1、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
2、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
3、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
4、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
5、、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
6、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 cal
7、pain 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
8、-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
9、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 Gastr
10、oenterology 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 ZQ Corvera JS，Halkos ME，et a1Inhibition of myocardial injury by ischemic postconditioning during reperfusion：compa
11、rison 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)：s116 85Ritter T，Kupiec-Weglinski JWGene therapy for the prevention of ischemiareperfusion injury in organ transplantation Curr Gene Ther 2005；5(1)： 101
12、96Lee HTMechanisms of ischemic preconditioning and clinical implications formultiorgan ischemicreperfusion injury J Cardiothorac Vase Anesth1999；13(1) ：78917 Sun HY，Wang NP，Kerendi F，et a1Hypoxic postconditioning reduces cardio myocyte loss by inhibiting ROS generation and intracellular Ca2+ overloa
13、d. Am J Physiol Heart Circ Physiol 2005；288(4)：H1900-88Halkos ME，Kerendi F，Corvera JS，et a1Myocardial protection withD0stconditioning is not enhanced by ischemic preconditioningArm Thorac Surg2004；78(3) ：9619：discussion 9699 Kin HZhao ZQ，Sun HY，et al Postconditioning attenuates myocardial ischemia-r
14、eperfusion injury by inhibiting events in the early minutes of reperfusion Cardiovasc Res2004；62(1)：74-8510 D0senho VENagibin VS，Tumanovskaya LV，et a1Proteasome inhibitors eliminate protective effect of postconditioning in cultured neonatal cardiomyocytesFiziol Zh, 2006；52(3)：152411Fantinelli JC，Mos
15、ca SMComparative effects of ischemic pre and postconditioning on ischemia-reperfusion injury in spontaneously hypertensive rats(SHR) Mol Cell Biochem 2006；25Epub ahead of print】12Zhu M， Fang J，Lucchinetti E，et al Ischemic postconditioning protects remodeled myocardium via the P13K-PKBAkt reperfusion
16、 injury salvage kinase pathway. Cardiovasc Res2006；72(1)：1526213 Iliodromitis EK，Zoga A，Vrettou A，et a1The effectiveness of postconditioning and preconditioning on infarct size in hypercholesterolemic and normal anesthetized rabbitsAtherosclerosis 2006；188(2) ：3566214Tang XL，Sato H，Tiwari S，et al Ca
17、rdioprotection by postconditioning in conscious rats is limited to coronary occlusions45 minutes. Am J Physiol Heart Circ Physiol2006；30【Epub ahead of print】15Wang HC， ，Zhang HF，Guo WY，et a1Hypoxic postconditioning enhance the survival and inhibits apoptosis of cardiomyocytes following reoxygenation
18、：role ofperoxynitrite formation. Apoptosis, 2006；11(8)：14531460-16Ye S，Tang L，xu J，et a1Postcondhionins protection of THSG on cardiacischemia-reperfosion injury and mechanism J Huazhong Univ Sci Tecimolog Med Sci 2006；26(1) ：13617 Kloner RA，Dow J，Bhandari APostconditioning markedly attenuates ventri
19、cular arrhythmias after ischemia-reperfusion J Cardiovasc Pharmacol Ther 2006；1 1(1)：556318Heusch G，Buchert A，Feldhaus S，et al. No loss of cardioprotection by postconditioning in connexin 43-deficient mice. Basic Res Cardiol 2006；101(4) ：354-619 Crisostomo PR，Wang M，Wairiuko GM，et al Postconditionin
20、g in females depends on injury severity. J Surg Res2006；134(2)：342720 Philipp S，Yang XM，Cui L，et a1Postconditioning protects rabbit hearts through a protein kinase Cadenosine A2b receptor cascade. Cardiovasc Res 2006；70(2)： 3081421Bopassa JC，Ferrera R，Gateau-Roesch O，et a1PI 3-kinase regulates the m
21、itochondrial transition pore in controlled reperfusion and postconditioning. Cardiovasc Res2006；69(1)：1788522Schwartz LM，Lagranha CJ Ischemic postconditioning during reperfusion activates Akt and ERK without protecting against lethal myocardial ischemia-reperfusion injury in pigs. Am J Physiol Heart
22、 Circ Physi012006；290(3)：H1011-823Staat P, Rioufol G，Plot C，et al. Postnconditioning the human heart . Circulation 2005：1 12(14)：2143-824Kerendi F，Kin H， Halkos ME，et al. Remote postconditioning. Brief renal ischemia and reperfusion applied before coronary artery reperfusion reduces myocardial infar
23、ct size via endogenous activation of adenosine receptorsBasic Res Cardiol 2005；100(5) ：404-1225 Darling CE，Jiang R，Maynard M，et a1 Postconditioning via stuttering reperfusion limits myocardial infarct size in rabbit hearts：role of ERKI2Am J Physiol Heart Circ Physi012005；289(4)：H16182626 Yang XM，Phi
24、lipp S，Downey JM，et a1Postconditionings protection is not dependent on circulating blood factors or ceils but involves adenosine receptors and requires P13kinase and guanylyl cyclase activationBasic Res Cardiol 2005；100(1) ：576327，Yang XM，Proctor JB，Cui L，et al. Multiple，brief coronary occlusions du
25、ring early reperfusion protect rabbit hearts by targeting cell signaling pathways J Am Coll Cardiol2004；44(5)：11 03-1028Tsaug AHausenloy DJ，Mocanu MM，et al. Postconditioning：a form of “modified reperfusion” protects the myocardium by activating the phosphatidylinositol 3-kinaseAkt pathway . Circ Res
26、2004；95(3)：230229 Galagudza M，Kurapeev D，Minasian S，et al . Ischemic postconditioning：brief ischemia during reperfusion converts persistent ventricular fibrillation into regular rhythmEur J Cardiothorac Surg2004；25(6) ：1006-1030 Loukogeorgakis SP，Panagiotidou AT，Yellon DM，et a1 Postconditioning prot
27、ects against endothelial ischemia-reperfusion injury in the human forearmCirculation 2006；113(7)：1015-931Sun k Liu ZS，Sun Q Role of mitochondria in cell apoptosis during hepatic ischemia-reperfusion injury and protective effect of ischemic posttconditioning World J Gastroenterol 2004：10(13)：1934-832
28、 Wang N，Ma QJ，Lu JG，et al. Protective effect of ischemic posttconditioning on ischemia-reperfusion injury of rat liver graft. Zhonghua Wai Ke Za Zhi 2005；43(23)：1533633 Zhan H，Sapolsky RM，Steinberg GKInterrupting reperfusion as a stroke therapy：ischemia postconditioning reduces infarct size alter fo
29、cal ischemia in ratsJ CerebBlood Flow Metab 2006；26(9)：1114-2134Vinten-Johansen J，Zhao ZQ，Zatta AJ，et a1Postconditioning-A new link inNatures armor against myocardial ischemia-reperfusion injury. Basic Res Cardiol 2005；100(4) ：295-31035 Hausenloy DJ，Yellon DMNew directions for protecting the heart a
30、gainst ischemiareperfusion injury：targeting the reperfusion injury salvage kinase pathway. Cardiovasc Res2004；61：44846036Kin H，Zatta AJ，Lolye MT，et al. Postconditioning reduces infarct size via adenosine receptor activation by endogenous adenosineCardiovasc Res 2005；67(1)： 124-3337Argaud L，Gateau-Ro
31、esch O，Raisky 0，et a1Postconditioning inhibits mitochondrial permeability transitionCirculation 2005；11l(2) ：194-738Gatean-Roesch 0，Argand L，Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovase Res2006 ；70(2)：2647339 Jaeschke HMechanisms of reperfusion injury after wa
32、rm ischemia of the liver. J Hepatobiliary Pancreat Surg1998；5(4) ：402-840Kang KJMechanism of hepatic ischemiareperfusion injury and protection against reperfusion injuryTransplant Proc2002；34(7)：26596141Pannen BHNew insights into the regulation of hepatic blood flow after ischemiaand reperfusionAnes
33、th Analg 2002；94(6)：14485742Sakon M，Ariyoshi H，Umeshita K，et aI Ischemia-reperfusion injury of the liverwith special reference to calcium-dependent mechanismsSurg Today 2002；32(1)： 1-1243 CasillasRamimz A， Mosbah 1 IB，Ramaiho F，et a1Past and future approaches to ischemia-reperfusion lesion associate
34、d with liver transplantationLife Sci2006；79(2 0) ：1881-1894 44 Lemasters JJ. Necrapoptosis and the mitochondrial permeability transition：shared pathways to necrosis and apoptosisAm J Physiol 1999；276(1 Pt 1)： G1-645Lemasters JJ ，Qian T，He L，et a1 Role of mitochondrial inner membranepermeabilization
35、in necrotic cell death，apoptosis，and autophagy. Antioxid RedoxSignal2002；4(5)：769-8146Jaeschke H，Lemasters JJ Apoptosis versus oncotic necrosis in hepatic ischemiareperfusion injuryGastroenterology 2003；125(4)：12465747Yin XM Bid, a BH3-only multi-functional molecule，is at the cross road of life and
36、deathGene 2006；369：71948 Griffiths EJ，Halastrap AP. Mitochondrial non-specific pores remain closed during cardiac ischaemia but open upon reperfusion Biochem J1995；307：939849Piper HM，Meuter K，Sehafer CCellular mechanisms of ischemia reperfusioninjuryAnn Thorac Surg 2003；75：$644 一$644850Di Lisa FMena
37、bo R，Canton M，et al Opening of the mitochondrial permeabilitytransition pore causes depletion of mitochondrial and cytosolic NAD+and is a causative event in the death of myocytes in postischemic reperfusion of the heartJBi01 Chem 2001；276：2571-257551Soeda JMiyagawa S，Sano K，et a1Cytochrome c release
38、 into cytosol withsubsequent caspase activation during warm ischemia in rat liverAm J PhysiolGastrointest Liver Physi012001；281(4) ：G1 1 152352Hirakawa A，Takeyama N，Nakatani T，et al. Mitochondrial permeability transition and cytochrome e release in ischemia-reperfusion injury of the rat liverJ Surg
39、Res 2003；1 1 1(2)：240753MOrin DPires F，Plin C，ct a1Role of the permeability transition pore incytochrome C release from mitochondria during ischemia-reperfusion in rat live. rBiochem Pharmac012004；68(10)：2065-7354Barone S，Okaya T，Rudich S，et al. Distinct and sequential upregulation of genes regulati
40、ng cell growth and cell cycle progression during hepatic ischemia-reperfusion injury. Am J Physiol Cell Physiol 2005；289(4)：C826-3555Xu WH，Ye QF，Xia SS. Apoptosis and proliferation of intrahepatic bile duct after ischemia-reperfusion injuryHepatobiliary Pancreat Dis Int2004；3(3)：428-3256Cai FG， Xiao
41、 JS，Ye QF Effects of ischemic preconditioning on cyclin D1 expression during early ischemic reperfusion in ratsWorld J Gastroenterol, 2006；12(18) ：29364057Jeay SSonenshein GE，Postel Vinay MC，et al. Growth hormone can act as a cytokine controlling survival and proliferation of immune cells：new insigh
42、ts into signaling pathwaysMol Cell Endocrinol, 2002；188(12)：1-758 Burgering BM，Kops GJ. Cell cycle and death control：long live Forkheads. Trends Biochem Sci20 。2；27(7)：3526059Liang J，Slingerland JM. Multiple roles of the P13KPKB(Akt)pathway in cellcycle progression. Cell Cycle2003；2(4) ：339-4560 Kim
43、 DChung J. Akt：versatile mediator of cell survival and beyond. J Biochem Mol Bi012002；35(1)：1061561 Yoon S，Seger R. The extracellular signal-regulated kinase：multiple substratesregulate diverse cellular functions. Growth Factors2006；24(1) ：21-4462Chuderland D，Seger R. Proteinprotein interactions in
44、the regulation of theextracellular signalregulated kinase. Mol Biotechnol 2005；29(1) ：57-7463SchumackerPT Hypoxlainducible factor-1 (HIF1). Crit Care Med2005；33(12) ：$42342564Koniaris LGMcKillop IH，Schwaz SI，Zimmers TALiver regeneration. J Am Coll Surg2003；197(4)：6345965 Zimmermann A Regulation of l
45、iver regeneration. Nephrol Dial Transplant2004；19 Suppl 4：iv6 1066 Taub RLiver regeneration：from myth to mechanismNat Rev Mol Cell Biol2004；5(10) ：836-47 67FauatoN. Liver regenerationJHepat012000；32(1 Suppl)：193168Kerem M，Bedirli A，Ofluoglu E，et a1Ischemic preconditioning improves liverregeneration
46、by sustaining energy metabolism after partial hepatectomy underischemia in rats Liver International 2006；26：99499969 Mehendale HMand Limaye PB Calpain： a death protein that mediates progression of liver injuryTRENDS in Pharmacological Sciences 2005；26(5)：23223670K0hli V，Gan W，Camargo CA Jr，et a1Calp
47、ain is a mediator of preservation-reperfusion injury in rat liver transplantationProc Natl Acad Sci1997；94：9354-5971Kohli V，Madden JF，Bentley RC，et al. Calpain mediates ischemic injury of the liver through modulation of apoptosis and necrosis. Gastroenterology 1999 1 16：168-78721nsene JGarcia Dorado
48、 D，RuizMeana M，et a1Ischemic preconditioningattenuates calpainmediated degradation of structural proteins through a protein kinase A-dependent mechanism. Cardiovascular Research 2004；64：10511473Harw00d SM，Yaqoob MM and Allen DACaspase and calpain function in celldeath：bridging the gap between apoptosis and necrosis. Ann Clin Biochem 2005；42： 415-43l74Chen M，Won DJ，Kr
