心脏停搏供体大鼠供肝热缺血对原位肝移植物的影响

目的:探讨来自心脏停搏供体大鼠肝移植供肝热缺血再灌注损伤对移植物的影响.方法:实验分为4组:对照组(C)和移植组,移植组根据供肝获取前经历供体心脏停搏时间的不同分为3组:热缺血0 min(W0组)、热缺血15min(W15组)和热缺血30 min(W30组),其后用建立大鼠动脉化原位肝移植模型,每组均为24只大鼠,分别测定术后3、7、14和30 d移植肝组织学、肝功能和细胞增殖核抗原Ki-67蛋白的变化,每个时间点各取6只大鼠处死.结果:随着供肝热缺血时间的延长,移植肝损伤加重,并且恢复过程也延长.移植组和对照组术后第3、7、14和30天血清ALT、AST均无显著性改变.肝移植术后早期肝细胞Ki-67表达水平随供肝热缺血时间的延长而增高,术后14 d恢复正常.各组的肝细胞Ki-67表达均与血清ALT、AST无关.结论:肝移植过程中供肝热缺血主要损伤肝细胞,并随着供肝热缺血时间的延长移植肝细胞损伤加重,肝细胞功能恢复早于其形态学恢复.     

粒细胞集落刺激因子促进大鼠移植肝的再生作用

目的:探讨部分肝移植后,粒细胞集落刺激因子(granulocyte colony-stimulating factor, G-CSF)对部分肝移植物再生的促进作用．方法:采用改良“二袖套法”建立SD大鼠 50％部分肝移植模型,受体鼠随机分为实验组和对照组,术后分别注射(sc)G-CSF和相同体积的生理盐水5 d．观察大鼠移植肝存活时间．术后1,3,5,7和14 d取血清和移植肝脏,测移植物与受体质量比(graft-recipient weight ratio,GRWR),检测血清生化指标,并采用免疫组织化学方法观察肝内的增殖细胞核抗原 (proliferating cell nuclear antigen,PCNA)的表达．结果:实验组移植肝存活率较对照组高(90％ vs 60％,X2=5．03,P<0．05),术后第3-5天,实验组GRWR较对照组明显增加(P<0．05)．两组肝再生均于移植后3 d达高峰．与对照组相比, 实验组肝坏死灶少,AST,ALT水平低(3 d:t= 17．61,P<0．05;t=20．16,P<0．05;5 d:t=15．64, P<0．05;t=23．08,P<0．05),白蛋白水平高(3 d: 36．2±4．7 vs 29．5±3．4,P<0．05;5 d:43．2± 4．1 vs 33．8±3．9,P<0．05),PCNA表达升高(t= 23．08,P<0．05)．结论:大鼠部分肝移植后G-CSF可以促进肝细胞再生,减轻肝损伤．     

大鼠移植肝热缺血再灌注损伤的研究

建立大鼠动脉化肝移植胆管外引流模型,术后24 h测胆汁酶学及血清肝功能指标、内皮素-1(ET-1)、超氧化物歧化酶(SOD)、丙二醛(MDA)、一氧化氮(NO)和移植肝细胞间黏附分子-1(ICAM-1)mRNA.结果显示,随着供肝热缺血时间的延长,肝移植后大鼠血清ALT、AST、乳酸脱氢酶(LDH)、GGT、直接胆红素(DBil)、MDA、NO及肝细胞ICAM-1 mRNA显著升高.认为肝移植中供肝热缺血主要损伤肝细胞,SOD、ET-1、NO、ICAM-1参与供肝热缺血造成的移植肝损伤,ICAM-1可能是导致移植肝缺血再灌注时肝细胞损伤的主要因素.     

苦参碱对大鼠小体积肝移植缺血再灌注损伤的保护作用

目的: 探讨苦参碱对大鼠小体积肝移植缺血再灌注损伤的保护作用及机制.方法:采用大鼠30%小体积肝移植模型, ♂SD大鼠322只随机分为假手术组、小体积肝移植对照组和高、低剂量苦参碱治疗组(60、40 mg/kg). 观察术后1 wk生存率, 检测移植术2h、4 h、1 d、2 d、3d、7 d后ALT、AST及LDH值. 光镜及电镜下评估移植肝病理形态学改变, ELISA法检测肝脏IL-6, TNF-α表达.结果: 与对照组比较, 苦参碱高、低剂量治疗组术后1 wk生存率显著增加(80%, 70% vs 50%, 均P<0.05), 术后2h、4h、1d ALT、AST及LDH明显降低(P<0.01). 苦参碱治疗组中肝细胞和肝窦内皮细胞凋亡减少、细胞形态明显改善, 苦参碱治疗组术后2 h、4 h、1 d肝脏组织中IL-6, TNF-α水平明显降低(P<0.01). 结论:苦参碱可减轻肝细胞及肝窦内皮细胞的损伤, 改善小体积肝移植术后缺血再灌注损伤, 其机制可能与苦参碱抑制肝移植术后IL-6、TNF-α等炎症因子的释放有关.     

改良大鼠离体肝脏灌注模型在供肝冷保存中的应用研究

背景:供肝冷保存和缺血再灌注损伤是影响肝移植患者预后的关键因素,建立稳定的冷保存再灌注动物模型是开展相关研究的基础。目的:建立适用于综合评价供肝质量的改良大鼠离体肝脏灌注模型,为供肝冷保存研究提供合适的离体再灌注模型。方法:12只Sprague-Dawley大鼠随机分为两组,对照组正常获取供肝后进行离体灌注90 min,实验组供肝经30 min热缺血后获取,冷保存24 h后再行离体灌注。离体灌注期间监测灌注液转氨酶水平、电解质浓度和pH值;以压力感受器监测供肝门静脉压力;以超微自由基探针监测肝组织过氧化氢(HPO)水平。灌注结束后记录胆汁流出量,检测供肝组织丙二醛(MDA)、超氧化物歧化酶(Cu/Zn SOD)水平及其组织病理学改变和肝细胞凋亡情况。结果:与对照组相比,实验组供肝AST、ALT水平、门静脉压力和HPO水平明显增高,胆汁流出量显著减少,肝组织Cu/Zn SOD水平显著降低。对照组供肝组织病理学损伤和肝细胞凋亡相对较轻。结论:经热缺血和冷保存后,供肝的肝功能和抗氧化能力均明显降低。本研究构建的改良大鼠离体肝脏灌注模型可有效监控和评估大鼠供肝冷保存再灌注损伤情况。     

骨髓间充质干细胞对大鼠减体积肝移植术后肝细胞再生与修复的作用

目的探讨骨髓间充质干细胞（BMSCs）对大鼠减体积肝移植术后肝脏再生与修复的影响。方法采用生化、免疫组化等方法检测术后不同时间的血清和肝脏标本。（1）取同周龄Wistar大鼠骨髓体外培养BMSCs,标记CSFE荧光后制备悬液;（2）建立大鼠50%减体积肝移植模型（供体鼠为SD大鼠,受体鼠为Wistar大鼠）,分为实验组（取BMSCs悬液经门静脉植入）和对照组（取等量生理盐水经门静脉注射）,观察大鼠生存状态,测量肝再生的相关指标。结果 （1）成功分离培养BMSCs;（2）术后2h,大鼠均自由活动饮水;（3）实验组肝组织中存在大量BMSCs,术后第2、3 d实验组肝体比显著高于对照组。病理显示：实验组肝细胞损伤较对照组轻微。结论植入BMSCs后,可以促进大鼠减体积肝移植术后肝细胞增殖和修复。     

白细胞介素-10与减体积大鼠肝移植后肝再生的关系

目的 探讨白细胞介素-10(IL-10)与减体积大鼠肝移植术后移植肝再生的关系。方法 建立减体积大鼠肝移植模型,实验分为:肝切除组、全肝移植组和减体积肝移植组,分别于术后1、2、4、7d取肝组织,免疫组织化学检测各组IL-10的表达,流式细胞仪检测移植肝的增殖活性。结果 肝切除组、全肝移植组和减体积肝移植组肝细胞增生活跃,术后4d增殖高峰分别为26.3±0.9、35.8±2.2、32.4±1.8。IL-10与移植后肝再生呈负相关(r=-0.58,P<0.01)。结论 减体积肝移植和全肝移植术后肝脏具有同样的增殖活性,但增殖峰值较肝切除延迟。IL-10对移植肝肝再生具有明显的调控作用,同时受免疫系统产生的其它细胞因子和激素的影响。     

磁共振氢质波谱成像评估高强度聚焦超声治疗原发性肝癌疗效的可行性及其价值

目的 通过比较原发性肝癌高强度聚焦超声(HIFU)治疗前后胆碱峰、脂质峰下积分面积及胆碱/脂质峰下积分面积比值的变化,探讨磁共振氢质波谱成像(1H-MRS)在HIFU治疗原发性肝癌疗效评估中的价值及可行性.方法 24例原发性肝癌患者于HIFU治疗前及治疗后2周内行常规磁共振平扫及动态增强扫描.以呼吸触发的横轴位T2WI...     

受体血预先经门静脉输入供体延长肝移植大鼠生存时间

目的:探讨受体全血预先经门静脉输入供体对大鼠肝移植排斥反应的作用．方法:采用异系大鼠肝移植模型,供体为ACI大鼠．受体为LEW大鼠．将实验组受体大鼠全血1 mL经门静脉输入供体肝内,7 d后将处理后的供体肝脏移植于受体．经门静脉将生理盐水1 mL输入对照组供体肝内,7 d后将此供体肝脏移植于受体．观察大鼠生存时间,分期分批处死大鼠,从移植肝及受体脾分离T淋巴细胞,测定移植肝及受体脾内中供体来源活化T淋巴细胞的比例,检测细胞因子mRNA的表达．结果:肝移植大鼠生存时间:实验组31．7±7．6 d,对照组11．1±2．1 d．两组差异显著(P< 0．05)．移植后7 d从移植肝分离的浸润T淋巴细胞分析结果:实验组OX76 CD4 细胞25．6%．OX76 CD8 细胞26．6％;对照组OX76 CD4 细胞6．2％,OX76 CD8 细胞7．4％．实验组移植肝内有大量供体来源的活化T淋巴细胞．实验组肝内OX76 CD8 细胞可见IL-10 mRNA及IFN-γmRNA的表达．结论:受体血预先经门静脉输入供体,可以延长异系大鼠肝移植生存时间．有限的移植物抗宿主反应,可能是这种肝移植排斥反应抑制的机制．     

Synergistic effect of cold and warm ischemia time on postoperative graft outcome in human liver transplantation

BACKGROUND/AIMS: Prolonged cold ischemia time (CIT) during graft preservation and warm ischemia time (WIT) defined as a rewarming time have been reported to cause postoperative graft dysfunction after orthotopic liver transplantation (OLT). However, a synergistic effect of both CIT and WIT on patients of graft survival has not been confirmed. The aim of this study was to determine whether simultaneously prolonged CIT and WIT was associated with early graft outcome after clinical OLT. METHODOLOGY: Between May 1997 and July 1998, 186 consecutive OLT cases were divided into 4 groups as follows: group A, CIT < or =12 hrs and WIT < or =45 min; group B, CIT >12 hrs and WIT < or =45 min; group C, CIT < or =12 hrs and WIT >45 min; and group D, CIT > 12 hrs and WIT >45 min. Liver graft survival within 90 days of OLT and early postoperative graft function were analyzed. RESULTS: The graft loss rates were 5.4% in group A, 9.8% in group B, 11.1% in group C, and 42.9% in group D. The mean highest aspartate aminotransferase (AST) values after OLT in group D (3352.3+/-569.4 U/L) was significantly higher than those in groups A (1411.7+/-169.2 U/L) and B (1931.3+/-362.6 U/L). CONCLUSIONS: The simultaneously prolonged cold and warm ischemia time significantly caused hepatic allograft injury and failure, suggesting some synergistic effects of CIT and WIT on postoperative graft function.     

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats

AIM: To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time (WIT) in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS: The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30, 45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation (OLT), the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS: The donor liver from non-heart-beating donors (NHBD) underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergoing warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic lobule first. In the group undergoing warm ischemia injury for 60 min, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothe-lial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder. CONCLUSION: The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.     

Dynamical changing patterns of histological structure and ultrastructure of liver graft undergoing warm ischemia injury from non-heart-beating donor in rats

AIM: To investigate the histological and ultra-structural characteristics of liver graft during different of warm ischemia time (WIT) in rats and to predict the maximum limitation of liver graft to warm ischemia. METHODS: The rats were randomized into 7 groups undergoing warm ischemia injury for 0, 10, 15, 20, 30,45 and 60 min, respectively. All specimens having undergone warm ischemia injury were investigated dynamically by light and electron microscopy, and histochemistry staining. After orthotopic liver transplantation (OLT), the recovery of morphology of liver grafts after 6, 24 and 48 h was observed. RESULTS: The donor liver from non-heart-beating donors (NHBD) underwent ischemia injury both in the warm ischemia period and in the reperfusion period. Morphological changes were positively related to warm ischemia injury in a time-dependent manner during the reperfusion period. The results demonstrated that different degrees of histocyte degeneration were observed when WIT was within 30 min, and became more severe with the prolongation of WIT, no obvious hepatocyte necrosis was noted in any specimen. In the group undergolng warm ischemia injury for 45 min, small focal necrosis occurred in the central area of hepatic lobule first. In the group undergoing warm ischemia injury for 60 min, patchy or diffused necrosis was observed and the area was gradually extended, while hepatic sinusoid endothe lial cells were obviously swollen. Hepatic sinusoid was obstructed and microcirculation was in disorder. CONCLUSION: The rat liver graft undergoing warm ischemia injury is in the reversible stage when the WIT is within 30 min. The 45 min WIT may be a critical point of rat liver graft to endure warm ischemia injury. When the WIT is over 60 min, the damage is irreversible.     

Energy metabolism and survival of liver grafts from non-heart-beating donor rats with warm ischemia injury

Introduction The quality of liver graft is a key factor for liver transplantation. Organs from non- heart-beating donors (NHBDs) seem to bean effective option to alleviate the problem of liver donor shortage.[1-3] However, the main obstacle to the use of livers from NHBDs is that warm ischemia injury to the liver is related to cardiac arrest.[4-6] Moreover, in liver transplantation, the allograft sustains inevitable cold ischemia in addition to re- warming injury during liver reperfusion. …     

Mouse livers with macrosteatosis are more susceptible to normothermic ischemic injury than those with microsteatosis

BACKGROUND/AIMS: Fatty livers are increasingly used for transplantation due to the dramatic organ shortage. While steatosis is an established risk factor for post-operative complications, the impact of macro- vs. microvesicular steatosis on ischemic injury is unclear. METHODS: The effects of ischemia and reperfusion were tested in two different models of steatotic mice: ob/ob as a model disclosing predominantly macrovesicular steatosis and choline deficient diet having mainly microvesicular steatosis. Steatotic and lean livers were subjected to 45 min of ischemia. Serial markers of hepatocellular injury, animal survival were measured. Hepatic tissue blood flow and portal vein perfusion were assessed. RESULTS: Ob/ob mice had a significantly lower tolerance to hepatic ischemia. with increased AST release and decreased survival in comparison to the choline deficient mice. No difference in ATP content was found between both steatosis models, but hepatic perfusion and portal vein flow were significantly lower after reperfusion in the ob/ob mice when compared to the choline deficient animals. Ischemic pre-conditioning significantly improved liver reperfusion and injury in both models of steatosis. CONCLUSIONS: Livers with macrovesicular steatosis have a lower tolerance to ischemic injury than those with microvesicular steatosis. Low intrahepatic and portal vein perfusion in macrovesicular fatty livers is, at least partially, responsible for the poorer outcome.     

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment eady after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin(UW) solution at 4 ℃ pre-implantation, IP was done by damp of the portal vein and hepatic artery of the donor liver for 10 minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragrafl: expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization, The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored,RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP-2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MIP-2 and TNF-α levels were significantly elevated in non-IP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservation-reperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.     

Effects of warm ischemia time on biliary injury in rat liver transplantation

AIM: To investigate the effect of different secondary warm ischemia time (SWIT) on bile duct injury in liver-transplanted rats.METHODS: Forty-eight male inbred Sprague-Dawley rats were randomly assigned into four groups: a sham-operation group and three groups with secondary biliary warm ischemia time of 0 min, 10 min and 20 min. A rat model of autologous liver transplantation under ether anesthesia was established, and six rats were killed in each group and blood samples and the median lobe of the liver were collected for assay at 6 h and 24 h after hepatic arterial reperfusion.RESULTS: With prolongation of biliary warm ischemia time, the level of vascular endothelial growth factor-A was significantly decreased, and the value at 24 h was higher than that at 6 h after hepatic arterial reperfusion, but with no significant difference. The extended biliary SWIT led to a significant increase in bile duct epithelial cell apoptosis, and a decrease in the number of blood vessels, the bile duct surrounding the blood vessels and bile duct epithelial cell proliferation in the early postoperative portal area. Pathologic examinations showed that inflammation of the rat portal area was aggravated, and biliary epithelial cell injury was significantly worsened.CONCLUSION: A prolonged biliary warm ischemia time results in aggravated injury of the bile duct and the surrounding vascular plexus in rat autologous orthotopic liver transplantation.     

Ischemic preconditioning decreases C-X-C chemokine expression and neutrophil accumulation early after liver transplantation in rats

AIM: Polymorphonuclear neutrophil (PMN) plays a major role in liver ischemia/reperfusion injury. Protective effect of ischemic preconditioning (IP) has been confirmed in liver ischemia/reperfusion injury. The purpose of this study was to investigate the effect of IP on C-X-C chemokine expression and PMNs recruitment early after liver transplantation.METHODS: Male Sprague-Dawley rats were used as donors and recipients of orthotopic liver transplantation (OLT). The donor liver was stored 24 hours in University of Wisconsin (UW) solution at 4℃ pre-implantation. IP was done by clamp of the portal vein and hepatic artery of the donor liver for 10minutes followed by reperfusion for 10 minutes before harvesting. The neutrophilic infiltration in liver was quantified using a myeloperoxidase (MPO) assay. Intragraft expression of macrophage inflammatory protein-2 (MIP-2) mRNA was investigated with in situ hybridization. The serum levels of MIP-2 and tumor necrosis factor (TNF)-α were also monitored.RESULTS: After liver transplantation without IP, the hepatic MPO increased significantly compared with sham operated group. In IP group, PMN in liver indicated by MPO was reduced significantly. In situ hybridization showed no MIP2 mRNA in sham group but dramatic expression in hepatocytes in non-IP group. In IP group, MIP-2 mRNA was significantly down-regulated. Similarly, serum MTP-2 and TNF-α levels were significantly elevated in non-TP group and both were reduced in IP group.CONCLUSION: IP might protect graft liver from preservationreperfusion injury after OLT through down-regulating C-X-C chemokine expression of hepatocytes, and alleviating PMNs recruitment after reperfusion.     

肝移植术后早期乳酸清除率与ALT和AST的相关性及对移植肝损伤的评估研究

目的分析肝移植术后早期乳酸清除率(early lactate clearance rate,ELCR)和初期肝特异性氨基转移酶水平变化的特点及相关性,探讨ELCR对移植肝损伤的评估价值。方法回顾性分析301例原位肝移植(orthotopic liver transplantation,OLT)病例资料,针对其术后转入重症监护室的初期ALT、AST、动脉血乳酸水平以及术后6 h动脉血乳酸水平计算ELCR,并与ALT、AST及AST/ALT比值进行相关性分析。结果 ELCR与ALT、AST呈中度负相关(r分别为-0.668、-0.602,P均0.001),而与AST/ALT比值无相关性(r=0.077)。经统计分析构建的3个ELCR等级分别为Ⅰ级25%、Ⅱ级为25%~50%、Ⅲ级≥50%。Ⅰ级、Ⅱ级和Ⅲ级对应ALT水平分别为(1670.20±897.41)U/L、(937.87±350.06)U/L和(556.16±173.56)U/L,AST为(2835.98±1549.06)U/L、(1642.87±1106.00)U/L和(827.44±260.92)U/L,3组间ALT和AST水平差异有统计学意义(P均0.001)。随着ELCR等级增大,ALT和AST值降低。结论 ELCR可方便快速地反映肝移植后初期肝缺血-再灌注损伤程度,其不同等级可对肝移植术后初期移植肝功能进行初步评估。