离体肝脏常温下保存的实验研究

为了探索常温下（２２℃）保存离体肝脏的可能性，本实验以ＨＴＫ液为基础，氟碳乳剂为氧载体，脂肪乳剂为能量，常温下充氧持续灌注离体猪肝门静脉。定时取灌注液测量肝生化指标，观察肝细胞线粒体大小及密度。发现含氧载体灌注液中氨浓度下降，尿素浓度升高，线粒体损伤较轻；加入脂肪乳剂则能维持葡萄糖的浓度。各项指标与对照组比较差异有显著意义。说明利用氧载体（氟碳乳剂）为离体肝提供足够的氧，同时给予足够的能量（脂肪乳剂）时，离体肝脏就能在较高温度下较长时间（４０小时）地维持它的主要解毒功能——摄取氨合成尿素，同时亦可维持肝细胞线粒体的稳定性。可见常温下保存离体肝脏是可能的。     

磷酸肌酸对离体大鼠肝脏冷保存的保护作用

目的探讨磷酸肌酸(CP)对大鼠离体肝脏冷保存的保护作用。方法建立大鼠肝脏单纯冷保存离体灌注模型,对照组予单纯威斯康星大学保存液(UW液)灌注肝脏,低剂量组以UW液为基液加入1 g/100 ml CP灌注肝脏,中剂量组以UW液为基液加入2 g/100 ml CP灌注肝脏;高剂量组以UW液为基液加入3 g/100 ml CP灌注肝脏。各组大鼠肝脏分别于4℃相应灌注液中冷保存后0、6、12、18、24 h共5个时间点,分别检测肝下下腔静脉内保存液的丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)含量,检测肝脏组织丙二醛(MDA)含量、髓过氧化物酶(MPO)活性,观察肝脏组织肝细胞的凋亡指数(AI)和肝脏组织核因子-κB阳性表达率,光学显微镜下观察肝脏组织的病理学变化。结果低、中、高剂量组大鼠肝脏在冷保存12 h后,ALT及LDH含量均低于对照组(均为P0.05);冷保存18 h后低、中、高剂量组大鼠肝脏组织的MDA、MPO含量均低于对照组(均为P0.05);在冷保存12 h及18 h时,低、中、高剂量组大鼠肝脏的肝细胞AI及核因子-κB阳性表达率均低于对照组(均为P0.05);冷保存24 h后,高剂量组保存液的ALT、MDA含量均明显高于对照组及低、中剂量组(均为P0.05)。病理检查结果显示,高、中、低剂量组大鼠肝脏的损伤明显轻于对照组,各剂量组之间比较无明显差别。结论在UW液中加入CP对大鼠离体肝脏冷保存有较好的保护作用,优于单纯应用UW液保存。     

硫化氢在兔离体心脏保存中的保护作用

目的 研究硫化氢(H2S)在兔离体心脏低温保存中的作用,并探讨其作用机制.方法 获取40只大白兔的离体心脏,应用Langendorff灌注装置对兔离体心脏分别进行灌注后保存.根据离体兔心灌注和保存液的不同,分为5组,每组8只.对照组:单纯Krebs-Henseleit(K-H)液;硫氢化钠(NariS)组:K-H液中加入1 μmol/L的NariS(H2S的供体);NariS+格列本脲(G1i)组:K-H液中加入1 μmol/L的NariS和10 μmol/L的Gli;G1i组:K-H液中加入10 μmol/L的Gli;STH组:单纯St.Thomas液.各组心脏在灌注和保存6 h后,留取心脏标本,检测各组兔离体心脏低温保存6 h后心功能恢复率、心脏复跳时间、心肌三磷酸腺苷(ATP)含量和含水量变化,使用电子显微镜观察心尖部组织的结构变化.结果 心脏保存6 h后,NariS组心脏复跳时间最短、心功能恢复率、心肌ATP含量最高,均优于STH组,但差异无统计学意义(P＞0.05);与NariS+Gli组、Gli组和对照组比较,差异均有统计学意义(P＜0.05).NariS组左心室功能和结构损伤较轻,其次是STH组,NariS+Gli组、Gli组及对照组的左心室功能及心肌组织结构损伤严重.结论 硫化氢在兔离体心脏低温保存6 h内具有良好的保护作用.开放KATP通道可能是硫化氢发挥保护作用的重要机制.     

保存液中加入硝酸甘油提高离体犬肺的保存效果

在研究不同器官保存液的基础上，我们先后开发了SDMC-1、2液，可有效应用于离体心脏的保存。为了提高肺保存的效果，我们将保存液中加入硝酸甘油（NTG），用于供肺的灌洗及保存，取得了较好的效果，报道如下。     

L－精氨酸在离体肺保存中的作用

目的：研究一氧化氮前体L－精氨酸（L-Arg)在离体兔肺保存中的保护作用。方法：将14只新西兰兔随机分为2组,对照组以改良的Euro-Collins(ECS)液灌注及保存供肺,实验组以L－精氨酸（3mmol/L)加入改良的ECS液中灌注及保存供肺。冷保存6h。以自体血再灌注1h后,做肺静脉血气分析（PvO2),并测定血及肺组织中一氧化 氮（NO）,超氧化物歧化酶（SOD）,脂质过氧化物（LPO）含量及肺组织的超微结构以评价肺保护的效果。结果：再灌注后,实验组比对照组血氧分压显著提高（P＜0．05）。实验组血及肺组织中NO及SOD含量较对照组高（P＜0．05）,LPO含量较对照组低（P＜0．05）,透射电镜检查实验组损伤轻于对照组,结论：肺保存不超过6h时,在改良的ECS液中加入L-Arg用于离体肺的灌注和保存,能减轻肺损伤。     

大鼠肝脏保存中不同器官保存液对肝细胞凋亡的影响

目的 研究３种目前国内常用的器官保存液对供肝细胞凋亡的影响。方法 分别用ＵＷ液、ＨＣ－Ａ液和ＷＭＯ－１号液灌洗并保存大鼠肝脏,于保存后０、１２、２４ｈ用原位末端标记法检测供肝细胞凋亡情况,并将保存２４ｈ的肝及行大鼠原位全肝移植,观察受者的３ｄ存活率。结果 ３种保存液保存的肝脏均在保存１２ｈ时出现细胞凋亡,ＨＣ－Ａ液级瑟ＷＭＯ－１号液组的凋亡指数（ＡＩ）高于ＵＷ液组（Ｐ〈０．００１）,而ＨＣ－Ａ液组     

保存温度对大鼠肝脏功能的影响

目的 研究温度对大鼠肝脏功能的影响,以确定大刀肝脏的是适保存温度。方法 采用单纯低温保存方法及建立大鼠原位肝移植模型研究保存温度对大鼠肝脏功能的影响。实验对0℃、4℃、8℃、12℃进行研究,保存时发为6,18及30h,保存18h后行肝移植术,检测指标包括肝脏酶学、能量指标及组织形态学改变。结果 ①保存18,30h的灌出液及移植后2h血清中AST、LDH水平为4℃＜0℃＜8℃＞12℃（P＜0.01或P＜0.05）。②保存18、30h及移植后20min的肝脏能量指标ATP、EC为4℃＞0℃＞8＞12℃（P＜0.01或P＜0.05）。③肝脏形态学示4℃组损害较其它组轻。结论 4℃对于大鼠肝脏是是适保存温度,它能够明显减轻大鼠肝脏的冷损伤程度。     

器官保存液对大鼠肝脏细胞凋亡的影响的研究

细血再灌注损伤的主要机制之一。氧自由基除直接损害细胞外 ,还可以间接激活细胞内核酸内切酶 ,导致缺血再灌注时的细胞凋亡 ,有研究表明再灌注时 ,氧自由基对线粒体的损害可以产生凋亡诱导因子造成细胞凋亡。Ｂｕｒｎｓ等〔1〕证实肾小管的凋亡在低温缺血尤其是长时间缺血损伤中起着重要作用。尽管一些临床移植表明细胞凋亡只发生在再灌注 1ｈ内〔2〕,但一些实验研究表明凋亡可在再灌注后相当一段时间内存在〔3〕。最近的两项研究提供了更加直接的佐证 ,Ｇａｏ等〔4〕发现低温保存的大鼠肝脏由于乏氧引发蛋白酶的释放 ,这些蛋白酶作用于肝…     

离体肾脏保存研究进展

离体肾脏保存是肾移植领域中急待解决的重大课题 ,近来离体肾脏保存研究取得了一定进展 ,本文从保存液、保存方式、延长肾脏保存的方法三个方面给以总结。     

改良FWM心肌保存液对离体鼠心低温保存效果

目的 探讨改良FWM心脏保存液对离体鼠心低温保存效果.方法 20只雄性Wistar大鼠随机分为4组:对照组、改良FWM液组、HTK液组和K-H组.除对照组外,其余各组取大鼠心脏后,立即分别用各自4℃保存液灌注至心脏停搏,并置于相应的4℃心肌保护液中保存8 h,使用Langendorff模型测定心脏功能、检测冠脉流出液的心肌酶、观察心肌的形态学改变以评价各组心肌保护效果.结果 与对照组比较,各实验组心功能指标均有明显下降.HTK组LVDP、dp/dt<,max>、dp/dt<,min>,均低于改良FWN组(P<0.01).而HTK组CF的高于改良FWM组(P<0.05)和K-H组(P<0.01).HTK组GOT、LDH、CK、CTNI、CK-MB、GPT漏出量也高于改良FWM组(P<0.01).电镜显示改良FWM组形态学无明显改变仅偶见内质网扩张;而HTK组心肌肌纤维疏松、线粒体轻度水肿、内质网轻度扩张、间质轻度水肿.结论 离体鼠心低温保存8 h后,心肌功能均有很大程度下降.改良FWM液对离体鼠心的低温保存效果略优于HIK液.     

Delivery of the bioactive gas hydrogen sulfide during cold preservation of rat liver: effects on hepatic function in an ex vivo model

The insults sustained by transplanted livers (hepatectomy, hypothermic preservation, and normothermic reperfusion) could compromise hepatic function. Hydrogen sulfide (H₂S) is a physiologic gaseous signaling molecule, like nitric oxide (NO) and carbon monoxide (CO). We examined the effect of diallyl disulfide as a H₂S donor during hypothermic preservation and reperfusion on intrahepatic resistance (IVR), lactate dehydrogenase (LDH) release, bile production, oxygen consumption, bromosulfophthalein (BSP) depuration and histology in an isolated perfused rat liver model (IPRL), after 48 h of hypothermic storage (4°C) in University of Wisconsin solution (UW, Viaspan). Livers were retrieved from male Wistar rats. Three experimental groups were analyzed: Control group (CON): IPRL was performed after surgery; UW: IPRL was performed in livers preserved (48 h—4°C) in UW; and UWS: IPRL was performed in livers preserved (48 h—4°C) in UW in the presence of 3.4 mM diallyl disulfide. Hypothermic preservation injuries were manifested at reperfusion by a slight increment in IHR and LDH release compared with the control group. Also, bile production for the control group (1.32 µL/min/g of liver) seemed to be diminished after preservation by 73% in UW and 69% in UW H₂S group at the end of normothermic reperfusion. Liver samples analyzed by hematoxylin/eosin clearly showed the deleterious effect of cold storage process, partially reversed (dilated sinusoids and vacuolization attenuation) by the addition of a H₂S delivery compound to the preservation solution. Hepatic clearance (HC) of BSP was affected by cold storage of livers, but there were no noticeable differences between livers preserved with or without diallyl disulfide. Meanwhile, livers preserved in the presence of H₂S donor showed an enhanced capacity for BSP uptake (k_ACON = 0.29 min^?1; k_AUW = 0.29 min^?1; k_AUWS = 0.36 min^?1). In summary, our animal model suggests that hepatic hypothermic preservation for transplantation affects liver function and hepatic depuration of BSP, and implies that the inclusion of an H₂S donor during hypothermic preservation could improve standard methods of preparing livers for transplant.     

Liver transplantation using liver grafts preserved under high pressure

To extend organ preservation time, we attempted to establish a unique method of maintaining a preservation solution in a stable unfrozen state below its freezing point by pressurizing the solution. Livers removed from Lewis rats (RT1l) were stored in UW solution pressurized at the prescribed pressure. After the termination of preservation, orthotopic liver transplantation was performed. Experiment 1: Liver grafts were pressurized up to 30, 40, 50, and 70 MPa and preserved at 0 degrees C for 60 min. Experiment 2: Liver grafts were compressed at a rate of 1.32 or 0.04 MPa/s to 35 MPa and preserved for 60 min at 0 degrees C. Experiments 3 and 4: Liver grafts were pressurized up to 5, 10, 20, and 30 MPa and preserved at -2 degrees C (Exp. 3), -3 degrees C or -4 degrees C (Exp. 4) for 5 h. All rats transplanted with livers pressurized up to 30 MPa (Exp. 1), all rats in the 5 MPa and control groups at -2 degrees C (Exp. 3), and all rats in the 5 MPa group at -3 degrees C (Exp. 4) survived for 2 weeks. In light microscopy, diffuse hemorrhage and vacuolar degeneration of hepatocytes were observed in a pressure-dependent manner. Liver grafts preserved under pressurized, subzero nonfrozen condition have sufficient function to sustain the life of rats after orthotopic transplantation.     

The impact of donor organ quality on postoperative liver function after orthotopic rat liver transplantation

The impact of donor factors for posttransplant liver function was evaluated in the model of orthotopic rearterialized liver transplantation in the rat. The effect of donor fasting, parenteral hyperalimentation, hypotension, warm ischemia and endotoxins on histology, clinical chemistry and MEGX test was analyzed in syngeneic and allogeneic recipients of livers stored for 4 hrs on ice. In syngeneic animals, 20 min of warm ischemia led to significantly elevated serum transaminase levels and degree 2 histological damage on POD 2. Endotoxins produced a grade 1 histological damage. All groups had a lower MEGX formation rate compared to controls. In allogeneic animals, warm ischemia was the single most detrimental parameter. The strength of the rejection response on POD 8 did not depend on the type of donor pretreatment. The major finding of this non-survival study is the deleterious effect of warm ischemia and endotoxin on the functional and structural integrity of liver grafts after 4 hrs of cold ischemia. Received: 9 June 1997     

Celsior液和UW液保存供肝的前瞻性随机对照研究

目的比较Celsior液和UW液保存供肝的效果。方法随机选取拟行肝移植的患者60例，平均分为两组，一组接受以Celsior液灌洗和冷保存的供肝（Celsior液组）移植，另一组接受以UW液灌洗和冷保存的供肝（UW液组）移植，两组在患者年龄、性别构成、肝功能分级以及原发病、肝移植术式等方面的差异无统计学意义。比较两组供肝组织学变化、术后早期肝功能恢复情况及术后3个月内缺血性胆道狭窄的发生率。结果Celsior液组供肝冷缺血时间为（8．83±1．53）h，UW液组为（9．08±1．85）h，差异无统计学意义（P〉0．05）。两组术后早期血清丙氨酸转氨酶、天冬氨酸转氨酶、γ-谷氨酰转移酶、胆红素总量、出血时间及胆汁量的差异无统计学意义（P〉0．05），术后3个月内，Celsior液组缺血性胆道狭窄发生率为6．7％（2／30），UW液组为13．3％（4／30），差异无统计学意义（P〉0．05）。两组移植肝的组织学改变相似。结论在冷缺血时间一致的情况下，Celsior液保存供肝的效果与UW液相同。     

体外持续灌注氧合血对离体猪肺的保护作用

目的 观察氧合血体外持续灌注对离体猪肺的保护作用.方法 将低钾右旋糖酐葡萄糖液(Perfadex液)单次经肺动、静脉灌注后的猪肺分为低温保存组(对照组)与氧合血体外持续灌注组(实验组),保存12 h后,将两组猪肺与体外灌注系统和呼吸机连接,模拟体内正常肺工作6h,检测肺血管阻力(PVR)、肺通气阻力(LR)、左下肺静脉血氧分压(PaO2)及肺含水量等肺功能指标,并观察肺组织损伤,检测肺组织中前炎症因子白细胞介素1β(IL-1β)的表达量.结果 两组肺PVR、LR及肺含水量随肺工作时间的延长均逐渐上升,PaO2逐渐下降;但相同时间点,实验组PVR及LR显著低于对照组(P＜0.05),PaO2高于对照组(P＜0.05).与正常肺组织比较,实验组和对照肺组织水肿分级的差异均有统计学意义(P＜0.01),实验组肺水肿分级显著低于对照组(P＜0.05);实验组和对照组肺组织中IL-1βmRNA的相对表达量分别为0.422±0.132和0.578±0.163,两组差异有统计学意义(P＜0.05).结论 单纯采用氧合血体外持续灌注是可行的供肺保存技术,可减轻低温保存所致供肺的时间依赖性缺血再注损伤,但仍需在完全生理状态下进行更系统的评价.     

还原型谷胱甘肽和供氧对伴有脂肪变性的供肝移植后肝细胞凋亡及超微结构的影响

目的探讨还原型谷胱甘肽 (GSH)和冷保存时供氧对脂肪变性供肝移植术后肝细胞凋亡及超微结构的影响。方法大鼠喂饲 79%标准饲料加 2 0 %猪油加 1%胆固醇 ,同时以 5 0 %酒精灌胃 1ml·10 0g-1·d-1,6周后诱导形成Ⅱ级脂肪变性供肝 ;GSH和静脉供氧预处理后行肝移植 ,正常供肝作对照 ;观察术后存活率、肝细胞凋亡、微循环及超微结构改变。结果大鼠脂肪变性供肝移植术后 3d肝细胞坏死率为 (38± 10 ) % ,凋亡率 (2 2± 11) % ;而给予预处理后坏死减少 (17± 6 ) % ,凋亡增多 (33± 8) % ;同时肝细胞内线粒体水肿减轻、肝窦内皮细胞损伤减少 ;微循环由 (5 96± 0 2 6 )伏 (相对值 )增为 (7 73± 0 4 3)伏 ;预处理后的脂肪变性供肝移植 1周存活率由 2 5 % (2 /8)提高到 6 2 % (5 /8) ,(χ2 =4 0 7,P =0 0 4 36 )。结论GSH和静脉供氧可通过减轻肝细胞线粒体损伤、减少肝窦破坏、改善微循环而提高大鼠脂肪变性供肝移植的存活率。     

保存不同时间的大鼠部分肝脏移植后的肝细胞再生

目的 探讨保存不同时间的大鼠部分肝脏移植后的肝细胞再生及其可能机制.方法 采用近交系雄性Lewis大鼠为供、受者,按照实验设计分别将供肝于4℃UW液中保存1 h(冷缺血1 h组)、8 h(冷缺血8 h组)和16 h(冷缺血16 h组).然后进行原位肝移植.移植肝恢复血流前,用3-0丝线结扎供肝左侧中央叶、左外叶及尾状叶,保留右侧的肝叶.即可制成大鼠50%体积肝脏(以下简称"半肝")原位移植模型.术后观察各组移植肝的存活情况和肝细胞再生情况;采用逆转录聚合酶链反应测定肝组织中自细胞介素-6(IL-6)和肿瘤坏死因子α(TNF_n)的表达情况;采用Western印迹法检测肝组织中信号传导与转录因子-3(STAT-3)表达情况;采用免疫组织化学染色检测移植肝组织中细胞周期素DI(Cyelin D1)的表达和肝细胞摄取溴脱氧尿核苷(BrdU)情况.结果 各组手术成功率均为100%.与冷缺血1 h组相比,冷缺血8 h组和冷缺血16 h组移植肝组织中TNF-a(F=67.45,P<0.05)和IL-6(F=287.73,P<0.05)的表达明显增加.STAT-3的表达也明显增强.肝移植后24 h.冷缺血8 h组在胞浆和细胞核内均有Cyclin D1的表达.而冷缺血16 h组移植肝组织中未见明显的Cyclin D1表达.移植后24 h.冷缺血16 h组的BrdU染色阳性的肝细胞数无明显增多,而在冷缺血8 h组可见BrdU染色阳性的肝细胞明显增多(t=19.40,P<0.05).结论冷保存一定时限的大鼠部分肝脏在移植后可获得肝细胞再生,此过程可能通过TNF-α/IL,16/sTAT-3/Cyclin D1/DNA合成的途径进行调节;当冷保存时间达16 h后,肝细胞不能对肝脏再生早期信号起反应.     

供肝冷缺血时间延长诱发大鼠原位肝移植术后早期急性排斥反应的研究

目的 研究供肝冷缺血时间延长对大鼠原位肝移植术后早期急性排斥反应的影响.方法 选取30只健康纯系清洁级BN大鼠和30只Lewis大鼠.分别作为纯系移植和同种异体移植的供者,受者均为健康纯系清洁级BN大鼠60只,建立纯系和同种异体原位肝移植模型.根据纯系移植和同种异体移植供肝冷缺血时间的不同,将供、受者分为A、B、C和D组,每组15对.A组:供肝冷缺血1 h后进行纯系移植;B组:供肝冷缺血18 h后进行纯系移植;C组:供肝冷缺血1 h后进行同种异体移植;D组:供肝冷缺血18 h后进行同种异体移植.术后观察受者的2周存活率、移植肝组织病理学及肝功能的改变,检测受者主要组织相容性复合物(MHC)-Ⅱ类分子和核转录因子κB(NF-κB)的表达水平.结果 肝移植术后2周,A、B、C和D组的存活率分别为83.3%、66.7%、16.7%和0%,不管是纯系移植组还是同种异体移植组中供肝的冷保存时间越短,受者的存活率越高,且经肝功能检查发现冷保存时间短的受者移植肝功能恢复较好,移植肝组织病理学损伤和急性排斥反应也明显较轻.B组受者术后移植肝大量表达MHC-Ⅱ类分子,明显高于A组(P＜0.05);两同种异体移植组MHC-Ⅱ类分子的表达量较两纯系移植组增加明显,D组增加最多.A组几乎不表达NF-κB,而B组NF-κB的表达显著增加(P＜0.05);两同种异体移植组受者NF-κB的表达峰值提前.结论 冷缺血时间的延长可以诱导发生和加重大鼠原位肝移植术后早期急性排斥反应,降低术后2周存活率.     

The effect of methylprednisolone on warm ischemia-reperfusion injury in the liver

Background

Liver ischemia-reperfusion (I-R) injury is a well-known cause of morbidity and mortality following liver surgery and transplantation. Further investigation is warranted to identify measures that reduce the untoward sequelae of liver ischemia.

Methods

Male Sprague-Dawley rats (wild-type) and Zucker rats (with hepatic steatosis) were subjected to 75 minutes of 70% hepatic ischemia and 3 hours of reperfusion. The ischemic periods were based on protocols of either continuous clamping (CC) or ischemic preconditioning (IP). Prior to ischemia induction, rats were pretreated with intravenous methylprednisolone (MP; 2 mg/kg) or normal saline. Warm I-R injury was evaluated using serum levels of aspartate aminotransferase (AST), serum interleukin-6 (IL-6), and hematoxylin and eosin staining.

Results

Histology, serum IL-6, and AST release revealed that MP treatment provided significant protection as compared with ischemic controls (both CC and IP groups) only in the normal, not steatotic, livers. The inflammatory response was considerably reduced in MP groups with normal livers but not in steatotic livers. In general, the IP groups showed decreased I-R injury compared to the CC group. However, MP was able to further reduce I-R injury only in normal, not steatotic, livers.

Conclusions

MP attenuated the postischemic and inflammatory response in the normal, and not steatotic, livers. MP pretreatment might be effective in reducing warm I-R injury to livers without steatosis. The mechanism of I-R-related hepatocellular damage in steatotic liver is different than in normal liver.