摘要
第一部分eNOS重组腺病毒载体的制备
目的：构建含有eNOS基因的重组腺病毒载体。
方法：（1）将重组腺病毒载体Ad-eNOS行基因测序。（2）将重组腺病毒载体Ad-eNOS用脂质体介导转染293细胞包装成病毒颗粒，应用合成的eNOS基因引物行Real-time PCR鉴定。（3）通过测定吸光值计算病毒滴度并分装。
结果：（1）经测序，Ad-eNOS基因序列与GeneBank中的eNOS序列一致，比对符合率100%。（2）Real-time PCR鉴定重组腺病毒Ad-eNOS包装成功。（3）经病毒滴度测定，Ad-eNOS滴度为9.35×109PFU/ml。
结论：成功构建了重组腺病毒载体Ad-eNOS质粒，采用HEK293细胞进行腺病毒包装，最终获得重组腺病毒Ad-eNOS。
第二部分eNOS基因转染对L02肝细胞缺复氧损伤的保护作用
目的：探讨eNOS基因转染对L02肝细胞缺复氧损伤的保护作用。
方法：采用L02肝细胞，放入低氧环境（含95%N2+5%CO2、PO2≤4Kpa）及复氧环境（85%O2+15%C O2，PO2≥13Kpa）孵箱中培养，建立缺复氧模型，实验分三组：实验组加入Ad-eNOS处理；对照组和正常组加RPMI-1640培养液。实验组和对照组放入缺复氧模型中培养，正常组在5%CO2、饱和湿度的孵箱内培养。检测培养液中ALT及NO的含量，Real-time PCR检测eNOS的基因表达，Western-blot检测eNOS的蛋白表达，流式细胞检测细胞凋亡。
结果：实验组细胞培养液中ALT含量(26.26±3.78u/l)明显低于对照组(48.42±5.31u/l)（P﹤0.05）,但高于正常组(17.20±2.64u/l)，差别有显著意义（P﹤0.05）；而NO的含量实验组（18.89±3.30umol／L）明显高于对照组（6.44±2.11umol／L）和正常组（8.85±2.40umol／L）（P﹤0.05）；Real-time PCR及Western-blot结果显示实验组eNOS在基因及蛋白水平的表达均明显高于对照组及正常组（P﹤0.05）；流式细胞结果显示实验组L02肝细胞凋亡明显低于对照组（P﹤0.05），而正常组L02肝细胞凋亡很少。
结论：（1）腺病毒载体成功将eNOS基因转染到L02肝细胞上，使其高表达eNOS基因及蛋白；（2）eNOS基因转染可减轻L02肝细胞缺复氧损伤的细胞凋亡；（3）eNOS基因转染可能通过升高NO途径对L02肝细胞缺复氧损伤有保护作用。
第三部分大鼠小体积肝脏移植模型的建立
目的：用改良二袖套法建立稳定的大鼠小体积肝脏移植模型。
方法：在Kamada二袖套法的的基础上，对大鼠原位肝移植术在取肝、灌注、修植肝等手术操作以及围手术期的处理进行改良，建立稳定的大鼠原位肝移植模；在此基础上再建立稳定的大鼠小体积肝脏移植模型。
结果：通过三阶段比较，正式实验组在手术时间、无肝期、手术成功率、并发症等方面均有明显提高。
结论：（1）改良的二袖套法具有无肝期短、手术成功率高、大鼠术后存活时间长的优点，是大鼠原位肝移植的理想术式；（2）在建立稳定的大鼠原位肝移植模型的基础上开展大鼠小体积肝移植模型有利于提高模型的稳定性。第四部分eNOS基因转染对大鼠小体积肝移植缺血再灌注损伤的保护作用
目的：探讨eNOS基因转染对大鼠小体积肝移植缺血再灌注损伤的保护作用。
方法：SD大鼠12对，随机分两组（n=6）行小体积肝移植。对照组供体用空载体进行腹腔注射，实验组供体用Ad-eNOS进行腹腔注射。36小时后取肝行小体积肝移植。门静脉复流后6小时后处死模型，采血送检肝功能；硝酸还原法检测NO浓度；免疫组化检测TNF-α、巨噬细胞；TUNEL法检测肝组织的细胞凋亡；流式细胞检测肝细胞凋亡率；Real-time PCR检测肝脏组织的eNOS基因表达；Western-blot检测肝组织中的eNOS蛋白表达。
结果：（1）实验组的ALT、AST、LDH显著低于对照组；细胞凋亡率显著低于对照组；（2）肝组织NO含量实验组明显高于对照组；（3）Real-time PCR及Western-blot结果显示实验组eNOS在基因及蛋白水平的表达均明显高于对照组；（4）免疫组化发现TNF-α及巨噬细胞在对照组中高表达，实验组中低表达。
结论：eNOS在大鼠小体积肝移植缺血再灌注损害中对肝细胞有保护作用，其作用机制可能与下调TNF-α，抑制巨噬细胞浸润有关。
Part One Construction of Recombinant Adenovirus of eNOS
Objective: To construct the recombinant adenovirus vector containing eNOS gene.
Methods:(1) The recombinant adenovirus vector Ad-eNOS using gene sequencing.(2) The recombinant adenovirus vector Ad-eNOS by liposome mediated transfection293cells packaged into viral particles, using eNOS gene primers for identification of PCRsynthesis.(3)By measuring the absorbance of the virus titer was calculated and packed.
Results:(1) By DNA sequencing, the Ad-eNOS gene sequence is consistent withGeneBank.(2) Identification of recombinant adenovirus Ad-eNOS packing is successfulby Real-time PCR.(3) The virus titer was9.35×109PFU/ml by the determination of virustiter.
Conclusion: To construct the recombinant adenovirus vector Ad-eNOS by usingHEK293cells.To obtain recombinant adenovirus Ad-eNOS.
Part Two Protective effects of eNOS gene transfer onhypoxia-reoxygenation injury in hepatocellular L02
Objective: To investigate the protective effect of eNOS gene transfer on hypoxia-reoxygenation injury in hepatocellular L02.
Methods:Using the L02liver cells, in the hypoxia model (including95%N2+5%CO2、PO2≤4Kpa) and reoxygenation model (85%O2+15%CO2，PO2≥13Kpa), constructinghypoxia-reoxygenation model, The experiments were divided into three groups:inexperimental group treated with Ad-eNOS; in control group and in normal group with RPMI-1640medium. The experimental group and the control group in hypoxia-reoxygenation model, in normal group were cultured in5%CO2, saturated humidityincubation box. The contents of ALT and NO was detected in the medium, the expressionof eNOS gene was detected by Real-time PCR, the expression of eNOS protein wasdetected by Western-blot, apoptosis cell was detected by FCM.
Results: In experimental group, the content of ALT (26.26±3.78u/l) lower than thatof in control group (48.42±5.31u/l)(P <0.05), but higher than that in normal group(17.20±2.64u/l)(P <0.05); and the content of NO in experimental group (18.89±3.30umol/L) was significantly higher than in control group (6.44±2.11umol/L) and innormal group (8.85±2.40umol/L)(P <0.05); Real-time PCR and Western-blot resultsshowed that the expression of eNOS in experimental group in the gene and protein levelswere significantly higher than in control group and in normal group (P <0.05); FCMshowed that in experimental group of L02liver cell apoptosis was significantly lower thanin control group (P <0.05), and in normal group L02apoptosis of hepatic cells rarely.
Conclusion:(1) Ad-eNOS gene was transfected into the L02liver cells, the highexpression of eNOS gene and protein;(2) eNOS gene transfer can reduce cell apoptosis onhypoxia-reoxygenation injury in hepatocellular L02;(3) eNOS gene transfer could haveprotective effect on hypoxia-reoxygenation injury in hepatocellular L02by rising the NOpathway.
Part Three To establish the model of small-for-size liver transplantationin rats
Objective: to establish the rat model of small-for-size liver transplantation bymodified two cuff method.
Methods: Based on the Kamada two cuff technique, improvement of the ratorthotopic liver transplantation in perfusion, repair, treatment with liver operation and perioperation period, to establish a stable rat orthotopic liver transplantation model; on thisbasis to establish the small-for-size liver transplantation in rat model.
Results: By comparison of the three phase, the formal experimental group in theoperation time, anhepatic phase, the success rate of operation, complication were obviouslyincreased.
Conclusion:(1)The modified two cuffed technique has the advantages of longsurvival time, short anhepatic phase operation, high success rate, which is an idealoperative method of orthotopic liver transplantation in rats;(2) Based on the stable modelof orthotopic liver transplantation in rats establishing of small-for-size liver transplantationin rats model is helpful to improve the stability of the model.
Part Four The protective effect of eNOS gene transfer on IRI of ratsmall-for-size liver transplantation
Objective: To investigate the protective effect of eNOS gene transfer on IRIsmall-for-size liver transplantation in rats.
Methods:12SD rats were randomly divided into two groups (n=6) for small-for-sizeliver transplantation.The control group were intraperitoneal injection of donor with emptyvector, the experimental group with Ad-eNOS donor. After36hours small-for-size livertransplantation is operated. Six hours after reperfusion, the recipients were sacrificed, NOwere measured, TNF-α、macrophage levels were detected by immunohistochemistry.Apoptosis of the hepatocytes was analyzed by TUNEL and FCM; the expression of eNOSgene was detected by Real-time PCR, the expression of eNOS protein was detected byWestern-blot.
Results:(1) In experimental groups ALT, AST, LDH were significantly lower thanin the control group; apoptosis rate was significantly lower than in control group;(2) thecontent of NO in liver tissue in experimental group was significantly higher than in controlgroup;(3) Real-time PCR and Western-blot results showed that in experimental groupeNOS at the level of gene and protein were significantly higher than in control group;(4)Immunohistochemistry showed that TNF-α and macrophages high expression in controlgroup and low expression in experimental group.
Conclusion: eNOS has a protective effect on IRI of small-for-size livertransplantation in rats, and its mechanism may be related to the down-regulation of TNF-α,inhibiting the macrophage infiltration.
引文
[1] Devlin J， O’Grady J． Indications for referral and assessment in adult1ivertransplantation：a clinical guideline[J]． Gut，1999：45(Suppl6)： Ⅵ1.
[2] Raia S，Nery JR，Mies S． Liver transplantation from live donors[J]． Lancet，1989，2(8661)：497．
[3]张会欣，张建新.一氧化氮在缺血性脑损伤中作用的实验研究进展[J],中国药理学通报.2004:20(10):1094-1097.
[4]吴剑华.内源性一氧化氮与肝硬变,国外医学.流行病学.传染病学分册,1995,22(4):156.
[5] Theruvath TP, Zhong Z, Currin RT etal. Endothelial nitric oxide synthase protectstransplanted mouse livers against storage/reperfusion injury: Role of vasodilatory andinnate immunity pathways[J].Transplant Proc.2006Dec;38(10):3351-7.
[1] Baltzer AM，Lattermann C，Whalen JD，el a1．Potential role of direct adenoviral genetransfer in enhancing fracture repair]-J]．Clin Orthop，2000，(379Supp1)：S120一S125．
[2] He GA, Luo JX, Zhang TY, et al. Canstatin N fragment inhibits in vitro endothelialcell proliferation and suppresses in vivo tumor grouth ［J］. Biochem Biophys ResCommun,2003,312(3):801-805.
[3] Rein DT, Breidenbach M, Cruiel DT. Current developments in adenovirus basedcancer gene therapy［J］. Future Oncol,2006,2(1):137-143.
[4]王玉泉，马立宾，于秋莲.动物腺病毒载体的研究进展[J].内蒙古民族大学学报，2009，24（2）：180-183.
[5] Tatsis N, Ertl H C J. Adenoviruses as vaccine vectors [J]. Mol Ther,2004,10(4):616-629.
[6] Morral N, O’Neal W, Rice K, et al. Administration of helperdependent adenoviralvectors and sequential delivery of different vector serotype for long-termliver-directed gene transfer in baboons [J]. Proc Nat Acad Sci, USA.1999,96,12816-12821.
[7]刘思景，郭伟韬，王辉.腺病毒载体的研究新进展[J].现代中西医结合杂志，2011，19（13）：1669-1670.
[1]Qian Y，Liu Z，Geng X．Lack of protection of ischaemic preconditioning in the ratmodel of major hepatectomy with ischaemia reperfusion injury[J]．Asian J Surg，2008，31(3)：140-147．
[2] Urakami H，Abe Y，Grisham MB．Role of reactive metabolites of oxygen and nitrogenin partial liver transplantation：lessons learned from reduced—size live ischaemia andreperfusion injury[J]．Clin Exp Pharmacol Physiol，2007，34(9)：912-919．
[3]Lan AK，Luk HN，Goto S，et a1．Stress response to hepatectomy in patients with ahealthy or a diseased liver[J]．World J Surg，2003，27(7)：761-764．
[4] Marcos A，Fisher RA，Ham JM，et a1．Liver regeneration and function indonor andrecipient after fight lobe adult living donor liver transplantation[J]．Transplantation，2000，69(7)：1375-1379．
[5]Li SQ.Liang LJ.Protective mechanism of L-arginine against liver ischemic-reperfusioninjury in rats[J].Hepatobiliar Pancreat Dis Int.2003.2(4):549-552.
[5]Gong JP.Tu B.Wang W.et a1.Protective effect of nitric oxide induced by ischemicpreconditioning on reperfusion injury of rat liver graft[J].World J Gastroenterol.2004.10(1)：73-76.
[7] Shah V，Kamath PS．Nitric oxide in liver transplantation：Pathobiology and clinicalimplications[J]．Liver Transpl，2003，9(1)：1-11
[8]Brunner F，Maler R，Andrew P，et a1．Attenuation of myocardial ischemia/reperfusioninjury in mice with myocyte-specific over expression of endothelial nitric oxidesynthasc[J]．Cardiovasc Res，2003，57：55．
[9]Kiziltepo U，Tunctan B，Eyileten ZB，et a1．Efficiency of L-arginine enrichedcardioplegia and non cardioplegic repefusion in ischemic hearts[J]．Mol cell Biochem，2004，260：39-47．
[10]左新华，欧阳文，段开明等，利多卡因对缺氧/复氧后肝细胞NO、MDA生成及细胞凋亡的影响[J].天津医科大学学报,2008,14(1):14-17。
[11]周少丽,黑子,郭娜等，瘦素对缺氧复氧培养L02肝细胞功能和凋亡的影响[J].中华实验外科杂志，2009，26（2）：197-198.
[12]徐小平,刘发全,张积仁等，辅酶Ⅰ对L02肝细胞缺血-再灌注损伤的保护作用[J],中华实验外科杂志，2002，19（2）：186
[13]黎一鸣，徐心，吉鸿等，一氧化氮对供肝肝动脉缺血后肝细胞凋亡的影响[J].肝脏，2002,7（1），20-27.
1. Lee S，Charters C，Chandler J，Orloff M A Technique for orthotopic livertransplantation in the rat．Transplantation1973；16：664-669
2. Lee S，Chaners C，Orloff M Simplified technique fbr Orthotopic liver transplantationin the rat． Am J Surg1975；130：38-40
3. Kamada N，Calne R, Orthotopic liver transplantation in the rat． Transplantation1979；28：47-50
4. Kamada N， Calne R， A surgical experience With five hundred thirty livertransplantation in the rat． Surgery l983；93：64
5. Zimmerman FA，Butcher GW, Davies HS，et al Techniques for orthotopic livertransplantation in the rat and some studies Of the immunologic responses to fullyallogenetic liver grafts．Transplant Proc1979；11：571-574
6. Miyata M， Fisher J， Fuhs M， et al A simple method for orthotopic livertransplantation in the rat．Transplantation1980；30：335-338
7. Engemann R，Thiede A，et al. The value of a physiological liver transplant model inthe rat．Transplantation1982；33：566
8.王军,庞利群,余斌等，120例大鼠原位肝移植报道.肝胆胰外科杂志，2006,18(3)。
9.吴建武，胡明政，杨小华等，不同术式大鼠原位肝移植模型的比较.肝胆胰外科杂志，2007,19(1)。
10.张伯，钱海鑫，秦磊，改良二袖套管法大鼠原位肝移植动物模型150例体会，当代医学，2010，16（10）。
11. Verhaegen M, Iaizzo PA, Todd MM, A comparison of the effects of hypothermia,pentobarbital, and isoflurane on cerebral energy stores at the time of ischemicdepolarization. Anesthesiology.1995;82(5):1209-15.
12.彭勇，龚建平，刘长安等,大鼠原位肝移植模型制作过程中麻醉方法的选择,中国普通外科杂志，2003,9,673-676.
13.余斌，秦磊，钱海鑫，大鼠肝移植的麻醉，苏州大学学报（医学版），2005，25（2）：24-247.
14. Bittard H, Chiche L, Moukarzel M， et al，Study of kidney and liver viability in the ratafter exclusive aortic perfusion using intracellular ATP measurement，Urol Res.1992;20(6):415-7
15.吴星宇，李江利，仇毓东等．大鼠原位肝移植的手术技巧［Ｊ］.肝胆外科杂志，2004，12(5)：376-378．
16. Fuller BJ, Lee CY． Hypothermic perfusion preservation: the future of organpreservation revisited? Cryobiology.2007Apr;54(2):129-45.
17. Tolboom H, Milwid JM, Izamis ML,etal, Sequential cold storage and normothermicperfusion of the ischemic rat liver. Transplant Proc.2008Jun;40(5):1306-9.
18. Schemmer P，Schoonhoven R，Swenberg JA，et al Gental in situ liver manipulationduring organ harvest decreases survival after rat liver transplantation．Role OfKupffer cells．Transplantation1998；65：1015.
19. Kamada N,Calne RY. A surgical experience with five hundred thirty liver transplantation in therat[J].Surgery.1983,93,(1):64-69。
20. Goto M，Takei Y, Kawano S，et al Tumor necrosis factor and endotoxin inthepathogenesis of liver and pulmonary injuries after orthotopic liver transplantation inrat. Hepatology1992；16：487。
21. Kamada N．Calne RY．Orthotopic liver transplantati0n in the rat[J]．Transplantation，1979,28(1)：47-50。
22. Xu HS, Pruett TL, Jones RS, et al Study of donor-recipient liver size match fortransplantation. Ann Surg,1994;219(1):46-50
23. Kwan M, Chung-Mau L, Irene O, et al. Liver transplantation in rats usingsmall-for-size grafts. Arch Surg.2001;136(2):280-285.
24. Ikegami T, Nishizaki T, Shimada M, et al. Changes of candate lobe, transplantedwith extended leftlobe liver graft from living donors. Surgery2001,129:76285.
25. Yang ZF, David WY, Patricia N, et al. Antiinflammgatory properties of IL-10rescuesmall-for-size liver gtafts. Liver Transpl.2007;13(3):558-565.
26.周学平，杜铭，李济宇等．大鼠原位肝移植模型制作的术后稳定存活率[J]．上海交通大学报，2006，26(6)：603-6．
27.刘静，李江，张升宁等，大鼠减体积肝移植腹腔出血的原因分析，南方医科大学学报，2010；30(5).
28.谈鼓，黄陈等，提高二袖套法大鼠原位肝移植成功率的手术技巧,中国普外基础与临床杂志，2004；5:405-407。
29. Meyer K, Brown MF，Zobari G，et a1．ICAM一1upregulation in distant tissue afterhepatic ischemia／reperfusion：a clue to the mechanism of multiple organ failure[j]．JPediatr Surg，1998；33：350。
[1]黄洁夫．肝脏移植的理论与实践：广东科技出版社，1998，1．
[2] Devlin J， O’Grady J． Indications for referral and assessment in adult1ivertransplantation：a clinical guideline[J]． Gut，1999：45(Suppl6)： Ⅵ1.
[3] Zhong Z，Schwabe RF，Kai Y，et al，Liver regeneration is suppressed in small-for-sizeliver grafts after transplantation: involvement of c-Jun N-terminal kinase, cyclin D1,and defective energy supply. Transplantation.2006Jul27;82(2):241-50.
[4]朱应乾，覃云，王茂旭，等．缺血预处理对高原大鼠肝脏缺血／再灌注损伤早期的影响．世界华人消化杂志2007，15：287-289
[5]吕平，刘芳，王春友等．亚硝酸盐还原成一氧化氮保护肝脏对抗缺血再灌注损伤．世界华人消化杂志，2005；13：346．350
[6] Lundberg JO, Weitzberg E. NO-synthase independent NO generation in mammals.Biochem Biophys Res Commun.201021;396(1):39-45.
[7]Katsumi H, Nishikawa M, Yasui H. Prevention of ischemia/reperfusion injury byhepatic targeting of nitric oxide in mice. J Control Release.2009Nov16;140(1):12-7.
[8]吴剑华.内源性一氧化氮与肝硬变,国外医学.流行病学.传染病学分册,1995,22(4):156.
[9]Theruvath TP, Zhong Z, Currin RT etal. Endothelial nitric oxide synthase protectstransplanted mouse livers against storage/reperfusion injury: Role of vasodilatory andinnate immunity pathways[J].Transplant Proc.2006;38(10):3351-7.
[10] Yamashiro S, Kuniyoshi Y, Arakaki K. Cardioprotective effects oftetrahydrobiopterin in cold heart preservation after cardiac arrest. Ann ThoracCardiovasc Surg.2006;12(2):95-104
[11] Ben Mosbah I, Roselló-Catafau J, Alfany-Fernandez I,et al. Addition of carvedilol toUniversity Wisconsin solution improves rat steatotic and nonsteatotic liverpreservation. Liver Transpl.2010;16(2):163-71.
[12]Peralta C，Hotter G,Closa D，et a1．Protective effect of preconditioning on injuryassociated to hepatic ischemic-reperfusion in the rat：role of niric oxide and adenosineHepatology,1997，26(4)：934-937.
[13] Ikebe N, Akaike T, Miyamoto Y,et al. Protective effect of S-nitrosylatedalpha(1)-protease inhibitor on hepatic ischemia-reperfusion injury. J Pharmacol ExpTher.2000Dec;295(3):904-11
[14]Galen FX, Cottart CH, Souil E,，et al. Role of nitric oxide synthase III and guanosine3':5'-cyclic monophosphate in the protection exerted by nitric oxide on hepaticischemia-reperfusion injury．C R Acad Sci III.1999Oct;322(10):871-7.
[15] Hsu CM,Wang JS，Liu CH,et al Kupffer cells protect liver from ischemia-reperfusioninjury by an inducible nitric oxide synthase-dependent mechanism.Shock，2002，17(4)：280-285.
[16] Meguro M,Katsuramaki T, Kimura H, et al. Apoptosis and necrosis after warmischemia-reperfusion injury of the pig liver and their inhibition by ONO-1714Transplantation，2003，75(4)：703-10.
[17] Meguro M，Katsuramaki T, Nagayama M,et a1．A novel inhibitor of induciblenitricoxide synthase(ONO--1714) prevents critical warm ischemia-reperfusion injuryin the pig liver．Transplantation，2002，73：1439-1448．
[18] Wang LM,Tian XF,Song QY,et al Expresion and role of inducible nitric oxidesynthase in ischemia-reperfusion liver in rats．Hepatobiliary Pancreat Dis Int，2003，2(2)：252-258．
[19] Lee VGJohnson ML，Baust J,et al. The role of iNOS in liver ischemia-reperfusioninjury. Shock，2001，16(5)：355-60．
[20]Kawachi S，Hines IN，Laroux FS，et al. Nitric oxide synthase and postischemic liverinjury. Biochem Biophys Res Commun，2000，276(3)：85l一854．
[21]Cottart CH’Do L，Blanc MC，et al. Hepatoprotective effect of nitric oxide duringischemia-reperfusion in rat．Hepatology,1999，29(3)：809-813
[22] Troisi R，Cammu G，Miiiterno G，etal. Moduiation of portal graft infiow：anecessity in aduit living-donor liver transplantation［J］？Ann Surg，2003，237（3）：429-436.
[23] Rokcey D C. Hepatic biood fiow reguiation by stellate cells in normal and injuredliver [J]. Semi Liver Dis，2001，2（13）：337-349.
[24]冷建军，韩本立，董家鸿等，小型猪小体积肝移植后血清ET-1和NO水平变化研究，第三军医大学学报，2005，27（6），535-537
[25] Peralta C，Closa D，Hotter G，et al. Liver ischemic preconditioning is mediatedby the inhibitory action of nitric oxide on endothelin. Biochem Biophys Res Commun，1996，229：264-270.
[26]Gottlieb RA,Burleson KO,et al,Reperfusion injury induces apoptosis in rabbitcardiomyocytes. J Clin Invest94:1621–1628,1994.
[27]Tanaka M, Ito H,et al.Hypoxia induces apoptosis with enhanced expression of fasantigen messenger RNA in cultured neonatal rat cardiomyocytes. Circ Res75:426–433,1994.
[28] Comstock KL, Krown KA,et al.LPS-induced TNF-alpha release from and apoptosis inrat cardiomyocytes: obligatory role for CD14in mediating the LPS response. J MolCell Cardiol30:2761–2775,1998.
[29]涂兵，龚建平，曾勇等，一氧化氮在缺血预处理保护大鼠移植肝脏再灌注损伤中的作用，中华实验外科杂志，2003，20（4），313-315.
[30] Theruvath TP, Zhong Z, Currin RT etal. Endothelial nitric oxide synthase protectstransplanted mouse livers against storage/reperfusion injury: Role of vasodilatory andinnate immunity pathways[J].Transplant Proc.2006Dec;38(10):3351-7.
1. Palmer GCJ, Ashton DS, Moncada S. Vascular endothelial cells synthesize nitricoxide from L-arginine. Nature,1988,333:664-666
2. Marlatta MA. Nitric oxide synthase: structure and mechanism. J Biol Chem,1993,268:12231-12234.
3. Lloyd RV, Jin L, Qian X, et al. Scheithauer. BW, Nitric oxide synthase in the hummanpituitary gland, Am J Pathol1995,146:86-94.
4. Natanson C, Hoffman WD, Suffredini AF, et al. Selected treatment strategies for septicshock based on proposed mechanisms of pathogenesis. Ann Intern Med.1994,120(9):771-783. Natanson C, Hoffman WD, Suffredini AF, et al.
5. Selected treatment strategies for septic shock based on proposed mechanisms ofpathogenesis. Ann Intern Med.1994,120(9):771-783.
6. Rajagopalan S, Harrison DG. Reversing endothelial dysfunction with ACE inhibitors.A new trend. Circulation,1996,94(3):240-243.
7. Jia L, Bonaventura C, bonaventura J, et al. S-nitriosohaemoglobin: a dynamic activityof blood involved in vascular control. Nature,1996,380:221-226.
8. Billiar TR, Curran RD, Stuchr DJ, et al. Evidence that activation of kupffer cellsresults in production of L-arginine met abolites that release cell-associated iron andinhibit hepatocyte protein synthsis. Surgery,1989,106:364-372.
9. Bohlinger I, Leist M, Brsig J, et al. Interleukin-1and nitric oxide protect against tumornecrosis factor alpha-induced liver injury through distinct pathways. Hepatology,1995,22:1829-1837.
10. Morales RM, Jimenez M, Ros J, et al. Nitric oxide production by peritonealmacrophages of cirrhotic rats: a host response against bacterial peritonitis.Gastroenterology,1997,112:2056-2064.
11. Gasini A, Ceni E, Salzano R, et al. Neutrophil-derived superoxide anion induces lipidperoxidation and stimulates collagen synthesis in human hepatic stellate cells: role ofnitric oxide. Hepatology,1997,25:361-367.
12. Sharara AI, Perkins DJ, Misukonis MA, et al. Interferon(IFN)-alpha activation ofhuman blood mononuclear cells in vitro and in vivo for nitric oxide synthase(NOS)type2mRNA and protein expression: possible relationship of induced NOS2to theanti-hepatitis C effects of IFN-alpha in vivo. J Exp Med,1997,186:1495-1502.
13. Kurose I, Higuchi H, Watanabe N, et al. Nitric oxide and tumor necrosis factor-alphamediate apoptosis of hepatoma cells induced by Kupffer cells. hepatology,1995,22(Pt2):228A.
14. Moriyama A, Masumoto A, Nanri H, et al. High plasma concentrations ofnitrite/nitrate in patients with hepatocellular carcinoma. Am JGastroenterol,1997,92:1520-1523.
15. Guo FH, De Raeve HR, Rice TW, et al. Continnous nitric oxide synthesis by induciblenitric oxide synthase in normal humman airway epithelium in vivo. Proc Natl Acad SciUSA,1995,92;7809-7813.
16. Kadowaki K, Kishimoto J, Leng G, et al. Up-regulation of nitric oxide synthase gengexpression together with NOS activity in the rat hypothalamo-hypophysial systemafter chronic salt loading-arginine vasopressin and oxytocin secretion. Endocrinology,1994,134:1011-1047.
17. Suschek C, Fehsel K, Kroncke KD, et al. Primary culture of rat islet capillaryendothelial cells: constitutive and cytokine-inducible macrophage-like nitric oxidesynthase are expressed and activities regulated by glucose concentration. Am J Pathol,1994,145:685-695.
18. Bredt DS, Hwang. PM, Snyder SH, et al. Localization of nitric oxide synthaseindicating a neural role for nitric oxide, Nature1990347:768-770.
19. Vincent SR, Kimura H. Histochemical mapping of nitric oxide synthase in the rat brain.Neuroscience1992,46:755-784.
20. Lloyd RV, Jin L, Qian X, et al. Nitric oxide synthase in the humman pituitary gland.Am J Pathol1995,146:86-94.
21. T. Gotoh, T. Sonoki, A. Nagasaki, et al. Molecular cloning of cDNA fornonhepaticmitochondrial arginase (arginase II) and comparison of itsinduction withnitric oxide synthase in a murine macrophage-like cell line. FEBS Lett.395,119–122(1996).
22. I. M. Abou-Soud and D. J. Stuehr. Nitric oxide synthases reveal a role for calmodulinin controlling electron transfer. Proc. Natl. Acad. Sci. USA90,10769–10772(1993).
23. S. Moncada and A. Higgs. The L-arginine–nitric oxide pathway. N. Engl. J. Med.329(27),2002–2012(1993).
24. T. Sonoki, A. Nagasaki, T. Gotoh,et al. Conduction of nitric-oxidesynthase andarginase I in cultured rat peritoneal macrophagesand rat tissue in vivo bylipopolysaccharide. J.Biol. Chem.272(6),3689–3693(1997).
25. J. A. Schmidt and E. Abdulla, Down-regulation of IL-1b biosynthesis by inducers ofthe heat shock response. J.Immunol.141,2027–2034(1988).
26. S. Useno, A. Sano, K. Kotani, et al. Distribution of free methylarginines in rat tissuesand the bovine brain. J. Neurochem.59,2012–2016(1992).
27. P. Vallance, A. Leone, A. Calver, et al. Accumulation of an endogenous inhibitor ofnitric oxide synthesis in chronic renal failure. Lancet339,572–575(1992).
28. S. A. Fickling, A. Leone, S. S. Nussey, et al. Synthesis of NWNW dimethylarginine byhuman endothelial cells. Endothelium1,137–140.
29. R. J. McAllister, S. A. Fickling, G. St J. Whitley et al, Metabolism of methylargininesby human vasculature:implications for the regulation of nitric oxide synthesis.Br. J.Pharmacol.112,43–48(1994).
30. A. M. El-Badry, A. Otani, and T. E. Mansour, Studies on heart phosphofructokinase.Role of fructose-1,6-diphosphate in enzyme activity. J. Biol. Chem.248,557–563(1973).
31. J. P. Filkins and R. P. Cornell. Depression of hepatic gluconeogenesis and thehypoglycemia of endotoxic shock.Am. J. Physiol.227,778–781(1974).
32. M. E. Kirtley, M. McKay. Fructose-1,6-biphosphate, a regulator of metabolism. Mol.Cell Biochem.18,141–149(1977).
33. D. L. Duval, D. R. Miller, J. Collier et al. Billings,Characterization of hepatic nitricoxide synthase: identification as the cytokine-inducible form primarily regulated byoxidants. Mol. Pharmacol.50,277–284(1996).
34. B. G. Harbrecht, M. Di Silvio, V. Chough, et al. Glutathione regulates nitric oxidesynthase in cultured hepatocytes. Ann. Surg.225(1),76–87(1997).
35. D. R. Miller, J. M. Collier and R. E. Billings, Protein tyrosine kinase activity regulatesnitric oxide synthase induction in rat hepatocytes. Am. J. Physiol.(Gastrointest. LiverPhysiol.)35, G207–G214(1997).
36. T. Gaillard, A. Mulsch, R. Busse, H. Klein and K. Decker,Regulation of nitric oxideproduction by stimulated rat kupffer cells. Pathobiology59,280–283(1991).
37. B. G. Harbrecht, E. A. McClure, R. L. Simmons et al. Prostanoids inhibit kupffer cellnitric oxide synthesis. J. Surg. Res.58,625–629(1995).
38. B. G. Harbrecht, Y. M. Kim, E. M. Wirant, et al. PGE2and LTB4inhibitcytokine-stimulated nitric oxide synthase type2expression in isolated rat hepatocytes.Prostaglandins52,103–116(1996).
39. S. Hortelo, A. M. Genaro and L. Bosca. Phorbol esters induce nitric oxide synthaseactivity in rat hepatocytes. J.Biol. Chem.267,24937–24940(1992).
40. Romero M, Garcia-Monzon C, Clemente G, et al. Intrahepatic expression of induciblenitric oxide synthase in acute liver allograft rejection: Evidence of modulation bycorticosteroids. Liver Transpl2001;8:16-21.
41. M. Menegazzi, C. Guerriero, A Carcereri de Proti, et al, TPA and cycloheximidemodulate the activation of NF-kB and the induction and stability of nitric oxidesynthase transcript in primary neonatal rat hepatocytes. FEBS Lett.379,279–285(1996).
42. C. A. Salkowski, G. Detore, R. McNally, et al, Regulation of inducible nitric oxidesynthase messenger RNA expression and nitric oxide production bylipopolysaccharide in vivo. J. Immunol.138,905–912(1997).
43. J. A. Spitzer, Cytokine stimulation of nitric oxide formation and differential regulationin hepatocytes and nonparenchymal cells of endotoxemic rats. Hepatology19,217–228(1994).
44. R. D. Curran, T. R. Billiar, D. J. Stuehr, et al. Multiple cytokines are required to inducehepatocytes nitric oxide production and inhibit total protein synthesis. Ann. Surg.212,462–471(1990).
45. R. A. Schroeder, J. S. Gu and P. C. Kuo, et al. Interleukin1bstimulated production ofnitric oxide in rat hepatocytes in mediated through endogenous synthesis of interferongamma. Hepatology27,711–719(1998).
46. J. M. Griscavage, S. Wilk and L. J. Ignarro et al. Inhibitors of the proteasomepathways interfere with induction of nitric oxide synthase in macrophages by blockingactivation of transcription factor NF-kB. Proc. Natl. Acad. Sci. USA93,3308–3312(1996).
47. Q. W. Xie, Y. Kashiwabara, C. Nathan, et al. Role of transcription factor NF-kB/Rel ininduction of nitric oxide synthase. J.Biol. Chem.269,4705–4708(1994).
48. C. E. Bryant, M. Perretti and R. J. Flower, Suppression by dexamethasone of induciblenitric oxide synthase protein expression in vivo. A possible role for lipocortin
1.Biochem. Pharmacol.55,279–285(1998).
49. M. E. De Vera, B. S. Taylor, Q. Wang, et al, Dexamethasone suppresses iNOS geneexpression by upregulating I-k Ba and inhibiting NF-kB. Am. J. Physiol. Gastrointest.Liver Physiol.36, G1290–G1296(1997).
50. Suzuki A, Kudoh S, Mori K, et al. Expression of nitric oxide and inducible nitric oxidesynthase in acute renal allograft rejection in the rat. Int J Urol.2004Oct;11(10):837-44.
51. Yamaguchi Y, Okabe K, Matsumura F, et al. Peroxynotrite formation during rathepaticallograft. Hepatology1999;90:679-683.
52. Langrehr J, Murase N, Markus P, et al. Nitric oxide production in host-versus-graft andgraftversus-host reactions in the rat. J Clin Invest1992;90:679-683.
53. Kuo P, Alfrey E, Abe K, et al. Cellular localization and effect of nitric oxide synthesisin a rat model of orthotopic liver transplantation. Transplantation1996;61:305-312.
54. Ioannidis I, Hellinger A, Dehmlow C, et al. Evidence for increased nitric oxideproduction after liver transplantation in humans. Transplantation1995;59:1293-1297.
55. Bhathal P, Grossman H. Reduction of the increased portal vascular resistance of theisolated perfused cirrhotic rat liver by vasodilators. J Hepatol1985;1:325-32
56. Matei V, Rodríguez-Vilarrupla A, Deulofeu R, et al. Three-day tetrahydrobiopterintherapy increases in vivo hepatic NOS activity and reduces portal pressure in CCl4cirrhotic rats. J Hepatol.2008Aug;49(2):192-7.
57. Vogl S, Junker U, Vogelsang H,et al. Macrophages from rat livers wit h micronodularand macronodular cirrhosis differ with respect to mediator release and DNA-synthesis.J Hepatol,1997,26:1093-1103.
58. Bories PN, Campillo B, Azaou L, et al. Long-lasting NO overproduction in cirrhoticpatients with spontaneous bacterial peritonitis. Hepatology,1997,25:1328-1333.
59. Loureiro-Silva MR, Cadelina GW, Iwakiri Y, et al. A liver-specific nitric oxide donorimproves the intra-hepatic vascular response to both portal blood flow increase andmethoxamine in cirrhotic rats. J Hepatology2003;39:940-946.
60. Fiorucci S, Antonelli E, Brancaleone V, et al. NCX-1000, a nitric oxide-releasingderivative of ursodeoxycholic acid, ameliorates portal hypertension and lowersnorepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver. JHepatology2003;39:932-939.
61. Kwon SY, Groszmann RJ, Iwakiri Y, et al. Increased neuronal nitric oxide synthaseinteraction with soluble guanylate cyclase contributes to the splanchnic arterialvasodilation in portal hypertensive rats.
62. Shah V, Toruner M, Haddad F, et al. Impaired endothelial nitric oxide synthase activityassociated with enhanced caveolin binding in experimental liver cirrhosis.Gastroenterology1999;11(7):1222-1228.
63. Gupta T, Toruner M, Chung M, et al. Endothelial dysfunction and decreasedproduction of nitric oxide in the intrahepatic microcirculation of cirrhotic rats.Hepatology1998;28:926-931.
64. Sarela A, Mihaimeed F, Batten J, Davidson B, Mathie R. Hepatic and splanchnic nitricoxide activity in patients with cirrhosis. Gut1999;44:749-753
65. Shah V, Hendrickson H, Cao S, Yao J, Katusic Z. Regulation of hepatic endothelialnitric oxide synthase by caveolin and calmodulin after bile duct ligation in rats. Am JPhysiol2001;280:G1209-1216.