摘要
小体积肝移植在历经缺血再灌注损伤后,还存在复流后的门静脉高灌注损伤,其移植后的肝再生过程也不同于全肝移植和普通肝切除术后的肝再生。因此要提高临床活体肝移植的疗效,必须进一步研究部分肝移植尤其是小体积肝移植的缺血再灌注损伤、血液动力学、肝再生机制及其影响因素。通过建立与临床肝移植相似的动物模型,来寻找减轻肝移植术后移植物的缺血再灌注损伤、促进部分移植物尤其是小体积供肝的快速再生的途径,这对于提高临床活体肝移植的疗效有着非常重要的意义。
线粒体膜上存在对ATP浓度敏感的钾离子通道——mitoKATP,二氮嗪(DA)可以特异性开放该通道,而5-羟基癸酸盐(5-HD)则能将其特异性关闭。大量研究提示,mitoKATP通道开放在心肌、脑、肺、肾缺血再灌注损伤中均有保护作用,但目前国内外有关mitoKATP对肝脏的缺血再灌注损伤影响的研究很少。
通过二氮嗪(DA)预处理供体,观察mitoKATP开放对大鼠60%比例部分肝移植和30%比例小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。
第一部分大鼠30%比例小体积肝移植模型的建立
目的:建立稳定的大鼠30%比例小体积肝移植模型,为后续的移植肝缺血再灌注损伤(IRI)和小体积供肝移植后肝再生的实验研究打下基础。
方法:本部分行100例大鼠原位肝移植术,其中预实验50例,60%比例部分肝移植25例,30%比例小体积肝移植25例,全部采用“二袖套法”完成。全部手术均采用经腹主动脉插管原位快速灌注获取供肝,冷缺血时间均设定为1小时。另外各取5只大鼠,行40%和70%比例肝叶切除,分别观察存活率。
结果:60%和30%比例移植组,供肝与原肝重比分别为59.70±3.21%和31.92±2.43%,手术成功率(术后存活2天以上)分别为92%(23/25)和72%(18/25),1周存活率分别为72%(18/25)和64%(16/25)。肝切除组中,切除肝重与理论原肝重比分别为42.51±3.62%和66.83±4.37%,1周存活率为100%。
结论:在以往动物模型的基础上,通过改进操作方法,成功建立了大鼠60%比例部分肝移植和30%比例小体积肝移植模型。该模型稳定,手术成功率较高,便于操作,容易复制。大鼠部分肝移植模型尤其是小体积模型,与临床部分肝移植如活体肝移植有着相似的手术操作过程和病理生理变化,是研究改善肝移植缺血再灌注损伤、促进肝再生的理想途径。
第二部分二氮嗪预处理对大鼠小体积肝移植缺血再灌注损伤的保护机制研究及对肝再生的影响
目的:研究线粒体ATP敏感的钾通道(mitoKATP)开放,对大鼠部分肝移植尤其是小体积肝移植缺血再灌注损伤的保护作用,以及对肝再生的影响。为临床部分肝移植如活体肝移植,寻找减轻缺血再灌注损伤、促进术后肝再生的治疗靶点和途径。
方法:大鼠肝移植模型的建立方法同第一部分。SD大鼠300只,体重220~300g,随机配对成150对,随机分成6组,每组25对,供体体重应比受体轻10~20g。分组为全肝移植组(A组),60%比例部分肝移植组(C组),60%比例部分肝移植+二氮嗪组(DAl组),60%比例部分肝移植+5-羟基癸酸盐+二氮嗪组(HD-DA组),30%比例小体积肝移植组(S组),30%比例小体积肝移植+二氮嗪组(DA2组)。DA组供体术前30分钟腹腔注射二氮嗪(5mg/kg),再行手术;HD-DA组供体术前45分钟腹腔注射5-HD(5mg/kg),15分钟后腹腔注射二氮嗪(5mg/kg),再行手术。另取SD大鼠50只,体重220~300g,随机分成2组,每组25只。分组为40%比例肝切除组(B1组)和70%比例肝切除组(B2组)。
各手术组均取5个时间点,分别为术后2小时、1天、3天、5天和7天,每个时间点各取5例处死取材(n=5)。检测血清肝功能指标(ALT、AST),肝组织湿重并计算肝再生率,光镜下HE染色病理检查,电镜下超微结构检查,增殖细胞核抗原标记指数(PCNA),TUNEL法肝细胞凋亡指数,血清、肝组织细胞因子TNF-α、IL-6、IL-1β(ELISA法)检测,肝组织NF-κB活性检测(ELISA法)。
结果:
1.光镜下病理检查:DA组与对照组~*比较,肝细胞肿胀变性坏死及空泡样变性、肝小叶结构排列紊乱、汇管区炎症都较轻。DA组在术后7d分裂相多见,而对照组~*则减少。
2.电镜下超微结构检查:DA组中细胞结构完整,线粒体肿胀及细胞间隙扩张程度较轻,肝窦无扩张,肝巨噬细胞(Kupffer)活跃度低,吞噬体不多见。而对照组~*中细胞间隙明显增宽,窦间隙显著扩张,线粒体肿胀明显,线粒体嵴减少或消失,内质网扩张并有包裹线粒体现象,微绒毛丧失,Kupffer细胞侵入肝实质,吞噬活跃,局部见坏死区。
3.肝再生测定:DA组与对照组~*比较,肝湿重、肝再生率明显增加,术后7d有显著性差异(P<0.05)。
4.血清肝功能检测:DA组与对照组~*比较,谷丙转氨酶(ALT)、谷草转氨酶(AST)值明显降低,术后2h、1d、3d均有显著性差异(P<0.05)。
5.免疫组化法检测细胞核增值抗原(PCNA):DA组与对照组~*比较,PCNA阳性率明显增多,术后3d和5d有显著性差异(P<0.05)。
6.细胞凋亡检测(TUNEL法):DA组与对照组~*比较,凋亡指数(AI)术后2h和1d明显减少(P<0.05)。
7.肝组织白介素-1β(IL-1β)测定:DA组与对照组~*比较,术后2h和1d明显降低(P<0.05)。
8.血清、肝组织肿瘤坏死因子-α(TNF-α)测定:血清中,TNF-α表达较肝组织明显降低;DA组与对照组~*比较,TNF-α水平有所降低,但多数无显著性差异(P>0.05)。肝组织中,DA组与对照组~*比较,TNF-α明显下降,术后2h、1d和3d均有显著性差异(P<0.05)。
9.血清、肝组织白介素-6(IL-6)测定:DA组与对照组~*比较,血清和肝组织中IL-6均明显升高,术后2h、1d和3d有显著性差异(P<0.05)。
10.肝组织NF-κB活性的变化:DA组与对照组~*比较,术后2h和1d明显降低(P<0.05)。
结论:
1.应用mitoKATP开放剂二氮嗪,可以减轻大鼠部分肝移植包括小体积肝移植术后缺血再灌注损伤,其机制可能是通过降低IL-1β和TNF-α等细胞因子的表达,抑制了NF-κB的活性来减轻肝组织损伤。
2.二氮嗪在改善移植物缺血再灌注损伤的同时,能够增加大鼠部分肝移植包括小体积肝移植的肝重、肝再生率和细胞核增值抗原(PCNA)的表达,其机制可能是通过升高IL-6的表达、并维持一定的TNF-α和NF-κB的水平,来促进肝再生。
3.应用二氮嗪所产生的改善缺血再灌注损伤、促进肝再生的效应,可以被5-羟基癸酸盐特异性关闭,证明其作用位点在于mitoKATP。提示mitoKATP可能成为改善小体积肝移植疗效的一个治疗靶点。
PartⅠEstablishing the Model of 30%Small-for-Size Liver Transplantation in Rats
Objective:To establish the model of 30%small-for-size liver transplantation in rats, as a foundation for the forthcoming research on ischemia reperfusion injury(IRI) in small-for-size liver transplantation and liver regeneration post-operatively.
Methods:100 pairs of Sprague-Dawley(SD) rats were performed orthotopic liver transplantation(OLT) by using Kamada's 2-cuff method,among which 50 pairs were performed 100%OLT before formal experiment,25 pairs were performed 60%OLT, and 25 pairs were performed 30%OLT.The graft liver was procured by situ rapid perfusion through abdominal aorta.The time for cold ischemia was set as 1 hour. Besides,we randomly selected 10 rats to perform hepatectomy,5 rats by 40%and 5 rats by 70%,and observed the survival rate.
Results:For the groups of 60%and 30%partial liver transplantation,the weight ratios of donated liver to receipt liver were 59.70±3.21%and 31.92±2.43%. 92%(23/25) and 72%(18/25) survived for 2 days or above after transplantation. 72%(18/25) and 64%(16/25) were still alive 1 week after transplantation.For the group of hepatectomy,the weight ratios of the excised liver to the theoretical original liver were 42.51±3.62%and 66.83±4.37%,and 100%were alive 1 week after transplantation.
Conclusion:Based on past animal experiments,we successfully established the model of 60%and 30%partial liver transplantation in rats with improvement in methods.This model is stable,workable and replicable with high probability of success.The rat model of partial liver transplantation has similar operation procedures and pathological and physiological change as compared with clinical partial liver transplantation,such as living donor liver transplantation;and is an ideal way for research on improving IRI in liver transplantation and promoting liver regeneration.
PartⅡExperiment Study on the Role of Diazoxide Preconditioning in Alleviation Ischemia Reperfusion Injury after Rat Small-for-Size Liver Transplantation and the Impact on Liver Regeneration
Objective:To investigate the protection mechanism of mitochondrial ATP-sensitive potassium channels(mitoKATP) opening on IRI after rat partial liver transplantation, especially small-for-size liver transplantation,and the impacts on liver regeneration; and offer a better practice to relieve IRI and promote liver regeneration for clinical partial liver transplantation,such as living donor liver transplantation.
Methods:The animal model was built as the same way of PartⅠ.300 SD rats with the average weight of 220-300g were randomized into 150 pairs,and divided to 6 groups with 25 pairs in each group.Receipts should be heavier than donators by 10-20g.The 6 groups were:100%orthotopic liver transplantation(Group A),60%partial liver transplantation(Group C),60%partial liver transplantation with diazoxide(Group DA1),60%partial liver transplantation with 5-hydroxidecaprate and diazoxide (Group HD-DA),30%small-for-size liver transplantation(Group S),and 30% small-for-size liver transplantation with diazoxide(Group DA2).For Group DA, receipts received diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.For Group HD-DA,receipts received 5-HD(5mg/kg) 45 min preoperatively,and diazoxide(5mg/kg) by intraperitoneal injection 30 min preoperatively.Besides,50 SD rats with the average weight of 220-300g were randomized into 2 groups with 25 rats in each group.The 2 groups were respectively performed 40%hepatectomy(Group B1) and 70%hepatectomy(Group B2).
For each experimental group,5 rats were killed(n=5) respectively at 5 points of time,which were 2 hours,1 day,3 days,5 days,and 7 days postoperatively. Observation and detection includes liver function index(ALT,AST),wet weight of liver tissue postoperatively and ratio of liver regeneration,pathological examination by light microscope,examination of ultra-micro structure of cell by electro-microscope,proliferating cell nuclear antigen(PCNA) index,apoptosis of liver cells by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL),cytokines such as tumor necrosis fator-alpha(TNF-α),interleukin-6(IL-6), and interleukin-1β(IL-1β) in liver tissue and serum by enzyme-linked immunosorbent assay(ELISA),and the activity of NF-κB in liver tissue by ELISA.
Results:
1.Pathological examination by light microscope:as compared with the control group ~*,the liver cells of Group DA showed less swelling,degeneration and necrosis;the vacuolus showed less regeneration;the structure of hepatic lobule was less disorderly; inflammation was less serious.More cell division was found in Group DA than the control group.
2.Ultra-micro structure of cell examined by electro-microscope:the cell structure of Group DA was complete,swelling of mitochondria was less,intercellular space and sinus hepaticus was less distending,Kupffer cell was less active,and phagosome was less observed.However,mitochondrial crista and microvilli of the control group was less observed,endocytoplasmic reticulum was more swelling,Kupffer cell was more active,and some parts were necrosis.
3.Index of liver regeneration:as compared with the control group,liver wet weight of Group DA is larger and the liver regeneration ratio increased distinctly.Significant difference was noted 7 days post-operatively(P<0.05).
4.Index of liver function via serum:as compared with the control group,alanine aminotransferase(ALT) and aspartate aminotransferase(AST) of Group DA decreased distinctly,and significant difference was noted 2 hours,1 day,and 3 days post-operatively(P<0.05).
5.Proliferating cell nuclear antigen(PCNA) by immunohistochemistry:as compared with the control group,positive ratio of PCNA of Group DA increased distinctly,and significant difference was noted 3 days and 5 days post-operatively(P<0.05).
6.Apoptosis index(AI) by TUNEL:as compared with the control group,AI of Group DA decreased significantly 2 hours and 1 day post-operatively(P<0.05).
7.Expression of interleukin-1β(IL-1β) in liver tissue:as compared with the control group,IL-1βof Group DA decreased significantly 2 hours and 1 day post-operatively(P<0.05).
8.Expression of tumor necrosis fator-alpha(TNF-α) in serum and liver tissue:TNF-αin serum significant decreased as compared with that in liver tissue.As compared with the control group,TNF-αof Group DA decreased but no significant difference (P>0.05).For liver tissue,TNF-ot of Group DA decreased significantly as compared with the control group and significant difference was noted 2 hours,1 day and 3 days post-operatively(P<0.05).
9.Expression of interleukin-6(IL-6) in serum and liver tissue:as compared with the control group,IL-6 in both serum and liver tissue of Group DA increased distinctly and significant difference was noted 2 hours,1 day and 3 days post-operatively (P<0.05).
10.Changes in activity of NF-κB in liver tissue:as compared with the control group, NF-κB of Group DA decreased significantly 2 hours and 1 day post-operatively (P<0.05).
Conclusion:
1.DA,which opens the mitoKATP,can relieve IRI after partial liver transplantation, including small-for-size graft,by reducing IL-1βand TNF-αand restraining the activity of NF-κB.
2.DA could relieve IRI after transplantation;in the meantime,it could increase the weight,regeneration ratio and PCNA of the transplanted liver,the mechanism of which may be promoting liver regeneration by increasing IL-6 and maintaining essential expression of TNF-αand NF-κB.
3.The effect of improving IRI and promoting liver regeneration by using DA could be specially blocked by 5-HD,and it proves that DA produces the effect through mitoKATP and implies that mitoKATP might be a target for improving the treatment effect of partial liver transplantation,including small-for-size graft liver.
引文
1. Kiuchi T, Kasahara M, Uryuhara K, et al. Impact of graft size mismatching on graft prognosis in liver transplantation from livingdonors. Transplantation. 1999 Jan 27;67(2):321-7.
2. Park KM, Lee SG, Lee YJ, et al. Adult-to-adult living donor liver transplantation at Asian Medical Center, Seoul, Korea. Transplant Proc. 1999 Feb-Mar;31(1-2):456-8.
3. Li J, Dahmen U, Dirsch O, et al. Modified sleeve anastomosis for reconstruction of the hepatic artery in rat liver transplantation. Microsurgery. 2002;22(2):62-8.
4. Lemasters JJ, Thurman RG Reperfusion injury after liver preservation for transplantation. Annu Rev Pharmacol Toxicol. 1997;37:327-38.
5. Shimizu H, Miyazaki M, Ito H, et al. Mechanism of cold ischemia-reperfusion-induced graft injury after orthotopic liver transplantation in rats. Hepatogastroenterology. 2001 Jan-Feb;48(37):216-9.
6. Man K, Lo CM, Ng IO, et al. Liver transplantation in rats using small-for-size grafts: a study of hemodynamic and morphological changes. Arch Surg. 2001 Mar;136(3):280-5.
7. Troisi R, Ricciardi S, Smeets P, et al. Effects of hemi-portocaval shunts for inflow modulation on the outcome of small-for-size grafts in living donor liver transplantation. Am J Transplant. 2005 Jun;5(6):1397-404.
8. Asakura T, Ohkohchi N, Orii T, et al. Portal vein pressure is the key for successful liver transplantation of an extremely small graft in the pig model. Transpl Int. 2003 Jun;16(6):376-82.
9. Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery. 1983 Jan;93 Pt 1):64-9.
10. Lo CM, Fan ST, Liu CL, et al. Minimum graft size for successful living donor liver transplantation. Transplantation. 1999 Oct 27;68(8):1112-6.
11. Lo CM, Fan ST, Liu CL, et al. Extending the limit on the size of adult recipient in living donor liver transplantation using extended right lobe graft. Transplantation. 1997 May 27;63(10):1524-8.
12. Hashikura Y, Kawasaki S, Terada M, et al. Long-term results of living-related donor liver graft transplantation: a single-center analysis of 110 transplants. Transplantation. 2001 Jul 15;72(1):95-9.
13. Lee S, Charters AC, Chandler JG, et al. A technique for orthotopic liver transplantation in the rat. Transplantation. 1973 Dec;16(6):664-9.
14. Miyata M, Fischer JH, Fuhs M, et al. A simple method for orthotopic liver transplantation in the rat. Cuff technique for three vascular anastomoses. Transplantation. 1980 Nov;30(5):335-8.
15. Webber EM, Godowski PJ, Fausto N. In vivo response of hepatocytes to growth factors requires an initial priming stimulus. Hepatology. 1994 Feb;19(2):489-97.
16. Liu ML, Mars WM, Zarnegar R, et al. Collagenase pretreatment and the mitogenic effects of hepatocyte growth factor and transforming growth factor-alpha in adult rat liver. Hepatology. 1994 Jun; 19(6): 1521-7.
17. Kountouras J, Boura P, Lygidakis NJ. Liver regeneration after hepatectomy. Hepatogastroenterology. 2001 Mar-Apr;48(38):556-62.
18. Greenbaum LE, Li W, Cressman DE, et al. CCAAT enhancer- binding protein beta is required for normal hepatocyte proliferation in mice after partial hepatectomy. J Clin Invest. 1998 Sep 1;102(5):996-1007.
19. Yamaguchi Y, Kikuchi N, Hisama N, et al. Orthotopic reduced-size hepatic transplantation in rats. Transplant Proc. 1995 Apr;27(2): 1617-9.
20. Xia R, Emond JC. Orthotopic partial liver transplantation in the rat: a model of 70% hepatectomy and reduced size liver transplantation. Transplantation. 1993 Oct;56(4):1041-3.
21. Franco-Gou R, Peralta C, Massip-Salcedo M, et al. Protection of reduced-size liver for transplantation. Am J Transplant. 2004 Sep;4(9): 1408-20.
22. Ito T, Kiuchi T, Yamamoto H, et al. Changes in portal venous pressure in the early phase after living donor liver transplantation: pathogenesis and clinical implications. Transplantation. 2003 Apr 27;75(8):1313-7.
23. Dahm F, Georgiev P, Clavien PA. Small-for-size syndrome after partial liver transplantation: definition, mechanisms of disease and clinical implications. Am J Transplant. 2005 Nov;5(11):2605-10.
24. Palmes D, Minin E, Budny T, et al. The endothelin/nitric oxide balance determines small-for-size liver injury after reduced-size rat liver transplantation.Virchows Arch. 2005 Oct;447(4):731-41.
25. Schemmer P, Schoonhoven R, Swenberg JA, et al. Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation: role of Kupffer cells. Transplantation. 1998 Apr 27;65(8):1015-20.
26. Kobayashi E, Kamada N, Goto S, et al. Protocol for the technique of orthotopic liver transplantation in the rat. Microsurgery. 1993;14(8):541-6.
27. Urata K, Nguyen B, Brault A, et al. Decreased survival in rat liver transplantation with extended cold preservation: role of portal vein clamping time. Hepatology. 1998 Aug;28(2):366-73.
28. Goto M, Takei Y, Kawano S, et al. Tumor necrosis factor and endotoxin in the pathogenesis of liver and pulmonary injuries after orthotopic liver transplantation in the rat. Hepatology. 1992 Aug;16(2):487-93.
29. Meyer K, Brown MF, Zibari G, et al. ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion: a clue to the mechanism of multiple organ failure. J Pediatr Surg. 1998 Feb;33(2):350-3.
1. Arii S, Teramoto K, Kawamura T. Current progress in the understanding of and therapeutic strategies for ischemia and reperfusion injury of the liver. J Hepatobiliary Pancreat Surg 2003; 10(3):189-94.
2. Jaeschke H. Molecular mechanisms of hepatic ischemia-reperfusion injury and preconditioning. Am J Physiol Gastrointest Liver Physiol 2003; 284(1):G15-26.
3. Arii S, Monden K, Adachi Y, et al. Pathogenic role of Kupffer cell activation in the reperfusion injury of cold-preserved liver. Transplantation 1994; 58(10):1072-7.
4. Rauen U, Reuters I, Fuchs A, de Groot H. Oxygen-free radical-mediated injury to cultured rat hepatocytes during cold incubation in preservation solutions. Hepatology 1997; 26(2):351-7.
5. Piratvisuth T, Dunne JB, Williams R, Tredger JM. Amlodipine improves hepatic hemodynamic and metabolic function in the isolated perfused rat liver after sequential cold and warm ischemia. Transplantation 1995; 60(1):23-8.
6. Hamamoto I, Hossain MA, Mori S, et al. Impact of adhesion molecules of the selectin family on liver microcirculation at reperfusion following cold ischemia. Transpl Int 1996; 9(5):454-60.
7. Yoshidome H, Kato A, Edwards MJ, Lentsch AB. Interleukin-10 suppresses hepatic ischemia/reperfusion injury in mice: implications of a central role for nuclear factor kappa B. Hepatology 1999; 30(1):203-8.
8. Vollmar B, Glasz J, Leiderer R, et al. Hepatic microcirculatory perfusion failure is a determinant of liver dysfunction in warm ischemia-reperfusion. Am J Pathol 1994; 145(6):1421-31.
9. Lichtman SN, Lemasters JJ. Role of cytokines and cytokine-producing cells in reperfusion injury to the liver. Semin Liver Dis 1999; 19(2): 171-87.
10. Kiuchi T, Kasahara M, Uryuhara K, et al. Impact of graft size mismatching on graft prognosis in liver transplantation from living donors. Transplantation 1999; 67(2):321-7.
11. Park KM, Lee SG, Lee YJ, et al. Adult-to-adult living donor liver transplantation at Asian Medical Center, Seoul, Korea. Transplant Proc 1999; 31(1-2):456-8.
12. Li J, Dahmen U, Dirsch O, et al. Modified sleeve anastomosis for reconstruction of the hepatic artery in rat liver transplantation. Microsurgery 2002; 22(2):62-8.
13. Asakura T, Ohkohchi N, Orii T, et al. Portal vein pressure is the key for successful liver transplantation of an extremely small graft in the pig model. Transpl Int 2003; 16(6):376-82.
14. Man K, Lo CM, Ng IO, et al. Liver transplantation in rats using small-for-size grafts: a study of hemodynamic and morphological changes. Arch Surg 2001; 136(3):280-5.
15. Troisi R, Ricciardi S, Smeets P, et al. Effects of hemi-portocaval shunts for inflow modulation on the outcome of small-for-size grafts in living donor liver transplantation. Am J Transplant 2005; 5(6): 1397-404.
16. Mann DV, Lam WW, Hjelm NM, et al. Human liver regeneration: hepatic energy economy is less efficient when the organ is diseased. Hepatology 2001; 34(3):557-65.
17. Kroemer G, Zamzami N, Susin SA. Mitochondrial control of apoptosis. Immunol Today 1997; 18(1):44-51.
18. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature 1983; 305(5930): 147-8.
19. Hu H, Sato T, Seharaseyon J, et al. Pharmacological and histochemical distinctions between molecularly defined sarcolemmal KATP channels and native cardiac mitochondrial KATP channels. Mol Pharmacol 1999; 55(6): 1000-5.
20. Inoue I, Nagase H, Kishi K, Higuti T. ATP-sensitive K+ channel in the mitochondrial inner membrane. Nature 1991; 352(6332):244-7.
21. Rajesh KG, Sasaguri S, Suzuki R, et al. Ischemic preconditioning prevents reperfusion heart injury in cardiac hypertrophy by activation of mitochondrial KATP channels. Int J Cardiol 2004; 96(1):41-9.
22. Kis B, Rajapakse NC, Snipes JA, et al. Diazoxide induces delayed pre-conditioning in cultured rat cortical neurons. J Neurochem 2003; 87(4):969-80.
23. Fukuse T, Hirata T, Omasa M, Wada H. Effect of adenosine triphosphate-sensitive potassium channel openers on lung preservation. Am J Respir Crit Care Med 2002; 165(11):1511-5.
24. Rahgozar M, Willgoss DA, Gobe GC, Endre ZH. ATP-dependent K+ channels in renal ischemia reperfusion injury. Ren Fail 2003; 25(6):885-96.
25. Thadhani R, Mutter WP, Wolf M, et al. First trimester placental growth factor and soluble fins-like tyrosine kinase 1 and risk for preeclampsia. J Clin Endocrinol Metab 2004; 89(2):770-5.
26. Yao A, Li X, Pu L, et al. Impaired hepatic regeneration by ischemic preconditioning in a rat model of small-for-size liver transplantation. Transpl Immunol 2007; 18(1):37-43.
27. Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemia-reperfusion injury. Am J Surg 2001; 181(2):160-6.
28. Togo S, Kubota T, Matsuo K, et al. [Mechanism of liver failure after hepatectomy]. Nippon Geka Gakkai Zasshi 2004; 105(10):658-63.
29. Olthoff KM. Molecular pathways of regeneration and repair after liver transplantation. World J Surg 2002; 26(7):831-7.
30. Said A, Einstein M, Lucey MR. Liver transplantation: an update 2007. Curr Opin Gastroenterol 2007; 23(3):292-8.
31. Debonera F, Aldeguer X, Shen X, et al. Activation of interleukin-6/STAT3 and liver regeneration following transplantation. J Surg Res 2001; 96(2):289-95.
32. Debonera F, Wang G, Xie J, et al. Severe preservation injury induces I1-6/STAT3 activation with lack of cell cycle progression after partial liver graft transplantation. Am J Transplant 2004; 4(12): 1964-71.
33. Que X, Debonera F, Xie J, et al. Pattern of ischemia reperfusion injury in a mouse orthotopic liver transplant model. J Surg Res 2004; 116(2):262-8.
34. Selzner N, Selzner M, Tian Y, et al. Cold ischemia decreases liver regeneration after partial liver transplantation in the rat: A TNF-alpha/IL-6-dependent mechanism. Hepatology 2002; 36(4 Pt l):812-8.
35. Bedirli A, Kerem M, Pasaoglu H, et al. Effects of ischemic preconditioning on regenerative capacity of hepatocyte in the ischemically damaged rat livers. J Surg Res 2005; 125(1):42-8.
36. Selzner M, Camargo CA, Clavien PA. Ischemia impairs liver regeneration after major tissue loss in rodents: protective effects of interleukin-6. Hepatology 1999; 30(2):469-75.
37. Inagaki N, Inazawa J, Seino S. cDNA sequence, gene structure, and chromosomal localization of the human ATP-sensitive potassium channel, uKATP-1, gene (KCNJ8). Genomics 1995; 30(1): 102-4.
38. Inagaki N, Gonoi T, Clement JPt, et al. Reconstitution of IKATP: an inward rectifier subunit plus the sulfonylurea receptor. Science 1995; 270(5239): 1166-70.
39. Deutsch N, Weiss JN. Effects of trypsin on cardiac ATP-sensitive K+ channels. Am J Physiol 1994; 266(2 Pt 2):H613-22.
40. Suzuki M, Kotake K, Fujikura K, et al. Kir6.1: a possible subunit of ATP-sensitive K+ channels in mitochondria. Biochem Biophys Res Commun 1997; 241(3):693-7.
41. Zhou M, Tanaka O, Sekiguchi M, et al. Localization of the ATP-sensitive potassium channel subunit (Kir6. l/uK(ATP)-l) in rat brain. Brain Res Mol Brain Res 1999; 74(1-2): 15-25.
42. Liu Y, Sato T, O'Rourke B, Marban E. Mitochondrial ATP-dependent potassium channels: novel effectors of cardioprotection? Circulation 1998; 97(24):2463-9.
43. Garlid KD, Paucek P, Yarov-Yarovoy V, et al. Cardioprotective effect of diazoxide and its interaction with mitochondrial ATP-sensitive K+ channels. Possible mechanism of cardioprotection. Circ Res 1997; 81(6): 1072-82.
44. Yagi S, Iida T, Hori T, et al. Optimal portal venous circulation for liver graft function after living-donor liver transplantation. Transplantation 2006; 81(3):373-8.
45. Shinoda M, Shimazu M, Wakabayashi G, et al. Tumor necrosis factor suppression and microcirculatory disturbance amelioration in ischemia/reperfusion injury of rat liver after ischemic preconditioning. J Gastroenterol Hepatol 2002; 17(11): 1211-9.
46. Vanden Hoek TL, Shao Z, Li C, et al. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J Mol Cell Cardiol 1997; 29(9):2441-50.
47. Mikkelsen RB, Wardman P. Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms. Oncogene 2003; 22(37):5734-54.
48. Halestrap AP, Kerr PM, Javadov S, Woodfield KY. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfiision injury of the heart. Biochim Biophys Acta 1998; 1366(1-2):79-94.
49. Bor MV, Durmus O, Cayci B, Turkozkan N. An alternative parameter for monitoring the therapeutic benefits of allopurinol simultaneously in renal ischaemia-reperfusion injury: MDA/ATP Ratio. Cell Biochem Funct 2000; 18(4):229-34.
50. Sun YJ, Wang Q, Pei GX, et al. Changes of serum MDA and SOD levels in ischemia-reperfusion injuries of the limbs in rats preconditioned with heat stress. Di Yi Jun Yi Da Xue Xue Bao 2002; 22(6):506-8.
51. Ardite E, Ramos C, Rimola A, et al. Hepatocellular oxidative stress and initial graft injury in human liver transplantation. J Hepatol 1999; 31(5):921-7.
52. Gulec B, Coskun K, Yigitler C, et al. Ischemia-reperfusion injury in the liver during renal transplantation: does perfusion solution play any role? Transplant Proc 2008; 40(1):59-62.
53. Zhu X, Qiu Y, Shi M, Ding Y. Matrine protects sinusoidal endothelial cells from cold ischemia and reperfusion injury in rat orthotopic liver transplantation. Ann Clin Lab Sci 2003; 33(2):216-25.
54. Su JF, Guo CJ, Wei JY, et al. Protection against hepatic ischemia-reperfusion injury in rats by oral pretreatment with quercetin. Biomed Environ Sci 2003; 16(1):1-8.
55. Jassem W, Armeni T, Quiles JL, et al. Protection of mitochondria during cold storage of liver and following transplantation: comparison of the two solutions, University of Wisconsin and Eurocollins. J Bioenerg Biomembr 2006; 38(1):49-55.
56. Czyz A, Szewczyk A, Nalecz MJ, Wojtczak L. The role of mitochondrial potassium fluxes in controlling the protonmotive force in energized mitochondria. Biochem Biophys Res Commun 1995; 210(1):98-104.
57. Halestrap AP. Regulation of mitochondrial metabolism through changes in matrix volume. Biochem Soc Trans 1994; 22(2):522-9.
58. Kowaltowski AJ, Seetharaman S, Paucek P, Garlid KD. Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria. Am J Physiol Heart Circ Physiol 2001; 280(2):H649-57.
59. Laclau MN, Boudina S, Thambo JB, et al. Cardioprotection by ischemic preconditioning preserves mitochondrial function and functional coupling between adenine nucleotide translocase and creatine kinase. J Mol Cell Cardiol 2001;33(5):947-56.
60. Holmuhamedov EL, Wang L, Terzic A. ATP-sensitive K+ channel openers prevent Ca2+ overload in rat cardiac mitochondria. J Physiol 1999; 519 Pt 2:347-60.
61. Crestanello J A, Doliba NM, Babsky AM, et al. Opening of potassium channels protects mitochondrial function from calcium overload. J Surg Res 2000; 94(2): 116-23.
62. Fryer RM, Eells JT, Hsu AK, et al. Ischemic preconditioning in rats: role of mitochondrial K(ATP) channel in preservation of mitochondrial function. Am J Physiol Heart Circ Physiol 2000; 278(1):H305-12.
63. Ferranti R, da Silva MM, Kowaltowski AJ. Mitochondrial ATP-sensitive K+ channel opening decreases reactive oxygen species generation. FEBS Lett 2003; 536(1-3):51-5.
64. Bilbao G, Contreras JL, Eckhoff DE, et al. Reduction of ischemia-reperfusion injury of the liver by in vivo adenovirus-mediated gene transfer of the antiapoptotic Bcl-2 gene. Ann Surg 1999; 230(2): 185-93.
65. Majno G, Joris I. Apoptosis, oncosis, and necrosis. An overview of cell death. Am JPathol 1995; 146(1):3-15.
66. Gao W, Bentley RC, Madden JF, Clavien PA. Apoptosis of sinusoidal endothelial cells is a critical mechanism of preservation injury in rat liver transplantation. Hepatology 1998; 27(6): 1652-60.
67. Nadig SN, Periyasamy B, Shafizadeh SF, et al. Hepatocellular ultrastructure after ischemia/reperfusion injury in human orthotopic liver transplantation. J Gastrointest Surg 2004; 8(6):695-700.
68. Schleimer K, Stippel DL, Kasper HU, et al. Portal hyperperfusion causes disturbance of microcirculation and increased rate of hepatocellular apoptosis: investigations in heterotopic rat liver transplantation with portal vein arterialization. Transplant Proc 2006; 38(3):725-9.
69. Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267(5203): 1456-62.
70. Ohkohchi N, Shibuya H, Tsukamoto S, et al. Kupffer's cells modulate neutrophile activity by superoxide anion and tumor necrosis factor-delta in reperfusion injury of liver transplantation-mechanisms of radical generation and reperfusion injury after cold ischemia. Transplant Proc 1999; 31(1-2):1055-8.
71. Cutrn JC, Perrelli MG, Cavalieri B, et al. Microvascular dysfunction induced by reperfusion injury and protective effect of ischemic preconditioning. Free Radic Biol Med 2002; 33(9): 1200-8.
72. Le Moine O, Louis H, Demols A, et al. Cold liver ischemia-reperfusion injury critically depends on liver T cells and is improved by donor pretreatment with interleukin 10 in mice. Hepatology 2000; 31(6): 1266-74.
73. Teoh N, Leclercq I, Pena AD, Farrell G Low-dose TNF-alpha protects against hepatic ischemia-reperfusion injury in mice: implications for preconditioning. Hepatology 2003; 37(1):118-28.
74. Boros P, Suehiro T, Curtiss S, et al. Differential contribution of graft and recipient to perioperative TNF-alpha, IL-1 beta, IL-6 and IL-8 levels and correlation with early graft function in clinical liver transplantation. Clin Transplant 1997; ll(6):588-92.
75. Teoh N, Field J, Sutton J, Farrell G Dual role of tumor necrosis factor-alpha in hepatic ischemia-reperfusion injury: studies in tumor necrosis factor-alpha gene knockout mice. Hepatology 2004; 39(2):412-21.
76. Lentsch AB, Yoshidome H, Kato A, et al. Requirement for interleukin-12 in the pathogenesis of warm hepatic ischemia/reperfusion injury in mice. Hepatology 1999; 30(6): 1448-53.
77. Ohta A, Sitkovsky M. Role of G-protein-coupled adenosine receptors in downregulation of inflammation and protection from tissue damage. Nature 2001;414(6866):916-20.
78. Ricciardi R, Kim RD, McDade TP, et al. NFkappaB expression during cold ischemia correlates with postreperfusion graft function. J Surg Res 2000; 93(1):35-40.
79. Zughaier SM, Tzeng YL, Zimmer SM, et al. Neisseria meningitidis lipooligosaccharide structure-dependent activation of the macrophage CD14/Toll-like receptor 4 pathway. Infect Immun 2004; 72(1):371-80.
80. Tanonaka K, Iwai T, Motegi K, Takeo S. Effects of N-(2-mercaptopropionyl)-glycine on mitochondrial function in ischemic-reperfused heart. Cardiovasc Res 2003; 57(2):416-25.
81. Rentsch M, Beham A, Sirek S, et al. Glycine but not gadolinium chloride or methyl palmitate reduces postischemic white blood cell accumulation and early graft nonfunction after liver transplantation in the rat. Transplant Proc 2002; 34(6):2389-90.
82. Koniaris LG, McKillop IH, Schwartz SI, Zimmers TA. Liver regeneration. J Am Coll Surg 2003; 197(4):634-59.
83. Fausto N, Riehle KJ. Mechanisms of liver regeneration and their clinical implications. J Hepatobiliary Pancreat Surg 2005; 12(3):181-9.
84. Zimmermann A. Regulation of liver regeneration. Nephrol Dial Transplant 2004; 19 Suppl 4:iv6-10.
85. Tanno M, Taguchi T. Proliferating cell nuclear antigen in normal and regenerating rat livers. Exp Mol Pathol 1999; 67(3): 192-200.
86. Campbell JS, Prichard L, Schaper F, et al. Expression of suppressors of cytokine signaling during liver regeneration. J Clin Invest 2001; 107(10): 1285-92.
87. Aldeguer X, Debonera F, Shaked A, et al. Interleukin-6 from intrahepatic cells of bone marrow origin is required for normal murine liver regeneration. Hepatology 2002; 35(1):40-8.
88. Taub R. Liver regeneration: from myth to mechanism. Nat Rev Mol Cell Biol 2004; 5(10):836-47.
89. Cressman DE, Greenbaum LE, DeAngelis RA, et al. Liver failure and defective hepatocyte regeneration in interleukin-6-deficient mice. Science 1996; 274(5291): 1379-83.
90. Palmes D, Minin E, Budny T, et al. The endothelin/nitric oxide balance determines small-for-size liver injury after reduced-size rat liver transplantation. Virchows Arch 2005; 447(4):731-41.
91. Nagano Y, Nagahori K, Yoshiro F, et al. Gene expression profile analysis of regenerating liver after portal vein ligation in rats by a cDNA microarray system. Liver Int 2004; 24(3):253-8.
92. Liu H, Lo CR, Czaja MJ. NF-kappaB inhibition sensitizes hepatocytes to TNF-induced apoptosis through a sustained activation of JNK and c-Jun. Hepatology 2002; 35(4):772-8.
93. Kisseleva T, Bhattacharya S, Braunstein J, Schindler CW. Signaling through the JAK/STAT pathway, recent advances and future challenges. Gene 2002; 285(1-2):1-24.
94. Li W, Liang X, Kellendonk C, et al. STAT3 contributes to the mitogenic response of hepatocytes during liver regeneration. J Biol Chem 2002; 277(32):28411-7.
95. Selzner M, Clavien PA. Failure of regeneration of the steatotic rat liver: disruption at two different levels in the regeneration pathway. Hepatology 2000;31(1):35-42.
96. Fausto N. Liver regeneration. J Hepatol 2000; 32(1 Suppl):19-31.
97. Fausto N. Liver regeneration: from laboratory to clinic. Liver Transpl 2001; 7(10):835-44.
98. Kato A, Bamba H, Shinohara M, et al. Relationship between expression of cyclin D1 and impaired liver regeneration observed in fibrotic or cirrhotic rats. J Gastroenterol Hepatol 2005; 20(8): 1198-205.
1.Omura T,Ascher NL,Emond JC.Fifty-percent partial liver transplantation in the rat.Transplantation,1996,62(2):292
2.成峰,王学浩,张峰,等.冷缺血对大鼠部分肝移植肝再生的影响.中华器官移植杂志,2004,25(4):221
3.梁廷波,郑树森,徐骁,等.两种不同方法的大鼠小体积肝移植模型建立的比较.中华肝胆外科杂志,2004,10(5):320
4.Kamada N,Calne RY.A surgical experience with five hundred thirty liver transplants in the rat.Surgery,1983,93(1 Pt 1):64
5.Steinbauer F,Fodra C,Muller V,et al.Partial orthotopic liver transplantation in rats.Eur Surg Res,1995,27(4):205
6.Schemmer P,Schoonhoven R,Swenberg JA,et al.Gentle in situ liver manipulation during organ harvest decreases survival after rat liver transplantation:role of Kupffer cells.Transplantation,1998,65(8):1015
7.Kobayashi E,Kamada N,Goto S,et al.Protocol for the technique of orthotopic liver transplantation in the rat.Microsurgery,1993,14(8):541
8.Urata K,Nguyen B,Brault A,et al.Decreased survival in rat liver transplantation with extended cold preservation:role of portal vein clamping time.Hepatology,1998,28(2):366
9.Goto M,Takei Y,Kawano S,et al.Tumor necrosis factor and endotoxin in the pathogenesis of liver and pulmonary injuries after orthotopic liver transplantation in the rat.Hepatology,1992,16(2):487
10.Meyer K,Brown MF,Zibari G,et al.ICAM-1 upregulation in distant tissues after hepatic ischemia/reperfusion:a clue to the mechanism of multiple organ failure.J Pediatr Surg,1998,33(2):350
11.Franco-Gou R,Peralta C,Massip-Salcedo M,et al.Protection of reduced-size liver for transplantation. Am J Transplant, 2004,4(9): 1408
12. Zhong Z, Connor HD, Froh M, et al. Free radical-dependent dysfunction of small-for-size rat liver grafts: prevention by plant polyphenols. Gastroenterology, 2005,129(2):652
13. Man K, Lo CM, Ng 10, et al. Liver transplantation in rats using small-for-size grafts: a study of hemodynamic and morphological changes. Arch Surg, 2001,136(3):280
14. Palmes D, Budny TB, Dietl KH, et al. Detrimental effect of sinusoidal overperfusion after liver resection and partial liver transplantation. Transpl Int, 2005,17(12):862
15. Franco-Gou R, Rosello-Catafau J, Peralta C. Protection against lung damage in reduced-size liver transplantation. Crit Care Med,2006, 34(5): 1506
16. Glanemann M, Eipel C, Nussler A K, et al. Hyperperfusion syndrome in small-for-size livers. Eur Surg Res, 2005,37(6):335
1 Charlton M,Seaberg E,Wiesner R,et al.Predictors of patient and graft survival following liver transplantation for hepatitis C.Hepatology,1998,28:823-830.
2 Rodriguez-Luna H,Vargas HE,Sharrma P,et al.Hepatitis C virus recurrence in living donor liver transplant recipients.Dig Dis Sci,2004,49:38-41.
3 Vargas HE,Laskus T,Wang LF,et al.Outcome of liver transplantation in hepatitis C virus-infected patients who received hepatitis C virus-infected grafts.Gastroenterology,1999,117:149-153.
4 Everson GT.Treatment of patients with hepatitis C virus on the waiting list.Liver Transpl,2003,9:S90-94.
5 Gane E. Treatment of recurrent hepatitis C. Liver Transpl,2002,8(suppl l):S28-37.
6 Schirren CA, Zachoval R, Gerlach JT, et al. Antiviral treatment of recurrent hepatitis C virus (HCV) infection after liver transplantation: association of a strong, multispecific, and long-lasting CD4~+ T cell response with HCV-elimination. J Hepatol,2003,39: 397-404.
7 Gane EJ, Naoumov NV, Qian KP, et al. A longitudinal analysis of hepatitis C virus replication following liver transplantation. Gastroenterology, 1996,110:167-177.
8 Wiesner RH, Sorrell M, Villamil F. Report of the first International Liver Transplantation Society expert panel consensus conference on liver transplantation and hepatitis C. Liver Transpl,2003, 9:S1-9.
9 Pinna AD, Ricordi C, Weppler D, et al. Treatment of recurrent hepatitis C after liver transplantation with IL-2r Ab. Transplant Proc,2001,33:1087-1089.
10 Zhu J, Wu J, Frizell E, et al. Rapamycin inhibits hepatic stellate cell proliferation in vitro and limits flbrogenesis in an in vivo model of liver fibrosis. Gastroenterology, 1999,117:1198-1204.
1.Starzl TE,Miller C,Broznick B,et al.An improved technique for multiple organ harvesting[J].Surg Gynecol Obstet,1987,165:343-348.
2.Nakazato PZ,Concepcion W,Bry W,et al.Total abdominal evisceration:an en bloc technique for abdominal organ harvesting[J].Surgery,1992,111:37-47.
3.Casavilla A,Ramirez C,Shapiro R,et al.Experience with liver and kidney allografts from non-heart-beating donors[J].Transplantation,1995,59:197-203.
4.樊嘉,朱岳,Fung J.肝移植供肝获取的现代方法与实践[J].中国临床医学,2001,8:198-200.
5.Marino IR,Doria C,Doyle HR,et al.Matching donors and recipients[J].Liver Transpl Surg,1998,4:S115-119.
6.Pascher A,Neuhaus P.Bile duct complications after liver transplantation[J].Transpl Int,2005,18:627-642.
7.Moser MA,Wall WJ.Management of biliary problems after liver transplantation [J].Liver Transpl,2001,7:S46-52.