热缺血供肝胆管耐受冷保存时限的实验研究

目的探讨有热缺血损伤的供肝及胆管组织耐受冷保存的安全时限,为临床肝移植术后早期肝内外胆管不可逆性坏死和原发性移植肝无功能的防范提供实验依据. 方法在小型猪同种异体原位肝移植模型上,观察供肝血流阻断10 min后移植肝及胆管组织在不同冷保存时间条件下的形态功能改变及其可逆性.结果热缺血10 min,冷保存时间少于16 h组,动物均存活1周以上,且无早期胆管坏死所致的动物死亡;一旦冷保存时间超过16 h,术后早期胆管坏死的发生率显著上升（P＜0.05）,出现因胆管坏死、胆漏所致的动物死亡;随着冷保存时间的进一步延长,将出现原发性移植肝无功能所致的术中、术后早期动物死亡,存活动物均发生胆管坏死.与冷保存16 h以内组比较,16 h以上组术中移植肝胆管组织病理形态学评分和上皮凋亡细胞数显著增高（P＜0.05）,而Na^＋-K^＋-ATP酶及Ca^2＋-ATP酶活力显著降低（P＜0.05）,术后丙氨酸氨基转移酶、天冬氨酸氨基转移酶、γ-谷氨酰转肽酶、碱性磷酸酶恢复较慢.相关分析结果显示,术后胆管坏死率与术中移植肝胆管细胞病理形态学评分及凋亡细胞数呈显著正相关（r值分别为0.931、0.972,P值均＜0.01）,与Na^＋-K^＋-ATP酶和Ca^2＋-ATP酶活力呈显著负相关（r值均为-0.973,P值均＜0.01）.结论有热缺血损伤的供肝及胆管组织,在限定的冷保存时间范围内是可以利用的.若热缺血时间在10 min以内,冷保存时间延长至16 h对移植肝胆管组织仍安全,延长至20 h移植肝仍能发挥功能.     

冷保存时间对肝脏移植大鼠供肝线粒体ATPase6、ATPase8基因表达的影响及意义

目的探讨冷保存时间对移植肝脏能量代谢的影响及机制。方法取Wistar大鼠186只,其中180只采用改良“二袖套法”制作大鼠肝移植模型并分为A、B、C组各60只,供肝冷保存时间分别为30min、6h、12h;另6只为D组,行假手术对照。分别于制模后12h、24h、3d、5d、7d采集肝组织标本检测各组肝脏线粒体AT-Pase6、ATPase8 mRNA表达及ATP含量。结果与D组比较,术后12hA、B、C三组ATPase6、ATPase8 mRNA表达及ATP含量均降低,且C组显著低于A、B组（P〈0．05）,且ATPase6、ATPase8 mRNA变化趋势一致;24h后A、B、C组ATPase6、ATPase8 mRNA表达及ATP含量开始升高,C组ATPase6、ATPase8mRNA表达升高较A、B组缓慢。结论肝脏移植过程中,供肝冷保存时间延长可导致能量合成障碍,机制可能为下调ATPase6、ATPase8基因表达。     

无心跳供体心脏移植热缺血时限的实验研究

目的探讨未经药物预处理的无心跳供体心脏移植成功的热缺血时限。方法实验犬30只,供体和受体组各5只,对照组：心脏以0℃组氨酸-色氨酸-酮戊二酸溶液（histidine—tryptophan—ketogluarate solution,HTK）500ml主动脉根部灌注,心脏停跳后切取供心,置于0℃HTK液中保存2h;热缺血16min组：心脏缺氧停跳后热缺血16min．用0℃ HTK液500ml灌注冲洗冠状动脉,切取供心．置于0ccHTK液中保存2h;热缺血18min组：心脏缺氧停跳后热缺血18min,灌注及保存方法同热缺血16min组。以标准心脏移植方法行原位移植,监测供体心脏移植前后的血流动力学指标、心脏质量,测定心肌酶等指标,电镜观察心肌组织超微结构改变。结果心脏移植实验中对照组及热缺血16min组均可成功复跳、脱离体外循环辅助,血流动力学指标差异无统计学意义（P〉0．05）。热缺血18min组仅有2例可以脱机,与对照组及热缺血16min组相比,左心室舒张末期压升高、-dp／dtmax下降较明显．与对照组差异有统计学意义（P〈0．05）,但与16min组比较,差异无统计学意义（P〉0．05）,心脏质量及心肌酶明显升高（P〈O．05）,电镜观察超微结构破坏明显。结论常温热缺血16min的供心有可能被成功用于心脏移植。     

鼠肝冷保存与移植肝原发性无功能的研究

目的探讨肝脏冷保存与移植肝原发性无功能的关系。方法采用具有免疫耐受性的同种大鼠原位肝移植模型进行研究。将鼠肝分别在４℃ＵＷ液中保存１，６和２４ｈ，分别在移植后１ｈ和２４ｈ取肝脏做病理观察。结果保存在ＵＷ液中的肝脏基本保存了正常的组织结构，而在移植后则不同程度地出现了变性或坏死，并且肝细胞的坏死以小叶中央静脉为中心。保存时间越长，病变越严重。结论肝脏冷保存时间与移植肝原发性无功能的发生密切相关。     

大鼠无肝期耐受门静脉血流阻断的安全时限研究

目的探讨大鼠无肝期耐受门静脉血流阻断的最大安全时限,为临床肝移植术提供依据。方法将50只雄性Wistar大鼠随机分为阻断40 min组、阻断35 min组、阻断30 min组、阻断25 min组及完全转流组各10只,按照前期研究方法制备门颈静脉转流下自体肝移植动物模型,分别阻断门静脉40、35、30、25和0 min。观察大鼠在体肝脏持续低温灌洗保存期间、恢复肝门血流后大体形态变化及胃肠道淤血情况;大鼠术后一般情况及1～7 d存活情况、总存活率。结果①各组肝脏在体持续低温灌洗保存期间体积均无明显变化,转流期胃肠道颜色红润、未见淤血,肝脏冷灌注后质软、色泽呈均一土黄色;阻断门颈静脉转流管5 min后开始见胃肠道轻度淤血,其后随门静脉阻断时间增加,胃肠道和脾脏淤血更加明显,肠系膜静脉曲张更加严重;门颈静脉转流管开放后约4 min胃肠道颜色恢复红润,脾脏色红润、质地变软。②术后大鼠精神差、无活动能力(尤以阻断40 min组、阻断35 min组、阻断30 min组为著),约4 h后可站立,24 h后能自由饮食及活动。③术后1周阻断40 min组、阻断35 min组、阻断30min组均出现死亡,且随门静脉血流阻断时间缩短大鼠死亡数降低;阻断40 min组、阻断35 min组、阻断30 min组、阻断25 min组及完全转流组总存活率分别为20%、30%、70%、100%、100%,前三组均显著低于后两组。结论大鼠无肝期耐受门静脉血流阻断的最长安全时限为25 min。     

肝移植研究进展——肝移植供肝的获取和保存

肝移植手术的最关键一步是获取新鲜健康 ,动静脉及胆管管道完备 ,且经过保存后能恢复良好功能的供体肝脏。供肝的选择、切取、保存、修整技术对于移植术后移植肝的存活、功能恢复及术中术后并发症的发生有至关重要的影响。1　供体的选择供移植用的肝脏可来自活体或尸体。活体主要是指有血缘关系的亲属 ,仅用作部分肝移植的供体。活体肝脏移植成功率比尸体肝脏移植成功率高 ,存活时间长 ,它不仅可以提供健康的肝脏 ,而且具有遗传和免疫学方面的优点。尸体供肝要求肝热缺血时间不超过 30分钟 ,最好是有心跳的脑死亡尸体。绝大多数致命性脑外伤…     

肝窦内皮细胞在心跳停搏供肝热缺血中的表现

肝窦内皮细胞（sinusoid endothe-lial　cell,SEC）不仅作为抗原提呈细胞参加移植肝免疫排斥反应,其损伤后还可导致供肝微循环紊乱,并最终引起原发性移植肝无功能（primary graft non-function,PGF）的发生。我们从免疫组织化学和细胞超微结构等方面对心跳停搏供肝（non-heart-beating donor,NHBD）热缺血中SEC的表现进行研究。     

移植肝的保存

如何减少肝脏移植物缺血再灌注损伤是肝移植领域待解决问题之一,而肝脏移植物缺血再灌注损伤轻重与保存过程密切相关.保存液的发展经历了一个漫长的过程,现阶段保存液通过改善配方能明显提高移植物的保存质量,减少相关并发症.移植肝的保存除需高质量的保存液,与之相关的其他保存因素也影响着移植物的保存质量和手术预后.本文通过分析比较了目前常用的保存液的利弊及其相关并发症,探讨更好保存供肝,降低供肝缺血再灌注损伤的方法.     

缺血再灌注期间供肝糖原水平对供肝细胞内游离钙的影响

目的 研究供肝糖原含量对缺血再灌注期间肝组织ＡＴＰ及胞浆游离 Ｃａ２＋的影响。方法 供体组兔（２１只）均分为 ３组：Ａ组（禁食 ２４ ｈ）、Ｂ组（正常喂食）和 Ｃ组（正常喂食＋葡萄糖补充），运用自创的兔肝保存－再灌注模型来观察冷保存、复温和再灌注期间供肝组织中糖原含量、ＡＴＰ、肝细胞膜Ｃａ２＋ＡＴＰ酶活性及胞浆内游离Ｃａ２＋浓度的变化。结果 经上述处理，于冷保存前各组供肝糖原含量两两间差异有显著性（质量分数分别为１５．３８±２．６１，５１．８０±６．６３和６３．２３±２．７５）；且于缺血再灌注期间供肝糖原含量越高则ＡＴＰ水平越高，肝细胞膜Ｃａ２＋“ＡＴＰ酶活性也越高，胞浆内游离Ｃａ２＋浓度则越低。结论 术前提高供肝糖原含量能为肝细胞在缺血再灌注期间提供相对充足的ＡＴＰ，使肝细胞膜Ｃａ２＋ＡＴＰ酶活性能保持在较高的水平，能维持细胞内外Ｃａ２＋浓度差，防止胞浆游离Ｃａ２＋超载，对减轻供肝缺血再灌注损伤可起到一定作用。     

供肝冷热缺血移植后损伤与炎症细胞的关系

目的 探讨冷、热缺血移植术后引起供肝损伤的炎性细胞是否相同。方法 雄性SD大鼠随机分成2组,每组各24只。供肝分别在乳酸林格氏中保存120或240min后行原位肝移植术;另雄性SD大鼠随机分成3组,每组各24只供肝分别经心跳停搏90、120和150min后行原位肝移植术。大鼠分别于术后1、3、6和24h处死采样。结果 随着冷、热缺血时间的延长,移植后血清ALT等值不断升高,如冷缺血120min和240min移植术后1h ALT值(U/L)分别为454.8±45.2和1063.0±166.1,3h分别为712.0±65.9和1639.0±241.2,6h分别为1702.0±169.2和4193.0±672.7,24h分别为1067.0±141.2和1316.7±205.2,热缺血90、120和150min移植术后1hALT值(U/L)分别为5035.2±786.7、6075.3±1613.1和 7449.5±1052.0,3h分别为5564.5±696、4、7900.7±863.0和8854.8±2089.3,6h分别为7363.8±616.7、10459.3±1573.7和10294.0±530.0,24h90min组为1942.5±188.5;且术后移植肝的病理损害也逐渐加重。冷缺血移植术后3、6h肝细胞出现坏死,且有大量中性粒细胞浸润;热缺血移植术后3、6h肝细胞也有大量坏死,但却出现淋巴细胞趋润。电镜表现与镜下基本一致,旦电镜证实热缺血移植后浸润的淋巴细胞为T淋巴细胞。结论 冷、热缺血移植术后供肝的损伤似乎是由两种不同的炎性     

Prolonging warm ischemia reduces the cold preservation limits of liver grafts in swine

Introduction Liver transplantation (LT) is widely accepted as an effective treatment for end-stage liver disease, but a serious shortage of donor organs limits itsclinical application. To increase the number of livers available for transplantation, graft procurement from non-heart-beating donors (NHBDs) has again become a focus of attention.[1-3] It is suggested that warm ischemia (WI), cold ischemia (CI) or ischemia/reperfusion injury of the graft are risk factors for postoperative graft …     

Dynamical changing patterns of glycogen and enzyme histochemical activities in rat liver graft undergoing warm ischemia injury

AIM: To investigate the changing patterns of glycogen and enzyme histochemical activities in rat liver graft under a different warm ischemia time (WIT) and to predict the tolerant time limitation of the liver graft to warm ischemia injury. METHODS: The rats were randomized into five groups, WIT was 0,15,30,45,60 min, respectively, and histochemical staining of liver graft specimens was observed. The recovery changes of glycogen and enzyme histochemistry activities were measured respectively 6 and 24 h following liver graft implantation. RESULTS: The activities of succinic dehydrogenase, cytochrome oxidase, apyrase (Mg++-ATPase) and content of glycogen were decreased gradually after different WIT in a time-dependent manner. The changes were significant when WIT was over 30 min. CONCLUSION: Hepatic injury is reversible within 30 min of warm ischemia injury. Glycogen and enzyme histochemistry activities of liver grafts and their recovery potency after reperfusion may serve as criteria to evaluate the quality of liver grafts.     

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.     

Establishment of a rat liver transplantation model with prolonged biliary warm ischemia time

AIM:To investigate the impact of different time points of secondary warm ischemia on bile duct in a rat autologous liver transplantation model with external bile drainage.METHODS:One hundred and thirty-six male inbred SD rats were randomly assigned to one of four groups(Ⅰ-Ⅳ) according to the secondary warm ischemia time of 0,10,20 and 40 min.A rat model of autologous liver transplantation with continuous external biliary drainage under ether anesthesia was established.Ten rats in each group were used to evaluate the one-week survival rate.At 6 h,24 h,3 d and 7 d after reperfusion of the hepatic artery,6 rats were killed in each group to collect the blood sample via the infrahepatic vena cava and the median lobe of liver for assay.Warm ischemia time of liver,cold perfusion time,anhepaticphase,operative duration for biliary external drainage and survival rates in the four groups were analyzed for the establishment of models.RESULTS:No significant difference was shown in warm ischemia time,anhepatic phase and operative duration for biliary external drainage among the four groups.Five of the 40 rats in this study evaluated for the one-week survival rate died,including three deaths of severe pulmonary infection in group Ⅳ.A significant decrease of one-week survival rate in group Ⅳ was noted compared with the other three groups.With the prolongation of the biliary warm ischemia time,the indexes of the liver function assessment were significantly elevated,and biliary epithelial cell apoptosis index also increased.Pathological examinations showed significantly aggravated inflammation in the portal area and bile duct epithelial cell injury with the prolonged secondary warm ischemia time.Microthrombi were found in the micrangium around the biliary tract in some sections from groups Ⅲ and Ⅳ.CONCLUSION:The relationship between secondary warm ischemia time and the bile duct injury degree is time-dependent,and 20 min of secondary warm ischemia time is feasible for the study of bile duct injury.     

冷缺血时间及组织相容性对胰岛细胞活性的影响

目的:探讨胰岛细胞移植中冷缺血时间、组织相容性对于胰岛细胞活性的影响方法:采用脑死亡自愿捐赠器官的供者胰腺 (已知血型和HLA配型);高渗枸橼酸盐嘌呤溶液经过经主动脉原位灌注后,肝、肾、胰腺联合及分别切取,测定不同的冷却血时间条件下胰岛细胞活性的变化．测定胰岛细胞和血液组织相容性,分析供受者的组织相容性和胰岛细胞存活的关系．结果:肝脏、胰腺、肾脏联合切取及各器官的单独切取顺利,在冷却血5 h以内胰岛细胞活性率都在80％以上,用于胰岛细胞移植的胰腺和其他器官的切取不会影响胰岛细胞的活性; 人类胰岛暴露于未经抗凝的人类血中,胰岛将诱发一个迅速血细胞消耗．进行血小板激、中性粒细胞和单核细胞计数,HLA错配组分别为(1．0±0．72)×109／L,(0．54±0．24)×109／L, (0．01±0．00)×109/L,HLA匹配组为(6．0±0．27 ×109／L,(0．63±0．19)×109／L,(0．03±0．01)× 109／L,无论HLA错配还是匹配血细胞都发生明显的消耗;加入肝素后血细胞计数HLA错配组分别为(57．2±21．10)×109／L,(1．74±0．87) ×109／L,(0．75±0．24)×109／L,HLA匹配组为 (67．9±19．0)×109/L,(3．42±0．61)×109/L,(0．47 ±0．08)×109／L,与对照组比较反应明显减轻 (P<0．05);胰岛细胞体外培养24 h后,匹配组活性胰岛细胞数量分别为(1．085±0．167)×105／L, 错配组为(0．697±0．193)×105／L,具有显著性差异(P<0．05)．结论:把握胰岛细胞移植过程中冷缺血时间及组织相容性关系能够提高胰岛细胞的存活．     

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.     

New preservation strategies for preventing liver grafts against cold ischemia reperfusion injury

BACKGROUND: In spite of improvements in University of Wisconsin (UW) preservation solution, the injury from grafts during cold storage is an unresolved problem in liver transplantation. The aim of the present study was to evaluate the beneficial effect on ischemia-reperfusion injury associated with liver transplantation of the inversion of K(+) and Na(+) concentrations and the replacement of hydroxyethyl starch (HES) by polyethylene glycol (PEG) in UW preservation solution. METHODS: Using an orthotopic liver transplantation model, the effects on rat liver preservation of a modified preservation solution (UW-PEG) were evaluated, based on the inversion of K(+) and Na(+) concentration and the replacement of HES by PEG 35 kDa (0.03 mmol/L) in UW preservation solution. RESULTS: The use of UW-PEG preservation solution ameliorated the biochemical and histological parameters of hepatic damage. Thus, at 24 h after transplantation, transaminase levels were reduced significantly when livers were preserved during 8 h in UW-PEG preservation solution compared with the original UW solution. In addition, histological findings revealed fewer and smaller areas of hepatocyte necrosis. The benefits of UW-PEG solution cannot be explained by modifications in oxidative stress or neutrophil accumulation associated with liver transplantation. However, the results of hepatic and portal blood flow indicated that the benefits of this modified preservation solution, UW-PEG were associated with improvements in the microcirculatory disorders after reperfusion. CONCLUSIONS: The UW-PEG solution, while retaining all the advantages of UW solution, improved hepatic ischemia-reperfusion injury associated with liver transplantation.     

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. …     

氢质子波谱成像对热缺血再灌注损伤大鼠移植肝细胞再生能力的评价作用

目的 探讨磁共振氢质子波谱(1H-MRS)评价大鼠原位肝移植(OLT)热缺血模型肝细胞再生能力的意义,以及热缺血对于移植肝再生能力的影响. 方法 以实验组热缺血10min的大鼠肝移植模型和对照组无热缺血大鼠肝移植模型各30只为研究对象,移植术后分6个时间点(6 h、1 d、3 d、7 d、14 d、30 d)对每只大鼠行肝脏常规T1WI、T2WI成像及氢质子波谱扫描.扫描后取肝脏,测定肝细胞的细胞增殖核抗原(PCNA)表达,同时检测肝酶代谢水平. 结果 术后5个时间点实验组的肝细胞PCNA阳性率、胆碱峰与水峰的峰高比值均高于对照组,实验组和对照组胆碱峰/水峰峰高比值均与相应PCNA的阳性率呈显著正相关(r实验=0.819,P＜0.01;r对照=0.543,P＜0.01).实验组和对照组的血清ALT、AST术后明显升高,尤以术后6 h～3 d最为显著,实验组的ALT、AST明显高于对照组. 结论 热缺血再灌注损伤对于移植后肝细胞的再生能力有明显的影响,1H-MRS的胆碱峰可以无创伤地评价移植肝的肝细胞再生能力.