摘要
一、研究背景
在临床上,一些恶性肿瘤如:胰头癌、胆管癌(Ⅰ型)等,常以黄疸为首发症状,肿瘤常早期侵犯门静脉,导致较低的根治性切除率。近年来,联合门静脉切除重建的胰十二指肠切除术得到了较为广泛的认同,极大地提高了这类肿瘤的切除率,但是术中门静脉血流急性阻断( portal vein occlusion,PVO )对合并有梗阻性黄疸( obstructive jaundice, OJ )的肝脏必然造成进一步损害。目前,对肝脏缺血再灌注损伤(ischemia reperfution injury,IRI)的研究较多,但对于合并有OJ的PVO对肝脏引起的IRI的研究还未见报道。因此深入研究OJ情况下,PVO后肝脏病理生理改变的分子机制以及肝脏耐受PVO的安全时限,对临床如何把握PVO时限、提高手术耐受性、安全性、促进术后恢复具有重要意义。
研究认为肝脏的IRI主要以肝细胞凋亡为特征的损害,为防止肝脏的IRI,原位低温灌注技术得到了广泛的应用,大大提高了肝脏耐受缺血的时限。常用的灌注液有乳酸林格氏液、HTK液及UW液等,取得了一定减轻IRI的效果,但以往的原位低温灌注技术也有许多不足之处:1、技术操作复杂难度大,2、易引起严重低血压、肺水肿、高钾血症、酸中毒、DIC(disseminated intravascular coagulation)等并发症,3、HTK和UW液不能进入体循环。由此我们自行配制了一种自制肝保护液( self-made liver solution,SMLS )以用于合并有OJ且侵犯门静脉的肿瘤切除术中,PVO期间进行在体经门静脉持续低温灌注,观察其是否具有肝保护作用并进一步探明其可能的机制。
二、目的
本课题拟通过对门静脉转流下, OJ大鼠PVO期间,用4℃SMLS经门静脉在体持续灌注肝脏,观察A20mRNA及蛋白的表达,以及MAPKs的活化与线粒体凋亡途径上各相关蛋白的表达情况的关系,以期阐明OJ条件下,PVO肝脏损伤机制以及SMLS对肝保护作用及其机制。
三、方法与结果
1、常温下OJ大鼠PVO模型的建立及其对肝脏能量代谢的影响:正常大鼠被随机分为SO(Shame Operation)组、OJ大鼠再随机分为A组(胆道再通组)、B组(胆道再通-PVO 30min)、C组(胆道再通-PVO 60min)及D组(胆道再通-PVO 90min)。结果:D组术后一周大鼠累计病死率60%,而SO、A、B及C组病死率为0%。D组ALT、AST、TBIL在术后各时相点都显著高于SO、A、B、C组,且在术后24h仍无下降趋势(P<0.05);通过反应肝细胞线粒体能量代谢的指标观察,D组RCR、P/O、ATP值在术后各时相点都明显低于SO、A、B及C组;肝组织HE染色光镜观察即显示,D组肝组织结构严重紊乱,肝细胞广泛空泡化及坏死,炎细胞浸润,淤胆较重,明显重于其它各组。
2、SMLS对OJ大鼠PVO肝损伤的保护:OJ大鼠被随机分为E组(胆道再通、门静脉周围游离、切除肝尾状叶,90 min后关腹)、F组(胆道再通、肝尾状叶分流门静脉、余肝PVO 90 min、恢复正常门静脉血流、切除肝尾状叶,关腹)、G组(F组+门静脉肝侧灌注4℃乳酸林格氏液)、H组(F组+门静脉肝侧灌注4℃SMLS );术后各组ALT、AST和TBIL水平均呈升高趋势,均在术后6 h达高峰,在术后24 h有所下降,其中F、G及H组升高较E组明显且有显著性差异(P<0.05),而F组及G组又显著高于H组,F组高于G组之间有显著性差异(P<0.05)。未缺血的E组大鼠术后因胆道再通其内毒素( ETX )水平,在术后各时相点呈逐渐下降趋势,说明胆道梗阻在本实验中是ETX增高的主要因素,而胆道再通是有利于消除ETX的重要措施;在受到PVO损伤的F组其ETX在术后各时相点呈大幅度的逐渐增高,到术后72 h亦无明显下降趋势;而在给予门静脉灌注的G组和H组在术后虽呈逐渐增高趋势,但增高的幅度不如F组大,有显著性差异(P<0.05),且G组和H组到术后24 h开始逐渐下降;而给予SMLS的H组其ETX在术后各时相点均低于给予乳酸林格氏液灌注的G组,有统计学意义(P<0.05)。肝组织HE染色光镜观察,F组病理改变明显重于其它各组;透射电镜观察,F组线粒体损伤亦明显重于其它各组;TUNEL凋亡检测,各组术后0 h即有肝细胞凋亡,但E组在术后各时相点呈逐渐下降趋势;相反,F组、G组和H组均呈逐渐升高趋势,在术后6 h达到高峰,在术后24 h有所下降,其中F组和G组明显高于H组并有显著性差异(P<0.05)。F组高于G组且有显著性差异(P<0.05)。
3、MAPKs信号转导途径在SMLS保护肝细胞中的作用及机制:采用逆转录PCR、Western blotting及TUNEL等技术分别检测A20mRNA及其蛋白、P-JNK、P-ERK、P-p38、bcl-2、Bax、caspase-3等蛋白以及肝细胞凋亡百分率的检测,同时给予MAPK信号转导途径的特异性抑制剂SP600125(JNK特异性抑制剂)、PD98059(ERK特异性抑制剂)和SB203580(P-38特异性抑制剂)在灌注SMLS前30min,经尾静脉注射作为预处理,再次观察各组肝细胞凋亡的变化;结果显示,H组A20mRNA,A20、P-ERK及bcl-2蛋白显著高于E、F及G组(P<0.05),其凋亡率却相反,G组又显著高于F组(P<0.05),而F组P-JNK、Bax、caspase-3蛋白及凋亡率显著增高,与E、G及H组比有显著性差异(P<0.05),当被给予特异性抑制剂后,给予SP600125时肝细胞凋亡进一步减少,而给予PD98059时肝细胞凋亡增加,给予SB203580时肝细胞凋亡无明显变化。
四、结论
1、梗阻性黄疸大鼠门静脉血流急性阻断引起肝细胞线粒体能量代谢障碍;
2、初步判定梗阻性黄疸大鼠在门静脉转流下,耐受门静脉血流阻断的安全时限为60 min;
3、肝细胞凋亡是梗阻性黄疸条件下,门静脉血流急性阻断对肝脏造成的缺血再灌注损伤引起肝损害的一种重要方式。线粒体途径可能介导了肝细胞的凋亡;
4、经门静脉在体灌注低温乳酸林格氏液或自制肝保护液,可以减轻梗阻性黄疸大鼠因门静脉血流急性阻断导致的肝细胞线粒体损伤及肝细胞凋亡。其中自制肝保护液的肝保护作用明显优于乳酸林格氏液;
5、梗阻性黄疸大鼠门静脉血流急性阻断激活JNK信号转导通路,介导线粒体途径bcl-2/bax蛋白平衡的偏移,活化下游的凋亡执行蛋白caspase-3,从而促进肝细胞凋亡;
6、自制肝保护液经门静脉在体持续低温灌注肝脏,可以促进A20mRNA及其蛋白的高表达,由此激活ERK信号转导通路,抑制JNK信号转导通路,反向调节线粒体途径bcl-2/bax蛋白平衡的逆转,降低了其下游的凋亡执行蛋白caspase-3的表达,从而起到抗凋亡、保护肝细胞功能的作用。
Background
Jaundice is always the most common symptom in patients with malignant tumors such as pancreatic carcinoma,cholaniocarcinoma and so on.The character of special location and early portal vein invasion always lead to low radical resection rate.In recent years,the application of pancreaticcoduodenectomy with portal vein resection in managing these cancers has been improved.But acute portal vein occlusion(PVO) can cause progressive damage to liver especially in patients with obstructive jaundice(OJ).Until now,there is no report about hepatic ischemical reperfusion injury caused by PVO in patients with OJ.Therefore ,profound research of hepatic pathophisiology changes after PVO and safe time limit to PVO in rats with OJ has important influence on promoting the safety and toleration of operation and the following recovery.
Previous studies showed that apoptosis was the main character of hepatic ischemical reperfusion injury.Orthotopic hypothermical perfusion was widely used to prevent ischemical reperfusion injury that can promote hepatic tolerance of ischemia.At present,mainly used perfusion solution included Lactated Ringer'S Solution,HTK solution,UW solution and so on.These solutions can lessen ischemical reperfusion injury to some extent.But whole hepatic blood flow blockage with continuous orthotopic hypothermic perfusion also had some disadvantages:First、this technique has big difficulty and multiplicity;Second、it can cause complication such as severe hypotension, edema of lung,depression of potassium,acidosis,DIC and so on.3、HTK and UW solutions can’t exist in systemic circulation.Then we used a hepatic preservation solution(self-made liver solution,SMLS) and assessed its effect and mechanism in protecting hepatocytes .
Objective
The mRNA level and expression of A20 along with the relation between the activation of MAPKs and the expression of related proteins in mitochondria mediated apoptosis pathway were observed in rats with OJ and hepatic continuous hypothermic perfusion with SMLS after PVO to elucidate the mechanisms of hepatic damage by PVO in rats with OJ and protection by SMLS.
Methods and results
1、Animal model and effect of PVO on hepatic energy metabolism:normal rats were randomly divided into four groups:(1)group SO:shame operation;(2)group A:biliary tract recanalization;(3)group B:biliary tract recanalization with PVO 30m;(3)group C:biliary tract recanalization with PVO 60m;(3)group D: biliary tract recanalization with PVO 90m.Summary mortality of group D was 60 percent which was obviously higher than that of other groups at 1 post operation week. ALT,AST and TBIL of group D were marked higher than those of other groups and the tendency of decreasing was not showed at 24h post operation.RCR,P/O and ATP of group D were also lower than those of other groups.Hepatic tissue disorder,extensive vacuolization and necrosis of hepatocyte,infiltration of inflammatory cells and cholestasis were still obvious in group D.
2、Protective effect of SMLS on hepatic function in rats with OJ and PVO:rats with OJ were randomly divided into four groups:group E(biliary tract recanalization,liberation of portal vein,resection of caudate lobe of liver and closure of abdomen at 90m ).group F(biliary tract recanalization,portal vein bypass in caudate lobe of liver,PVO of left hepatic tissue 90m,then recovery of portal vein flow and resection of caudate lobe of liver).group G(group F with perfusion of 4℃Lactated Ringer'S Solution).group H(group F with portal vein perfusion of 4C SMLS).The level of ALT,AST and TBIL of all groups were heightened post operation and got to peak at 6h post operation,and then declined after 24h post operation.The increasing value of ALT,AST and TBIL in group F,group G and group H were obviously higher than that of group E.Because of no PVO,the level of ETX of group E gradually descended which means obstruction of biliary tract is the main reason for increasing of ETX. and biliray tract recanalization is of advantage to decrease ETX.In group F,the level of ETX markedly increased post operation and no tendency of decreasing was showed even at 72h post operation.In group G and group H,the level of ETX also increased post operation but amplitude of increasing was lower than that of group F(p<0.05).The level of ETX in group H was lower than that of group G at every time point post operation(p<0.05).All groups showed hepatic apoptosis post operation by TUNEL.Group E showed a tendency of decreaing but group F,groupG and group H had the adeverse tendency.
3、MAPK signal transduction pathway mediating protection of hepatocyte by SMLS: reverse transcription PCR,Western blotting and TUNEL were used respectively to determine A20 mRNA,A20,P-JNK,P-ERK,P-p38,bcl-2,Bax, caspase-3 and hepatocyte apoptotic rate.Specific inhibitor of MAPK signal transduction pathway SP600125(specific inhibitor of JNK), PD98059(specific inhibitor of ERK)and SB203580(specific inhibitor of p38)were also used in the experiments.The results showed that A20 mRNA and protein level of A20,P-ERK and bcl-2 were obviously higher than those of other groups(p<0.05).But the apoptotic index showed an adverse tendency in which the apoptotic index of group G was marked higher than that of group F(p<0.05).P-JNK,Bax,caspase-3 and apoptotic index of group F were different from those of other groups(p<0.05).
Conclusion
1、Acute occlusion of portal venous flow in rats with obstructive jaundice can cause mitochondrial energy metabolic block in liver cells.
2、The safe tolerant time limit to portal vein ligation in rats with obstructive jaundice and portal flow bypass is sixty minutes.
3、Apoptosis of hepatic cells is the mainly representation of ischemia reperfusion injury to liver by portal vein ligation in rats with obstructive jaundice.
4、Hepatic apoptosis caused by portal vein ligation in rats with obstructive jaundice is decreased by portal vein perfusion in low temperature with SMLS.
5、Acute portal vein ligation can promote hepatic apoptosis by activating JNK signal transduction pathway,mediating the offset balance of bcl-2/bax protein and activating downstream apoptosis-related protein caspase-3.
6、The expression of anti-apoptosis protein A20 is activated by hepatic continuous hypothermical perfusion with SMLS,then ERK signal transduction pathway is activated to regulate the balance of bcl-2/bax protein and decrease the expression of apoptosis executive protein caspase-3.
引文
1. Lebrec-D, et al. Hemodynamic Changes in patients with portal venous obstruction .Hepatology, 1983,3(4):550
2.赵建勇,董家鸿,詹国清等。猪单纯门静脉阻断安全时限的研究。第三军医大学学报2001,23(8):965-967
3.王万铁,徐正,林丽娜。家兔门静脉阻断和再开放对心脏血液动力学的影响。温州医学院学报1995,25(1):4-6
4. Farkouh EF, Daniel AM, Beandoin JG, et al. Predictive value of liver biochemistry in acute hepatic ischemia [J].Surg Gynecol Obstet ,1971,321:832-838
5.陈孝平,吴在德,裘法祖.家兔常温下阻断肝门最大耐受时限的实验研究[J].中华外科杂志,1984,22:153-155
6.董家鸿,何效东,李昆等,大鼠门静脉转流下耐受入肝血流阻断的安全时限[J].消化外科2002,1(1):20-24
7. Pichlmayr R, Hauss HG, Vbernatis G ,et al .Technique and preliminary results of extracorporeal liver surgery ( bench procedure) and of surgery on the insitu perfused liver [J] . Br J Surg ,1990,77 (1) :21~26
8.曹绣虎,黄洁夫.常温与低温灌注下的全肝血流阻断比较实验[J].中山医科大学学报,1990,2(1):49~51
9. Yamanaka N, Okamoto E. Topical cooling as an adjunct for hepatectomy with inflow occlusion[J]. Nippon Geka Gakkai Zasshi. 1998 Apr;99(4):223-228
10. van Gulik TM, de Graaf W, Dinant S. Vascular Occlusion Techniques during Liver Resection. Dig Surg. 2007;24(4):274-81. Epub 2007 Jul 27.
11.董家鸿.全肝血液转流下的肝切除术中华普通外科杂志,2000,1:54
12. Barakat O, Hoef J, Ozaki CF. Extended right trisegmentectomy using in situ hypothermic perfusion with modified HTKsolution for a large intrahepatic cholangiocarcinoma. J Surg Oncol. 2007 Jun 1;95(7):587-92.
13. Yamanaka N, Okamoto E, Furukawa K. Hepatic resection under in situ hemihepatic hypothermic perfusion with hepatoprotective agents. Hepatogastroenterology. 1995 Feb;42(1):1-4.
14. Kaiho T, Tanaka T, Tsuchiya S,. Hepatic resection under in situ hypothermic hepatic perfusion. Hepatogastroenterology. 2003 May-Jun;50(51):761-5.
15. Takeuchi T, Egawa H, Yamaoka Y. A prolongation of hepatic vascular exclusion by in situ hypothermic perfusion in dogs. Arch Surg. 1992 Apr;127(4):427-31.
16. Gruttadauria S,Marsh JW,Bartlett DL, et al,Ex situ resection techniques and liver autotransplantation last resource for otherwise unresectable malignancy[J]. Surgery.2000,127(5):520-527.
17. van Gulik TM, de Graaf W, Dinant S. Vascular Occlusion Techniques during Liver Resection. Dig Surg. 2007;24(4):274-81. Epub 2007 Jul 27.
18.高昌俊,李开宗,管文贤.维拉怕米对大鼠肝脏低温保存再灌注损伤的防护机制。第四军医大学学报,2001,22(19):1744-1747
19. Tazzi B, et al. Oxygen eadical injury and loss of highenergy compoumds in anixic and reperfused rat heaet:prevention by exogenous fructose -1,6-bisphosphate.Free Radic Res Commun,1990;10:167
20. Weiss J, Heltbrand E. Function compartmentation of glycolytic versus oxidative metabolism in isolated rabbit hesrt.J Clin Invest,1985;75:436
21. Sasaki H, Matsuno T, Tanaka N. Activation of apoptosis during the reperfusion phase after rat liver ischemia [J]. Transplant Proced,1996,289:1908-1909.
22. Gao W, Bentley RC, Madden JF, Clavien JF,Clavien PA. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation [J].Hepatology,1998,27:1652-1660.
23. Yue XJ, Song WD, Jiang XJ. The mitochondrial localization of tumor suppressor PTEN promotes apoptosis in A431 cells. Wei Sheng Wu Xue Bao. 2007 Dec 4;47(6):968-72.
24. Gateau-Roesch O, Argaud L, Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res. 2006 May 1;70(2):264-73.
25. Xu M, Wang Y, Ayub A, Ashraf M. Mitochondrial K(ATP) channel activation reduces anoxic injury by restoring mitochondrial membrane potential. Am J Physiol Heart Circ Physiol. 2001 Sep;281(3):H1295-303
26. Wang XD. Cory S,Adama JM. Bcl-2 gene promotes haemopoietic ceU survivaland cooperstes withc-myc to immortalize pre-B cells. Nature,1988,335:440~442
27. Arvelo MB et al.Hepatology.2002,35(3):535-543
28. He KL, Ting AT.A20 inhibits tumor necrosis factor (TNF) alpha-induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in Jurkat T cells.Mol Cell Biol,2002,22(17):6034-6045.
29. Lu GD, Shen HM, Chung MC,et al. Critical role of oxidative stress and sustained JNK activation in aloe-emodin-mediated apoptotic cell death in human hepatoma cells. Carcinogenesis. 2007 Sep;28(9):1937-45.
30. Wada T, Penninger JM. Mitogen-activated protein kitein kinases in apoptosis regulation [J].Oncogene,2004,23(16):2838-2849.
31. Seger R,Krebs E G The MAPK signaling cascade [J].FASEB J,1995,9(9):726-735.
32.端木德强,李中海,王敬泽.MAPK信号转导通路研究进展[J].细胞生物学杂志,2002,24(3):151-155。
33.姜勇,龚小卫.MAPK信号转导通路对炎症反应的调控[J].生理学报,2002,52(4):267-271.
34.牟金叶,陈晓光.促分裂原激活的蛋白激酶(MAPK)信号转导通路的研究进展[J].生命科学,2002,14(4):208-210.
35.黄轶峰,龚开政,张振刚.p38MAPKα/β和ERK1/2在心肌缺氧预处理信号传递中的不同作用[J].生理学报,2003,55(4):454-458.
36. Isozaki H, Okbayashi M, et al.Experimental study of liver injury after partial hepatectomy with intermittent or continous hepatic vascular occlsion [J].Eur Surg Res,1995;27(2)313
37. Gonzalez-Elecha B,Cutrin JC, Boveris A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion [J].J Clin Invest,1993;91(2)456
38.何效东,董家鸿,蔡景修等,门静脉转流下入肝血流阻断模型的建立及评价[J].西北国防医学杂志,1999;20(3):175
39. Delva E,Barberousse JP, Nordlinger B ,et al. Hemodynamic and biochemical monitoring during major liver resection with use of hepatic vascular exclusion [J].Surgery,1984;95(3):309
40.沈世强,王竹平,严际慎,犬肝切除时部分门静脉低温灌注对肝脏的保护作用[J].湖北医科大学学报,1999;20(4):319-321
41. Ferri M ,Gabriel S, Gavelli A, et al. Bacterial translocation during portal clamping for liver resection: a clinical study [J].Arch Surg, 1997;132(FEB):162
42. Lebrec-D ,et al .Hemodynamic Changes in patients with portal venous obstruction.Hepatology,1983,3(4):550
43.王万铁,等.门静脉阻断与开放对心脏血流动力学的影响.温州医学院学报,1995,25(1):4
44. Deitch EA,et al .AM J Surg,1990;159:79
45. Greve JW, et al .Gastroenterology,1990;98:478
46. Scotter-conner CEH ,et al. Am J Surg,1989;157:210
47. Mckeown CMB ,Edwards V ,Philips MJ, et al .Sinusoidal lining cell damage:the critcal injury in cold preservation of liver allograft in the rat .transplantation,1988,46:178-191.
48. Galdwell-kenkel JC,Thurman RG,Lemasters JJ.Selective loss of nonparenchymal cell viability after cold ischemic strrage of rat liver. Transplantation ,1998,45:834-837
49.王立明,刘浩,刘永峰,等.肝脏低温保存时肝窦内皮细胞损伤的研究.中华器官移植杂志,1997,18:221-222.
50. Rokicki M, Rokicki W .Liver regeneration in rats after complete and partial occlusion of the portal blood influx [J].Res Exp Med (Berl),1993;193(5)305
51. Edgcomb LP, Eckhauser FE ,Portacaval transposition on hepatocelluar and hepatic reticuloendothelial cell activity in the dog [J].J Surg Res,1984;36(5)446
52. Ding WX ,Yin XM, Dissection of the multiple mechanisms of TNF-alph-induced apoptosis in liver injury [J].J Cell Mol Med,2004,8:445-454
53. Neuman MG.Apoptosis in diseases of the liver [J]. Crit Rev Clin Lab Sci 2001,38:109-166
54. Jiang H,Meng E,Li I, et al Anti-apoptosis effects of oxymatrine protect the liver from warm ischemia reperfusiong injury in rats [J].World J Surg 2005,29:1397-1401.
55. Lebrec-D ,et al .Hemodynamic Changes in patients with portal venous obstruction.Hepatology,1983,3(4):550
56. Kaufmann SH, Earnshaw. Induction of apoptosis by cancer chemotherapy. Exp Cell Res,2000,256(1):42-49.
57. Lemasters JJ,Qian T, He L,et al.Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis ,and autophagy .Antioxid Redox Signal 2002;4:769-781
58. Zhai SQ, Wang DJ, Wang JL, et al. Basic fibroblast growth factor protects auditory neurons and hair cells from glutamate neurotoxicity and noise exposure. Acta Otolaryngol. 2004 , 124(2):124-129
59. Zhai SQ, Cheng JC, Wang JL,et al. Protective effect of basic fibroblast growth factor on auditory hair cells after noise exposure. Acta Otolaryngol. 2002, 122(4):370-373
60. Sekiya T, Shimamura N, Yagihashi A, et al. Effect of topically applied basic fibroblast growth factor on injured cochlear nerve. Neurosurgery. 2003, 52(4):900-907;
61.王大君,韩东一,杨伟炎等.碱性成纤维细胞生长因子对耳蜗螺旋神经节细胞谷氨酸钠毒性作用的影响.中华耳鼻咽喉科杂志, 1998, 33(3):165-168.
62. Devarajan P,Savoca M, Castaneda MP, et al .Cisplatin-induced apoptosis in auditory cell:role of death receptor and mitochondrial pathways.Hear Res.2002,174(1-2):45-54.
63. Aran D, Sanchez Y, Fernandez C, et al. Arsenic trioxide sensitizes promonocytic leukemia cells to TNF alpha-induced apoptosis via p38-MAPK-regulated activation of both receptor-mediated and mitochondrial pathways.
64. Hong HY,Kim BC. Mixed lineage kinase 3 connects reactive oxygen species to c-Jun NH2-terminal kinase-induced mitochondrial apoptosis in genipin-treated PC3 human prostate cancer cells.Biocen Biophys Res Commun .2007;362:307-312.
65. Park EJ, Zhao YZ, Kim YC, et al. Bakuchiol-induced caspase-3-dependent apoptosis occurs through c-Jun NH2-terminal kinase-mediated mitochondrial translocation of Bax in rat liver myofibroblasts.Eur J Pharmacol.2007;559(2-3):115-23.
66. Okada Y.Experimental study on the interruption of hepatic blood flow in obstructive jaundice,with special reference to the cause of death and prolonged decompression [J].Nippon Geka Hokan ,1989,58(3):275-288.
67. Ers?z S,Cakmakh S, Gecim E,et al.The safety of total hepatic vascular occlusion in rabbits with acute extrahepatic cholestasis [J]. Eur Surg Res,1996,28(2):104-110.
68. Itoh H,Miyazaki M,Udagawa I,et al, Exerimental study on cell damage of ischemic liver in obstructive jaundiced rats[J].Nippon Shokakibyo Gakkai Zasshi,1991, 88(5):1200-1203.
69. Schnabl B,Bradham CA,Bennett BL.TAK1/JNK and p38 have opposite effects on rat hepatic stellate cells.Hepatopogy,2001,34:953-963.
70. Bennett BL,Sasaki DT,Murray BW,et al.SP600125,an anthrapyrazolone inhibitor of Jun N-terminal kinase.Proc Natl Acad Sci USA,2001,98:13681-13686.
71. Bruggen TV,Ni jenhuis S,Ruaij EV,et al.Lipopolysaccharide induced necrosis factor alpha production by human monocytes involves the Raf/MEK1-MEK2/ERK1-ERK2 pathway.Infect Immun,1999,67(8):3824-3829.
1. Garcea G,Gescher A,Steward W,et al Oxidactive scress in humans following the Pringle manoeuvre[J].Hepatobiliary Pancreat Dis Int,2006,5(2):210-214
2. Lemasters JJ,Qian T, He L,et al.Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis ,and autophagy .Antioxid Redox Signal 2002;4:769-781
3. Kaibori M,Yanagida H, Uchida Y, et al. Pirfenidone protects endotoxin-induced liver injury after hepatic ischemia in rats. Transplant Proc2004;36(7):1973-1974
4.王磊,于丽萍,窦科峰,等。内毒素在肝脏缺血再灌注中的作用。中国医师杂志,2004,6(11):1467-1469
5. Travis S, Menzies I. Intestinal permeability :functional assessment and significance[J]. Clin Sci Colch,1992,82(5):471-488.
6. Ferri M,Cavelli A,et al .Bacterial translocation during portal clamping for liver resection : a clinical study [J].Arch Surg ,1997,132(2):162-165.
7. Deitch E A ,Bridges W ,Baker J ,et al .Hemorragic shock induced bacterial translocation in reduced by xanthine oxidase inhibition of inactivation [J]. Surgery,1988,104(2):191-198.
8. Shibayama Y. The relation of endotoxaemia of death and fetal hepatic damage following obstruction of the portal vein [J]. J Pathol,1990,160(4):355-360.
9. Willerms D,Cadranel S, Jacobs W. Measurement of urinary sugars by HPLC in the estimation of intestinal permeability:evaluation in pediatric clinical practice[J]. Clin Chem,1993,39(5):888-890.
10. Dumas F,Aussel C,Pernet P,et al. Cas chromatography applied to lactulose-mannitol intestinal permeability test[J]. J Chromatogr B Biomed Sci appl,1994,654(2):276-281.
11. Shippee R L, Johnson A A,Cioffi W G,et al .Simultaneous determination of lactulose and mannitol in urine of burn patients by gas-liquid chromatoggaphy[J].Clin Chem,1992,38(3):343-345.
12. Ryan C M,Yarmush M L,Burke J F,et al .Increased gut permeability early after burns correlates with the extent of burn injury [J]. Crit Care Med ,1992,20(11):1508-1512.
13.韩德五.肠源性内毒素血症所致“继发性肝损伤”的临床依据.世界华人消化杂志,1999,7(12):1055-1058.
14. Wang F, Wang L Y, Wright D, et al .Redox imbalance differentially inhibits lipopolysaccharide-induced maerophage activation in the mouse liver .Infect Immun,1999,67:5490-5416.
15. Eubanks J W, Sabek O, Koth M, et al .Acute pancreatitis induces cytokine production endotoxirr-resistant mice[J].Ann Surg,1998,227(6):904.
16. Kawamoyo S, Tashiro S, Miyauchi Y,Inoue M Mechanism for enterohepatic damage cansed by crirculatory disturbance of hepatioc vessels in the rat.Proc Soc Exp Bio Med,1991,198:629-635.
17. Lei M G, Stimpson SA, Morrison DC.Specific endotoxic lipopolysacc-haride-binding receptors on murine splenocytesⅢbinding specificity and characterization .J Immunol,1991,147:1925-1932.
18.张效杰,曹晖,凌伟,等.缺血预处理对肝脏缺血再灌注损伤中内毒素血症的影响.中华普通外科杂志,2000,15(1):50-53.
19. Curran RD, Billiar TR,Studehr DJ, et al.Multiple cytokines are required to indude hepatocyte nitric oxide production and inhibit total protein synthesis .Ann of Sur,1990,212(4):462-470.
20.王景华,孙兆黎,杨贵贞,等.一氧化氮与自身免疫病.国外医学生理、病理科学与临床分册;1997,17(1):58-60.
21. Davies M G ,Hagen PO. Systemic inflammatory response syndrome. Br J Surg ,1997;84(7):920.
22.王宇明.内毒素所致肝损害机理的研究进展.国外医学流行病学传染病学分册,1994,21(1):12.
23. Lebrec-D, et al. Hemodynamic Changes in patients with portal venous obstruction.Hepatology,1983,3(4):550
24. Lebrec D , Bataille C, Bercoff E,et al.Hemodynamic changes in patients with portal venous obstruction .Hepatology 1983;3:550-553
25. Sahni P, Pande GK,Nundy S. Extrahepatic portal vein obstruction .Br J Surg1990;77:1201-1202
26. Omokawa S, Asanuma Y,koyama K. Evaluation of hemodynamics and hepatic mitochondrial function on extrahepatic portal obstruction in the rat .World J Surg1990;14:247-254
27. Nightingale LM, Tambolini WP ,Kish P, et al .Depression of left ventricularperformance during canine splanchnic artery occulusion shock .Circ Shock1984; 14(2):93-106
28. Jakobsson B , el Hag IA ,Stenram U. Nucleotide profile in rat liver and intestine at portal vein obstruction and reflow. Biomed Biochim Acta.1991;50(8):1001-1006
29.王万铁,徐正,林丽娜.家兔门静脉阻断和再开放对心脏血流动力学的影响.温州医学院学报1995,25(1):4-6
30.徐正,王万铁,林丽娜,等.家兔门静脉阻断和再开放肺循环血流动力学动态变化的实验研究.温州医学院学报1995,25(1):1-3
31.王万铁,等.家兔门静脉阻断对家兔血液气体的影响.温州医学院学报1995,(2):1
32. Shibayama Y. The relation of endotoxaemia to the cause of death and fetal hepatic damage following obstruction of the portal vein J Pathol,1990,160(4):355
33. Swank GM,Deitch EA.Role of the gut in multiple organ failure:bacterial translocation and permeability changes. World J. Surg1996:20:411-417
34.郭占林,李政香,王金龙,等.大鼠肝切除时阻断肝门血流对细菌易位的影响.中华普通外科杂志,1999,14(6):458-460.
35. Koike K,Moore FA,moore EE,et al. Endotoxin after gut ischemia/reperfusion causes irreversible lung injury .J Surg Res 1992:52(6):656-662
36. Koike K,Moore EE,Moore FA.Gut ischemia/reperfusion produces lung injury independent of endotoxin .Crit Care Med 1994,22(9):1438-1444
37.时开网,王晓颖,井清源,等.门静脉阻断对肝脏及肠道结构功能的影响.南京医科大学学报2000,20(5):356-358
38. Baykal A, Kavuklu B, Iskit AB ,et al .Experimental study of the effect of nitric oxide inhibition on mesenteric blood flow and interleukin-10 levers with a lipopolysaccharide challenge . World J Surg2000,24(9):1116-1120
39. Kudo Y , Egashira T,Takayama F,et al. Investigation of the renal injury caused by liver ischemia-reperfusion in rats.Arch toxicol 1993,67(7):502-509
1. Kaufmann SH, Earnshaw. Induction of apoptosis by cancer chemotherapy. Exp Cell Res,2000,256(1):42-49.
2. Qin L F ,Ng IO .Induction of apoptosis by cisplatin and its effect on cell cycle-related proteins and cell cycle changes in hepatoma cell.Cancer Lett ,2002,183(1):147-153.
3. Jiang X , Wang X .Cytochrome C-mediated apoptosis[J].Annu Rev Biochem , 2004,73:87-106.
4. Purring-Koch C , McLendon G. Cytochrome c bingding to Apaf-1: the effects of dATP and ionic strength [J].Proc-Natl-Acad-Sci-U-S-A .2000,97(22): 11928-31.
5. Mootha VK, Wei MC, Buttle KF .A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous ctyochrome c[J].EMBOJ,2001,20(4):661-671.
6. Zhivotovsky B ,orrenius S, Brustugun OT. Injected cytochrome c induces apoptosis [J].Nature,1998,391(6666):449-450.
7. Yuste VJ, Sanchez-Lopez I, Sole C.The contribution of apoptosis-inducing factor ,caspase-activeated DNase,and inhibitor of caspase-actived DNase to the nuclear phenotype and DNA[J].J-Biol-Chem.2005,280(42):35670-83.
8. Purring-Koch C,McLendon G. Cytochrome c binding to Apaf-1 : the effects of dATP and ionic strength [J].Proc-Natl-Acad-Sci-U-S-A.2000,97(22):11928-31.
9. Hatai T, Matsuzawa A, Inoshita S. Execution of apoptosis signal-regulation kinasel (ASK1)-induced apoptosis by the mitochondria-dependent caspase activation [J]. J Biol Chem.2000,275(34) :26576-26581.
10. Wu YH ,Shih SF ,Lin JY .Ricin triggers apoptotic morphological changes through caspase-3 cleavage of BAT3[J].J-Biol-Chem .2004,279(18):19264-19275.
11. Pereverzev MO,Vygodina TV , Konstantinov AA. Cytochrome c ,an ideal antioxidant [J].Biochem-Soc-Trans .2003,31(Pt6):1312-1315.
12. Barros MH, Netto LE ,Kowaltowski AJ.H202 generation in Saccharomyces cerevisiac respiratory pet mutants :effect of cytochrome c [J]. Free Radic Biol Med .2003, 35(2):179-188.
13. Susin SA ,Daugas E, Ravagnan L. Two distinct pathways leading to nuclear apoptosis[J].Infect Immun .2002,70(1):55-61.
14. Perfettini JL, Reed JC, Israel N. Role of Bcl-2 family members in caspase-independent apoptosis during Chlamydia infection T. Mitochondrial permeability transition and cytochrome c release in ischemia-reperfusion injury of the rat liver[J]. J-Surg-Res. 2003,111(2):240-247.
15. Dawson VL ,Dawson TM..Deadly conversations:Nuclear-Mitochondrial cross-talk. J Biomembr,2004,36(4):287-294.
16. Patterson SD ,Spahr CS, Daugas E , Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition .Cell Death Differ,2000,7(2):137-144.
17. He L, Poblens AT , Medrano CI.Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore .J Biol Chem ,2000,275(16): 12175-12184.
18. Lemasters JJ,Nieminen AL , Qian T, et al .The mitochondrial permeability transition in cell death a common mechanism in necrosis and autophagy.Biochim Biophys Acta, 1998,1366(1-2):177-196.
19. Ma J, Fu NY ,Pang DB, et al . Ca2+ store depletiorroperated Ca2+ influx determines the apoptosis of MGG-803 cells induced by extract of glycyrrhiza uralensis fisch. Chin J Biochem Mol Biol ,2001,17(2):230-236.
20. MUCHMORE S W, SATTLER M ,LIANG H, et al, X-ray and NMR structure of human Bcl-Xl, an inhibitor of programmed cell death[J]. Nature,1996,381(6580):335-341.
21. VANDER HEIDEN M G, CHANDEL N S , LI X X,et al.Outer mitochondrial membrane permeability can regulate coupled respiration and cell survival [J].Proc Natl Acad Sci,2000,97(9)4666-4671.
22. MAURER M, TSAI M, METZ M, et al .A role for Bax in the regulation of apoptosis in mouse mast cells [J].Invest Dermatol ,2000,114(6):1205-1206.
23. KUDLA G ,MONTESSUIT S, ESKES R,et al.destabilization of lipid membranes induced by the C-terminal fragment of caspase 8-cleaved bid is inhibility by the N-terminal fragment [J]. J Biol Chem ,2000,275(30):22713-22718.
24. BRUSTOVETSKY N, BRUSTOVETSKY T, JEMMERSON R, et al. Calcium-induced Cytochrome c release from CNS mitochondria is associated with the permeabilitytransition and rupture of the outer membrane [J].Oncogene ,2000,19(3):329-336.
25. BRENNER C, CADIOU H,VIEIRA H L, et al .Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator [J].2000,19(3):329-336.
26. VON AHSEN O , RENKEN C,PERKIN C, PERKINS G,et al.Preservation of mitochondrial structure and function after Bid-or Bax-mediated cytochrome c release [J]. J Cell Biol ,2001,150(5):1027-1036.
27. WEI M C,LINDSTEN T, MOOTHA V K, et al .tBID , a membrane targeted death ligand ,oligomerizes BAK to release cytochrome c [J].Denes DEV,2000, 14(16): 2060-2071.
28. KUWANA T, MACKEY M R, PERKINS G,et al .Bid,bax,and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane [J].Cell,2002, 111(3):331-342.
29. MINN A J, VELEZ P, SCHENDEL S L,et al .Bclx(L)forms an ion channel in synthetic lipid membrane [J].Nature,1997,385(6614):353-357.
30. VANDER HEIDEN M G,CHANDEL N S, WILLIAMSON E K,et al.Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria[J]. Cell,1997, 91(5):627-637.
31. MATSUYAMA S, LLOPIS J,DEVERAUX QL ,et al .Changes in intramitochondrial and cytosolic pH:early events that modulate caspase activation during apoptosis [J]. Nat Cell Biol,2000,2(6):318-325.
32. MEISENHOLDER G W, MARTIN S J, GREEN D R, et al. Events in apoptosis:acidification is downstream of protease activation and Bcl-2 protection [J]. J Biol Chem, 1996,272(27):16260-16262.
33. CORY S,Regulation of lymphocyte survival by the Bcl-2 gene family [J].annu Rev Immunol ,1995,13:513-543.
34. YANG E, KORSMEYER S J. Molecular thanatopsis :a discourse on the Bcl-2 family and cell death [J].Blood,1996,88(1-2):386-401.
35. WELIHINDA A A,TIRASOPHON W,KAUFMAN R J .The cellular response to protein misfolding in the endoplasmic reticulum [J].Gene Expr,1999,7(4-6):293-300.
36. SABORY J, HERMAN M M, GHRIBI O. Intracellular mechanisms underlying aluminum-induced apoptosis in rabbit brain [J]. j Inorg Biochem,2003,97(I):151-154.
37. HACKI J, EGGER L, MONNEY L, et al. Apoptotic crosstalk between the endoplasmic reticulum and mitohondria controlled by Bcl-2[J].Oncogene ,2000,19(19):2286-2295.
38. KORSMEYER S J, YIN X M, OLTVAI Z N ,et al. Reactive oxygen species and the regulation of cell death by the Bcl-2 gene family[J].Biochim Biophys Acta, 19951271(1):63-66.
39. GHRIBI O, DEWITT D A, FORBES M S, et al .Co-involvement of mitochondria and endoplasmic reticulum in regulation of apoptosis:changes in cytochrome c,Bcl-2 and Bax in the hippocampus of alumimum-treated rabbits [J].Brain Res ,2001, 903(1-2):66-73.
40. Rosewicz S, Detjen K,Scholz A,et al. Interferon-alpha:regulatory effects on cell cycle and angiogenesis .Neuroendocrinology effects on cell cycle and angigenesis. Neuroendocrinology,2004,80(Suppl l): 85-93.
41. Yamaguchi YY, Kado JO,Kasukabe T, et al. Induction of apoptosis by combined treatment with differentiation-inducing agents and interferon-alpha in human lung cancer cells. Anticancer Res,2003,23(3B): 2537-2547.
42. Kessel D, Sun HH.Enhanced responsiveness to photAynamic therapy-induced apoptosis after mitochondrial DNA depletion. Photochem Photobiol ,1999,70(6) : 937-940.
43. Singh KK, Russell J,Sigala B, et al .Mitochondrial DNA determines the cellular response to cancer therapeutic agents .Oncogene,1999,18(48):6641-6646.
44. Wang TW,Yu HY, Kong SK, et al. The decrease of mitochondrial NADH dehydrogenease and drug in apoptosis in doxorubicin resistant A431 cell.Sci, 2000,67(9):1111-1118.
1、Barakat O, Hoef J, Ozaki CF, et al. Extended right trisegmentectomy using in situ hypothermic perfusion with modified HTK solution for a large intrahepatic cholangiocarcinoma. [J] Surg Oncol. 2007 Jun 1;95(7):587-592
2、Mckeown CMB ,Edwards V ,Philips MJ, et al .Sinusoidal lining cell damage:the critcal injury in cold preservation of liver allograft in the rat .transplantation, 1988,46:178-191.
3、Galdwell-kenkel JC,Thurman RG,Lemasters JJ.Selective loss of nonparenchymal cell viability after cold ischemic strrage of rat liver. Transplantation ,1998,45:834-837
4、Rokicki M,Rokicki W.Liver regeneration in rats after complete and partial occlusion of the portal blood influx [J].Res Exp Med(Berl)1993;193(5)305
5、Edgcomb LP,Eckhauser FE Porter-Fink VL,et al .Effects of portacaval shunt and portacaval transposition on hepatocelluar and hepatic reticuloendothelial cell activity in the dog [J].J Surg Res,1984;36(5)446