信号传导通路。线粒体在necrapoptosis通路中发挥关键作用，细胞的最终命运取决于缺血刺激的严重程度和细胞ATP的合成能力。关于IpostC能否像IpreC一样具有减轻其他器官的I-R损伤作用及其机制，目前研究甚少，仅见于IpostC对内皮细胞30和脑组织33的抗I-R损伤作用的报道。肝脏I-R损伤常常发生在肝脏手术、肝移植以及出血性休克等过程中。缺血性肝损伤的基本原因是缺氧，缺氧使线粒体呼吸功能丧失，ATP产生减少，能量依赖性的代谢途径和转运过程被破坏：含氧血再灌注则进一步加剧了缺氧或缺血性的损伤。因此，研究IpostC对I-R大鼠肝脏的保护作用及其机制，为IpostC在肝脏外科临床应用方面提供理论基础。 因此，本申请项目以细胞死亡和切入点，研究Necrapoptosis通路中的相关分子caIpain和caspase-3的变化，探讨在IpostC抗I-R损伤中肝细胞线粒体膜通透性和膜电位的变化，阐明IpostC的抗肝I-R损伤作用及其分子机制。特色与创新之处 到目前为止，对肝脏缺血后处理研究的文献报道甚少，后处理方案不一致，尤其是缺血后处理对I-R肝的抗损伤作用机制的研究在国内外尚属空白(参见附表)。本课题首次从整体、组织和分子水平上，从细胞死亡和增殖的角度入手，全面地研究缺血后处理对I-R大鼠肝的抗损伤作用及其分子机制，为抗肝I-R损伤的基础研究奠定一定的理论和实验依据，为临床外科防治肝I-R损伤提供新策略。 【参考文献】1 Selzner N，Rudiger H，Graf R，et a1Protective strategies against ischemic injury of the liver Gastroenterology 2003；125(3)：917362Murry CE，Jernnings RB，and Reimer KAPreconditioning with ischemia：a delay of lethal cell injury in ischemic myocardiumCirculation 1986；74：1 1241 136，3Zha0 ZQCorvera JS，Halkos ME，et a1Inhibition of myocardial injury by ischemic postconditioning during reperfusion：comparison with ischemic preconditioning Am J Physiol Heart Circ Physiol 2003；285(2)：H579884陶凌、李源、高峰、王跃民、龚卫琴缺血后处理对急性心肌缺血再灌注兔心 脏的保护作用，第四军医大学学报， 2000，21(6)：s11685Ritter T，Kupiec-Weglinski JWGene therapy for the prevention of ischemiareperfusion injury in organ transplantation Curr Gene Ther 2005；5(1)：10196Lee HTMechanisms of ischemic preconditioning and clinical implications for multiorgan ischemicreperfusion injury J Cardiothorac Vase Anesth 1999；13(1)：78917 Sun HY，Wang NP，Kerendi F，et a1Hypoxic postconditioning reduces cardio myocyte loss by inhibiting ROS generation and intracellular Ca2+ overload. 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Hepatology1999；30：12231231二研究内容1探讨IpostC抗I-R肝脏损伤的最佳保护作用模式建立大鼠I-R模型；研究不同的IpostC模式的抗I-R肝损伤作用；研究IpreC与IpostC联合应用的抗I-R肝损伤作用；2研究IpostC在I-R肝脏细胞死亡中的作用分子机制研究促凋亡分子Calpain和caspase-3在IpostC抗I-R损伤中的作用；研究在IpostC抗I-R损伤中肝细胞线粒体膜通透性和膜电位的变化三研究目标 以细胞死亡切入点，探讨Necrapoptosis通路中的相关分子calpain和caspase3的变化，揭示Ip