初始灌注液对离体低温保存大鼠肝脏的影响

目的 探讨缺血再灌注过程中初始灌注液对离体低温保存大鼠肝脏的影响。方法采用离体大鼠肝脏低温缺血保存再灌注模型90例,分别选择UW液、HC-A液和乳酸林格液作为不同的初始灌注液,观察大鼠肝脏在低温保存0、3、6、12、24 h后再灌注流出液中ALT、AST、LDH和ET的水平,同时比较3组之间肝脏细胞形态和细胞凋亡。结果 使用HC-A液作为初始灌注液的大鼠肝脏再灌注流出液中ALT、AST、LDH和ET水平较其他两组低(P<0.05),肝脏形态和细胞凋亡的发生3组差异无显著性。另外,上述指标均受低温保存时间的影响。结论 初始灌注液可影响离体低温保存大鼠肝脏的质量,细胞凋亡可能参与了肝脏的缺血再灌注损伤。     

Celsior液与UW液对无心跳大鼠供肝保存效果的比较

目的 比较Celsior(CS)液与UW液对大鼠无心跳供者(NHBD)供肝的保存效果.方法 选取健康雄性SD大鼠作为肝移植的供、受者.通过阻断大鼠主动脉和膈上下腔静脉10 min的方法,制备和获取NHBD供肝,并采用不同的器官保存液灌注和冷保存供肝.随机将受者分为4组.CS8 h组:受者采用经CS液灌注和冷保存8 h的供肝移植;UW8 h组:受者采用经UW液灌注和冷保存8 h的供肝移植;CS16 h组:受者采用经CS液灌注和冷保存16 h的供肝移植;UW16 h组:受者采用经UW液灌注和冷保存16 h的供肝移植.受者门静脉开放前、开放后1、3及6 h,取各组受者的静脉血检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、内皮素1(ET-1)、白细胞介素1(IL-1)及肿瘤坏死因子α(TNF-α)水平;观察和比较各组受者的胆汁生成量、移植肝组织病理学改变及术后7 d内的存活率.结果 NHBD供肝经UW液灌注后呈"花斑"状,肝叶边缘灌注不良,经CS液灌注后肝叶边缘灌注良好.CS8 h组和UW8 h组受者的胆汁生成量分别为(0.21±0.01)ml和(0.10±0.02)ml(P<0.05).门静脉开放后1、3及6 h,CS8 h组受者的血清ALT及AST水平明显低于UW8 h组(P<0.05),门静脉开放后1、3h,CS8 h组受者的血清ET-1、IL-1及TNF-α水平均明显低于UW8 h组(P<0.05);CS8 h组受者移植肝肝窦扩张、门静脉充血及炎症细胞浸润等病理学改变明显轻于UW8 h组,CS8 h组和UW8 h组受者术后7 d的存活率分别为58.3%和25.0%(P<0.05).CS16 h组和UW16 h组受者各时点的胆汁分泌量、血清ALT、AST、ET-1、IL-1及TNF-α水平的比较,差异均无统计学意义(P>0.05),两组受者均在术后3 d内死亡,两组受者移植肝组织病理学改变无明显差异.结论 CS液对大鼠NHBD供肝的保存效果优于UW液,这可能与UW液较CS液粘稠及CS液能够减少枯否细胞的激活有关;NHBD供肝的冷保存时间不宜超过16 h.     

丹参对大鼠供肝冷保存缺血再灌注损伤中肝细胞凋亡的影响

目的 探讨丹参对大鼠原位肝移植供肝冷保存缺血再灌注损伤中肝细胞凋亡的影响.方法 将40只SD大鼠分为对照组、实验组及假手术组,建立原位肝移植模型.供肝灌注冷保存以4℃乳酸林格氏液为基液,实验组加丹参注射液(60 ml/L),对照组不加丹参.保存5 h后植入受体.移植后6 h处死大鼠取样,检测血浆中ALT、AST水平;采用TUNEL法检测移植术后肝细胞凋亡情况;流式细胞仪检测凋亡相关基因Bcl-2、FasL蛋白的表达;光镜下观察移植肝脏病理形态学的改变.结果 再灌注后实验组ALT、AST显著低于对照组.与对照组比较,实验组肝细胞凋亡指数明显下降(F=133.802,P<0.05);肝组织中Bcl-2蛋白表达增加(F=91.063,P<0.01);FasL蛋白表达无明显变化(F=1.329,P>0.05);实验组与对照组比较肝组织形态学的再灌注损害程度明显减轻.结论 丹参可通过促进抑制凋亡基因Bcl-2蛋白的表达来抑制冷保存再灌注导致的肝细胞凋亡,对原位肝移植肝脏的缺血再灌注损伤有保护作用.     

常温机械灌注对大鼠心脏死亡器官捐献供肝微循环保护作用的研究

目的探讨常温机械灌注(normothermic machine perfusion,NMP)对大鼠心脏死亡器官捐献(donation after circulatory death,DCD)供肝微循环发挥的作用。方法采用夹闭SD大鼠胸主动脉热缺血30 min获取DCD供肝;在体外建立大鼠NMP系统。根据不同的保存供肝方式,将实验分为:正常(Normal)组(n=6),留取血清及肝脏待用;NMP组(n=30),保存4、6和8 h后收集肝脏标本,灌注后0、2、4、6 h和8 h收集流入道、流出道灌注液待检;静态冷保存(SCS)组(n=6),肝脏以20 ml 4℃UW液冲出肝内血液,并于UW液中4℃SCS 6 h后收集肝脏标本。采用生物化学方法检测流出道灌注液的肝功能;HE染色观察肝组织病理学改变;透射电镜观察肝细胞超微结构;TUNEL检测肝脏细胞凋亡情况;Western blot检测肝脏组织内皮素-1(endothelin-1,ET-1)、内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、血管性血友病因子(von Willebrand factor,vWF)、细胞间黏附分子(intercellular adhesion molecule-1,ICAM-1)和血管间黏附分子-1(intervascular adhesion molecule-1,VCAM-1)的表达情况。结果与SCS比较,NMP能显著改善DCD供肝组织学损伤,Suzuki评分NMP组(3.40±0.55)显著低于SCS组(7.00±0.71,F=229.75,P<0.05);减轻肝细胞凋亡,NMP组凋亡细胞数(9.80±1.48)显著低于SCS组(33.40±4.39,F=166.58,P<0.05);同时能修复肝细胞线粒体损伤,NMP组不可逆损伤线粒体数量(1.60±0.55)显著低于SCS组(2.80±0.45,F=36.29,P<0.05)。进一步发现,NMP可改善DCD肝脏微循环:①抑制细胞间黏附,改善内皮细胞损伤,与SCS比较,NMP显著抑制肝内ICAM-1(F=1728.45,P<0.05)、VCAM-1(F=254.72,P<0.05)和vWF(F=595.30,P<0.05)的表达;②改善肝脏ET-1/NOS平衡和微循环灌注,与SCS相比,NMP显著抑制肝内ET-1(F=1372.51,P<0.05)、iNOS(F=1102.20,P<0.05)的表达,促进eNOS(F=271.66,P<0.05)的表达。结论NMP能够改善大鼠DCD供肝质量,其机制可能通过抑制细胞间黏附,改善窦内皮损伤及微循环灌注发挥保护作用的。     

肝脏低温保存-再灌注期间肝细胞凋亡及其与Bax基因蛋白相关性的研究

目的　研究临床肝移植过程中供肝低温保存-再灌注期间肝细胞凋亡及其相关机制。方法　应用细胞凋亡原位末端标记法并结合电镜观察,检测低温保存时间分别为0(A组)、3(B组)、6(C组)、9(D组)h的4组兔肝脏在低温保存-再灌注过程中肝细胞凋亡情况。同时,对各组肝脏再灌注前后肝细胞内Bax基因蛋白表达进行测定。结果　A、B、C、D各组肝脏于低温保存后的再灌注60min时,组织内可见明显的肝实质细胞凋亡；其凋亡细胞数量(1×10     

潘生丁对大鼠移植肝缺血再灌注损伤保护作用的实验研究

目的探讨潘生丁对大鼠移植肝缺血再灌注损伤的保护作用及其相关机制。方法采用改良Kam ada“二袖套法”制作肝移植模型,48只雄性SD大鼠随机分成3组:假手术组(A组),同基因大鼠肝移植组(B组)和潘生丁预处理 同基因大鼠肝移植组(C组)。于供肝再灌注2 h后测定各组PAGT、ALT、AST、SOD、MDA含量,比较肝细胞凋亡和组织形态学改变。结果移植肝再灌注2 h后C组较B组肝组织损害轻,SOD、MDA、ALT、AST、PAGT变化及肝细胞凋亡差异有显著性(P<0.05)。结论潘生丁可通过改善肝脏微循环状况,改善缺血肝脏组织的能量代谢,提高肝组织抗氧化能力,抑制细胞凋亡和减少肝脏细胞的变性坏死程度而对肝脏热缺血再灌注起保护作用。     

前列地尔预处理对移植肝TNF-α和细胞凋亡的影响

目的探讨前列地尔预处理对移植肝组织中肿瘤坏死因子α(TNF-α)水平的变化和细胞凋亡的影响。方法将30例终末期肝病患者随机分为实验组和对照组,对照组供体摘取后常规以4℃UW液灌注保存,实验组供体摘取后以UW液灌注同时以10μg前列地尔经门静脉灌注,4℃保存。实验组和对照组均在供肝恢复血流后3小时抽取外周静脉血检测肝功能,并取部分肝组织检测TNF-α水平和肝细胞凋亡指数。结果实验组肝组织中TNF-α水平低于对照组(P<0.05),实验组肝细胞凋亡指数低于和对照组。结论前列地尔预处理可减少供肝TNF-α释放,抑制炎症反应,减少细胞凋亡,减轻肝细胞损伤,对供肝有保护作用。     

离体肝脏低温保存-再灌注期间肝细胞凋亡现象观察

目的 探讨临床肝移植过程中离体供肝低温保存－再灌注损伤与肝细胞凋亡的关系。方法 采用细胞凋亡原位末端标记法并结合电镜观察,检测低温保存时间分别为０、３、６、９小时的４组免肝脏在低温保存－再灌注过程中肝细胞凋亡发生情况。结果 在低温保存后的再灌注期间,４组动物的肝组织内均可见明显的肝实质细胞凋亡现象,而且低温保存时间越长的肝脏,其再灌注后产生的肝实质凋亡细胞数量越多,但各组肝脏于低温保存末,其组织内     

低氧与去铁敏对移植供肝作用的对比性研究

目的 对比研究低氧预处理及去铁敏预处理对移植供肝保护作用的特点,探索一种适合指导临床的供肝保护方法.方法 SD大鼠,随机分为假手术组(S组)、自体肝移植组(AT组)、去铁敏预处理组(DP组)与低氧预处理组(HP组).检测各组术后24 h血清中谷丙转氨酶(ALT),谷草转氨酶(AST),碱性磷酸酶(ALP)、肝组织形态学改变并检测肝组织中低氧诱导因子-1(HIF-1a)及丙二醛(MDA)的含量.结果 与S组相比AT组、DP组、HP组术后血清ALT、AST、ALP明显升高,DP组、HP组与AT组相比血清ALT、AST、ALP显著降低,肝形态异常变化减轻,肝组织中HIF-1a蛋白含量升高,MDA含量下降(P<0.05).结论 在肝移植术前行DP,可以起到与HP相似的供肝保护效果,但DP更适合指导临床.     

海藻糖对肝脏缺血再灌注损伤的保护作用及机制研究

目的探讨海藻糖是否对肝脏缺血再灌注损伤具有保护作用及其相关机制。方法C57BL/6J小鼠数字随机分为无缺血组、缺血再灌注组、海藻糖处理组和生理盐水对照组,缺血90 min后于再灌注的0h和6h,收集血液和肝组织,通过分离血清测丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)肝功能指标及肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)和白细胞介素-2(IL-2)炎症因子水平和肝组织病理改变研究海藻糖在肝脏缺血再灌注损伤中的作用;AML12小鼠肝细胞系构建缺糖缺氧-复糖复氧细胞模型,分为实验组和对照组,实验组根据给予的海藻糖浓度不同分为低剂量组和高剂量组,对照组无海藻糖,收集细胞,流式细胞仪检测凋亡水平以研究海藻糖对肝脏缺血再灌注损伤诱导的细胞凋亡的影响,蛋白印迹法检测Caspase-3、Cleaved Caspase-3和Bcl-2蛋白水平以研究海藻糖在肝脏缺血再灌注损伤诱导的细胞凋亡中的分子机制。结果体内动物实验显示肝脏缺血再灌注后,缺血再灌注组ALT、AST及TNF-α、IL-1β和IL-2等肝功能指标及炎症因子水平升高(P<0.05),且肝组织发生坏死;而在给予海藻糖处理后,ALT、AST、TNF-α、IL-1β和IL-2等水平较生理盐水对照组降低且肝组织坏死面积也减少(P<0.05)。体外细胞实验显示与对照组相比,实验组肝细胞凋亡水平下降;且实验组活化的促凋亡蛋白Cleaved Caspase-3水平下降、抗凋亡蛋白Bcl-2水平升高。结论在体内和体外条件下海藻糖对肝脏缺血再灌注损伤有保护作用,其机制可能是通过抑制肝脏缺血再灌注损伤诱导的炎症发生和抑制Caspase-3的活化并促进Bcl-2的表达,减轻细胞凋亡,从而保护肝脏缺血再灌注损伤。     

Combined Use of Subnormothermic Extracorporeal Support and Hypothermic Oxygenated Machine Perfusion for Liver Graft After Cardiac Death in Pigs

BackgroundThe use of grafts from donors after cardiac death (DCD) would greatly contribute to the expansion of the donor organ pool. This study aims to determine the benefits of extracorporeal membrane oxygenation (ECMO) and hypothermic oxygenated machine perfusion (HOPE) in a large animal model of DCD liver.MethodsAfter cardiac arrest, the abdominal aorta and the inferior vena cava were cannulated and connected to an ECMO circuit. Porcine livers were perfused in situ with ECMO at 22°C for 60 minutes after 45 minutes of cardiac death. Then, the livers were perfused for 4 hours by cold storage (CS) or HOPE. In group 1, non-in situ ECMO and grafts were preserved by HOPE. In group 2, in situ ECMO and grafts were preserved by HOPE. In group 3, in situ ECMO and grafts were preserved by CS. After preservation, all grafts were evaluated using an isolated reperfusion model (IRM) with autologous blood for 2 hours.ResultsDuring HOPE, aspartate aminotransferase (AST) levels and hepatic arterial pressure in group 2 tended to be lower than in group 1. Hematoxylin–eosin staining findings after HOPE showed more massive sinusoidal congestion and hepatocyte cytoplasmic vacuolization in group 1 than in group 2. The AST and LDH levels in group 2 at the start-up of IRM tended to be lower than in group 1.ConclusionsThe combined use of in situ subnormothermic ECMO and HOPE is essential for the functional recovery of DCD liver grafts.     

Hypothermic oxygenated machine perfusion alleviates liver injury in donation after circulatory death through activating autophagy in mice

Hypothermic oxygenated machine perfusion (HOPE) is a safe and reliable method that could alleviate liver injury in donation after circulatory death (DCD). This study focuses on the role of autophagy in HOPE’s protective effect on DCD liver injury. A 30‐minute warm ischemic liver model was established in mice. After 4 hours of cold storage (CS), 1 hour of hypothermic machine perfusion (HMP) with 100% O₂ or 100% N₂ was employed. During 2 hours of reperfusion, liver tissue and perfusate were collected to evaluate liver function, oxidative stress level, apoptosis, and necrosis. Western blotting was used to explore the level of autophagy. When the liver experienced warm ischemic injury, LC3B‐II expression was significantly enhanced. Compared with the CS, HOPE induced lower release of AST and ALT, as well as lower oxidative stress levels, apoptosis, and necrosis cell numbers, and led to higher tissue ATP content. Meanwhile, expression of autophagy‐related proteins, such as ULK1, Atg5, and LC3B‐II, increased. When oxygen was completely replaced by nitrogen, the washout effect of HMP did not activate autophagy and did not relieve DCD liver injury. When the autophagy inhibitor 3‐methyladenine was used in HOPE, the protective effect of HOPE was attenuated. In conclusion, DCD liver injury activated autophagy compared with healthy liver, while HOPE alleviated DCD liver injury by increasing autophagy levels further in this mouse model. However, HMP with 100% of N₂ had no beneficial effect on DCD liver injury or on autophagy levels compared with CS. The research on autophagy may provide a new strategy for alleviating DCD liver injury in clinical practice.     

Hypothermic oxygenated machine perfusion of the human pancreas for clinical islet isolation: a prospective feasibility study

Due to an increasing scarcity of pancreases with optimal donor characteristics, islet isolation centers utilize pancreases from extended criteria donors, such as from donation after circulatory death (DCD) donors, which are particularly susceptible to prolonged cold ischemia time (CIT). We hypothesized that hypothermic machine perfusion (HMP) can safely increase CIT. Five human DCD pancreases were subjected to 6 h of oxygenated HMP. Perfusion parameters, apoptosis, and edema were measured prior to islet isolation. Five human DBD pancreases were evaluated after static cold storage (SCS). Islet viability, and in vitro and in vivo functionality in diabetic mice were analyzed. Islets were isolated from HMP pancreases after 13.4 h [12.9–14.5] CIT and after 9.2 h [6.5–12.5] CIT from SCS pancreases. Histological analysis of the pancreatic tissue showed that HMP did not induce edema nor apoptosis. Islets maintained >90% viable during culture, and an appropriate in vitro and in vivo function in mice was demonstrated after HMP. The current study design does not permit to demonstrate that oxygenated HMP allows for cold ischemia extension; however, the successful isolation of functional islets from discarded human DCD pancreases after performing 6 h of oxygenated HMP indicates that oxygenated HMP may be a useful technology for better preservation of pancreases.     

Impact of Machine Perfusion Preservation of Liver Grafts From Donation After Cardiac Death

Because of the critical shortage of deceased donor grafts, using a donation after cardiac death (DCD) donor is an important resource. However, the ischemic damage of those DCD grafts jeopardizes organ viability during cold storage. Maintaining organ viability after donation until transplantation is important for optimal graft function and survival. This review describes the effective preservation in transplantation for DCD livers. Concepts and development of machine perfusion for DCD liver grafts to reduce ischemia/reperfusion injury are discussed. Despite the fact that hypothermic machine perfusion might be superior to static cold preservation, DCD livers are exposed to hypothermia-induced damage. Recently, some groups introduced the beneficial effects of normothermic or subnormothermic machine perfusion in DCD liver preservation and transplantation.     

他克莫司对脂肪肝供体大鼠肝移植围手术期移植物功能保护作用的研究

目的探讨他克莫司对脂肪肝供体大鼠肝移植围手术期移植物功能的保护作用及机制。方法建立脂肪肝供体Wistar大鼠肝移植模型,实验组与对照组受体大鼠分别在术中、术后注射他克莫司(0.3mg/kg)或生理盐水,在移植物再灌注0h、24h、72h和1周后分别检测两组受体大鼠的血清肝酶[丙氨酸氨基转移酶(ALT)、天冬氨酸氨基转移酶(AST)、乳酸脱氢酶(LDH)]、内皮素(ET)和脂质过氧化指标[超氧化物歧化酶(SOD)、过氧化氢酶(CAT)和丙二醛(MDA)],同时取肝组织制成匀浆检测其SOD、CAT和MDA水平,取小块肝组织制成切片在电镜下观察肝脏超微结构,采用原位末端标志法(TUNEL)检测肝组织的细胞凋亡情况。结果实验组大鼠的血清ALT、AST、LDH、ET和MDA水平明显低于对照组,SOD和CAT高于对照组(P0.05),实验组大鼠的肝组织匀浆中MDA水平也显著低于对照组,SOD和CAT高于对照组(P0.05)。实验组肝脏超微结构的损伤程度和细胞凋亡程度均较对照组有所减轻。结论他克莫司能在一定程度上保护脂肪肝供体大鼠肝移植围手术期移植物的功能,其作用机制可能与其抑制脂质过氧化、细胞凋亡和减轻再灌注损伤有关。     

Normothermic Acellular Ex Vivo Liver Perfusion Reduces Liver and Bile Duct Injury of Pig Livers Retrieved After Cardiac Death

We compared cold static with acellular normothermic ex vivo liver perfusion (NEVLP) as a novel preservation technique in a pig model of DCD liver injury. DCD livers (60 min warm ischemia) were cold stored for 4 h, or treated with 4 h cold storage plus 8 h NEVLP. First, the livers were reperfused with diluted blood as a model of transplantation. Liver injury was determined by ALT, oxygen extraction, histology, bile content analysis and hepatic artery (HA) angiography. Second, AST levels and bile production were assessed after DCD liver transplantation. Cold stored versus NEVLP grafts had higher ALT levels (350 ± 125 vs. 55 ± 35 U/L; p < 0.0001), decreased oxygen extraction (250 ± 65 mmHg vs. 410 ± 58 mmHg, p < 0.01) and increased hepatocyte necrosis (45% vs. 10%, p = 0.01). Levels of bilirubin, phospholipids and bile salts were fivefold decreased, while LDH was sixfold higher in cold stored versus NEVLP grafts. HA perfusion was decreased (twofold), and bile duct necrosis was increased (100% vs. 5%, p < 0.0001) in cold stored versus NEVLP livers. Following transplantation, mean serum AST level was higher in the cold stored versus NEVLP group (1809 ± 205 U/L vs. 524 ± 187 U/L, p < 0.05), with similar bile production (2.5 ± 1.2 cc/h vs. 2.8 ± 1.4 cc/h; p = 0.2). NEVLP improved HA perfusion and decreased markers of liver duct injury in DCD grafts.     

Hydrogen Gas Ameliorates Hepatic Reperfusion Injury After Prolonged Cold Preservation in Isolated Perfused Rat Liver

Hydrogen gas reduces ischemia and reperfusion injury (IRI) in the liver and other organs. However, the precise mechanism remains elusive. We investigated whether hydrogen gas ameliorated hepatic I/R injury after cold preservation. Rat liver was subjected to 48‐h cold storage in University of Wisconsin solution. The graft was reperfused with oxygenated buffer with or without hydrogen at 37° for 90 min on an isolated perfusion apparatus, comprising the H₂(+) and H₂(?) groups, respectively. In the control group (CT), grafts were reperfused immediately without preservation. Graft function, injury, and circulatory status were assessed throughout the perfusion. Tissue samples at the end of perfusion were collected to determine histopathology, oxidative stress, and apoptosis. In the H₂(?) group, IRI was indicated by a higher aspartate aminotransferase (AST), alanine aminotransferase (ALT) leakage, portal resistance, 8‐hydroxy‐2‐deoxyguanosine‐positive cell rate, apoptotic index, and endothelial endothelin‐1 expression, together with reduced bile production, oxygen consumption, and GSH/GSSG ratio (vs. CT). In the H₂(+) group, these harmful changes were significantly suppressed [vs. H₂(?)]. Hydrogen gas reduced hepatic reperfusion injury after prolonged cold preservation via the maintenance of portal flow, by protecting mitochondrial function during the early phase of reperfusion, and via the suppression of oxidative stress and inflammatory cascades thereafter.     

去铁敏预处理对移植供肝保护作用的研究

目的：探索去铁敏对移植供肝的保护作用及可能机制。方法：SD大鼠,随机分为假手术组（S组）、自体肝移植组（AT组）、自体肝移去铁敏预处理组（DP组）与自体肝移植去铁敏灌肝组（DI组）。检测各组术后24 h血清中谷丙转氨酶（ALT）、谷草转氨酶（AST）和碱性磷酸酶（ALP）,观察肝组织形态学改变并检测肝组织中低氧诱导因子-1（HIF-1α）及丙二醛（MDA）的含量。结果：与S组相比,AT组、DP组和DI组术后血清ALT,AST和ALP明显升高;但DP组、DI组较AT组血清ALT,AST和ALP明显降低,肝形态异常变化减轻,肝组织中HIF-1α蛋白含量升高,MDA含量下降(P﹤0.05)。结论：在肝移植术前行去铁敏预处理或术中行去铁敏溶液灌肝,对供肝有保护作用,且前者优于后者。其机制可能与提高组织HIF-1α含量及减轻脂质过氧化有关。