FK506对肝脏保存再灌注中肝窦内皮细胞损伤的保护作用

目的 探讨他克莫司（FK506）对肝脏低温保存再灌注中肝窦内皮细胞损伤的保护作用。方法 应用离体鼠肝脏保存再灌注模型,测定保存不同的时间后肝脏灌注流出液中内皮素（ET）及丙氨酸转氨酶（ALT）的含量以及透明质酸（HA）的摄取量,光镜及电镜下观察肝窦内皮细胞的形态学改变,比较分析FK506对上述指标的影响。结果 对照组随肝脏保存再灌注时间延长,灌注流出液中ET含量明显升高（P＜0．05）,HA摄取明显降低（P＜0．05）,并伴随ALT活性显著增高（P＜0．05）和肝窦内皮细胞形态学的异常改变;保存液和灌注液中均加入FK506后,上述指标异常变化均明显减轻,其差异有显著性（P＜0．05）。结论 FK506对离体大鼠肝脏肝窦内皮细胞的再灌注损伤有保护作用。     

尿胰蛋白酶抑制剂对肝脏保存再灌注中肝窦内皮细胞损伤的保护作用

目的　探讨尿胰蛋白酶抑制剂 (UTI)对肝脏低温保存再灌注中窦内皮细胞损伤的保护作用。方法　应用离体大鼠肝脏保存再灌注模型 ,测定保存 12 h、2 4 h,再灌注 30 min流出液中内皮素 (ET)、丙氨酸转氨酶 (AL T)、天冬氨酸转氨酶(AST)、乳酸脱氢酶 (L DH)的活性、透明脂酸 (HA)的摄取量以及肝组织中髓过氧化物酶 (MPO)水平 ,光镜下观察肝窦内皮细胞的形态学改变 ,比较分析 U TI(10 0 U/ m l)对上述指标的影响。结果　对照组肝脏保存再灌注流出液中 ET含量明显升高 (P<0 .0 5 ) ,HA摄取明显降低 (P<0 .0 5 ) ,并伴随 AL T、AST、L DH、MPO水平显著增高和肝窦内皮细胞形态学的异常改变 ;保存液中加入 UTI后 ,上述指标异常变化均明显减轻 ,其差异具有显著性 (P<0 .0 5 )。结论　 UTI对离体大鼠肝脏窦内皮细胞的保存再灌注损伤有保护作用     

药物和缺血预处理对肝硬化肝脏缺血再灌注损伤的协同保护作用

目的探讨阿霉素和缺血联合预处理对肝硬化肝脏缺血再灌注损伤的保护作用及其可能机制方法(1)诱导大鼠肝硬化模型；(2)缺血再灌注损伤前3组肝硬化大鼠分别行阿霉素预处理、缺血预处理、阿霉素 缺血联合预处理，比较3组和对照组AST、ALT、LDH和盯、TNF-α、NO、热休克蛋白70(HSP0)差异有显著性。结果阿霉素预处理、缺血预处理、阿霉素 缺血联合预处理能明显抑制AST、ALT、LDH水平升高，其中以阿霉素 缺血联合预处理作用最显著；缺血预处理能显著降低ET、TNF-α水平；阿霉素预处理和缺血预处理使N0显著升高；阿霉素预处理能使肝细胞HSP70显著增加。结论阿霉素和缺血预处理都能减轻肝硬化肝脏缺血再灌注损伤程度；阿霉素 缺血联合预处理对月十硬化肝脏缺血再灌注损伤有协同保护作用。     

川芎嗪对大鼠肝脏缺血-再灌注损伤保护作用的机理研究

目的　研究川芎嗪注射液对大鼠肝脏缺血再灌注损伤的保护作用。方法　96 只SD大鼠被随机均分为假手术组、肝脏缺血再灌注组(I/R组)和肝脏缺血+川芎嗪再灌注组(简称治疗组)3 组,分别于术前及术后30 min、6 h及24 h检测血浆丙氨酸氨基转移酶(ALT)、天门冬氨酸氨基转移酶(AST)及乳酸脱氢酶(LDH),观察每组大鼠1周存活情况及肝脏病理组织学变化,并行肝细胞凋亡指数检测。结果　治疗组术后1 周大鼠存活情况好于I/R组(P<0.05); 治疗组及I/R组血浆ALT、AST及LDH均明显高于假手术组(P<0.05),但治疗组低于I/R组(P<0.05)。光镜下见,缺血再灌注后肝血窦和中央静脉明显瘀血,肝细胞坏死I/R组重于治疗组。肝细胞凋亡指数I/R组高于治疗组(P<0.05)。结论　川芎嗪对大鼠肝脏缺血再灌注损伤具有明显的保护作用。     

自制KYL液和UW液保存大鼠肝脏效果的比较

目的比较自制的KYL液和UW液对大鼠肝脏的保存效果。方法采用大鼠肝脏非循环离体灌注模型(noncirculatedisolatedperfusionofratliver),随机以KYL液和UW液对大鼠肝脏保存0、4、8、16、24和48h,记录胆汁流出量,测定灌注流出液天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、乳酸脱氢酶(LDH)和氧自由基代谢产物丙二醛(MDA)及超氧化物歧化酶(SOD)含量,检测肝细胞内钙离子浓度,观察肝脏组织形态学变化。同时设生理盐水保存阴性对照组,了解器官保存液对大鼠肝脏有无保护作用。结果KYL液保存的大鼠肝脏在保存16h以内各时相胆汁流出量均较UW液保存者高(P<0.01),灌注流出液AST、ALT和LDH含量与UW液保存者相近,肝细胞内钙离子浓度较UW液保存者低(P<0.01);KYL组保存24及48h时,MDA含量低于UW组,SOD含量高于UW组(P<0.01);光、电镜观察两者形态学变化基本一致。两组所有指标均较生理盐水保存组好,提示KYL保存与UW液一样对大鼠肝脏具有保护作用。结论自制的KYL液对大鼠肝脏的保存效果总体上与UW液相当,在再灌注后肝细胞胆汁分泌方面和钙拮抗方面略优于UW液,而在防止细胞水肿方面较UW液稍差。     

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。     

细胞穿透肽PEP-1介导血红素加氧酶-1对大鼠离体心脏缺血再灌注损伤的影响

目的 探讨细胞穿透肽PEP-1介导血红素加氧酶-1(HO-1)对大鼠离体心脏缺血再灌注损伤的影响.方法 雄性SD大鼠,体重220～280g,制备Langendorff离体心脏灌注模型,选取模型制备成功的离体心脏18个,随机分为3组(n=6):假手术组(S组)、缺血再灌注组(IR组)和PEP-1/HO-1处理+缺血再灌注组(HO-1组).IR组K-H液平衡灌注30 min后,采用停灌40 min再灌注50 min的方法制备缺血再灌注模型.HO-1组在停灌前用含50 μmol/L融合蛋白PEP-1/HO-1的K-H液平衡灌注15 min,S组采用K-H液持续灌注120 min.再灌注50 min时,收集冠脉流出液,测定肌酸激酶(CK)和乳酸脱氢酶(LDH)的活性;取心肌组织,采用Western blot法测定HO-1蛋白表达水平,采用硫代巴比妥酸比色法测定MDA含量,黄嘌呤氧化酶法测定SOD活性.结果 HO-1组心肌组织HO-1蛋白表达水平较IR组升高(P＜0.01).与S组比较,IR组和HO-1组冠脉流出液CK和LDH活性及心肌组织MDA含量升高,心肌组织SOD活性降低(P＜0.01);与IR组比较,HO-1组冠脉流出液CK和LDH活性及心肌组织MDA含量降低,心肌组织SOD活性升高(P＜0.01).结论细胞穿透肽PEP-1可将HO-1蛋白成功导入心肌组织,并减轻大鼠心肌缺血再灌注损伤.     

川芎嗪在大鼠肝脏缺血-再灌注损伤中的保护作用

目的 探讨中药川芎嗪对大鼠肝脏缺血.再灌注损伤是否具有保护作用.方法 132只SD大鼠随机分为3组:A组(空白对照组)、B组(缺血-再灌注组)、C组(治疗组),于术前及再灌注后30 min、6 h、24 h抽血检测ALT、AST及LDH,同时观察组织病理学变化,对比每组大鼠1周累积生存情况:免疫组化检测肝细胞凋亡指数.结果 C组累积生存率高于B组(P<0.05).B组及C组ALT、AST及LDH均明显高于A 组(P<0.05)且C组低于B组(P<0.05),光镜下,缺血-再灌注后肝细胞坏死情况B组重于C组,B组肝细胞凋亡指数高于C组(P<0.05).结论 川芎嗪对大鼠肝脏缺血-再灌注损伤具有明显的保护作用.     

17-β雌二醇对大鼠肝切除肝缺血再灌注损伤肝细胞凋亡和Bcl-2、Bax表达的影响

目的 观察17-β雌二醇预处理对肝切除肝缺血再灌注损伤肝脏组织细胞凋亡及Bcl2、Bax表达的影响,并探讨其肝保护的机制.方法 建立大鼠肝切除肝缺血再灌注损伤模型,75只雄性SD大鼠随机分为3组:假手术组(Sham组)、缺血再灌注组(IR组)和17-β雌二醇预处理组(E2+ IR组).检测各组大鼠再灌注后lh、3h、6h、12 h、24 h肝功能变化.光镜下观察肝组织病理学改变.TUNEL法观察再灌注后12 h大鼠肝细胞凋亡情况、流式细胞学方法测定再灌注后12h肝细胞凋亡率.Western blot法检测再灌注后12 h Bcl-2和Bax的表达情况.结果 与Sham组相比,在IR组各时间点均可见ALT和AST增高,且在再灌注后的12h达到了最高值;病理学检查可见肝细胞肿胀,肝窦变窄,嗜中性粒细胞浸润和片状坏死等变化;在再灌注后12h,凋亡细胞增多及细胞凋亡率明显升高;肝脏组织Bcl 2表达减少,Bax的表达增加.17-β雌二醇预处理组在灌注后各时间点ALT和AST值明显下降,肝脏病理损伤改善;在再灌注后12h,凋亡细胞减少及细胞凋亡率明显降低,肝脏组织Bcl-2表达增加,Bax的表达减少.结论 17-β雌二醇对大鼠肝缺血再灌注损伤有明显的保护作用,其可能通过促进Bcl-2表达及抑制Bax表达,从而抑制肝细胞凋亡.     

羟丁酸钠对大鼠肝脏热缺血再灌注损伤的保护作用

目的探讨羟丁酸钠（γ-hydroxybutyrate,GHB）对肝脏热缺血再灌注损伤的保护作用.方法采用大鼠肝脏热缺血再灌注模型,观察肝脏组织形态学、酶学、脂质过氧化、细胞凋亡及血浆内皮素的变化.结果肝脏缺血再灌注损伤时,血清ALT、AST、LDH、血浆内皮素、TNF-α及肝组织MDA水平均显著增高,细胞凋亡增加,肝组织SOD明显减少.再灌注前使用GHB可以明显减轻上述改变,钠络酮可以部分阻断GHB的作用.结论GHB可能通过抗脂质过氧化,下调肝脏内皮素的表达,改善微循环,减少肝细胞凋亡,从而减轻热缺血再灌注对肝脏的损伤作用.     

Effect of glycine on isolated,perfused rabbit livers following 48-hour preservation in University of Wisconsin solution without glutathione

Glycine has been shown to decrease membrane injury in isolated cells due to hypoxia or cold ischemia. The mechanisms of action of glycine are not known, but glycine may be useful in organ preservation solutions or in treating recipients of liver transplantation. In this study the isolated, perfused rabbit liver was used to measure how glycine affected liver performance after 48-h preservation in University of Wisconsin (UW) solution without added glutathione. UW solution is less effective for 48-h liver preservation when glutathione is omitted. Rabbit livers stored for 48 h without glutathione show a large increase in enzyme release (LDH and AST) from the liver and a reduction in bile production. The addition of 15 mM glycine to UW solution, in place of glutathione, did not improve bile production or reduce enzyme release. However, infusion of 10 mM glycine into the reperfused liver lowered LDH release significantly (from 2383±562 units/100 g to 1426±286 units/100 g) during the initial reperfusion of the 48-h preserved liver. Hepatamine, a parenteral nutrition solution containing glycine, as well as other amino acids, was also effective in lowering LDH release from the preserved liver. Although glycine reduced LDH release, it did not decrease the amount of AST released from the liver, nor did it improve bile production. Thus, we conclude that glycine, either in UW solution or given to the liver upon reperfusion, has no significantly beneficial effect as tested in this model. Further testing of glycine, however, should be conducted in an orthotopic transplant model in the rat or dog.     

Subnormothermic machine perfusion protects against rat liver preservation injury: a comparative evaluation with conventional cold storage

Hypothermic machine perfusion (MP) of the liver has been reported to improve graft function reclaiming marginal livers, such as those from non-heart-beating donors. Livers from obese donors often have fatty infiltrates and are more susceptible to hypothermic conditions. No data exist about MP at temperatures >4 degrees C. This study evaluated liver function after organ preservation by comparing MP at 20 degrees C with conventional cold storage. METHODS: For MP, rat livers were perfused for 6 hours using an oxygenated Krebs-Henseleit (KH) solution at 20 degrees C (pH 7.4). For cold storage, livers were perfused in situ and preserved with Celsior solution at 4 degrees C for 6 hours. The reperfusion period with KH (2 hours at 37 degrees C) was performed under the same conditions both among livers preserved by MP or cold storage. Hepatic enzyme release (aspartate aminotransferase [AST], alanine aminotransferase [ALT], lactate dehydrogenase [LDH], and gamma-glutamyl transferase [GGT]), bile production, and ATP levels were measured during MP and reperfusion. RESULTS: At the end of reperfusion, livers preserved by MP showed significantly decreased liver damage compared with cold storage: AST, 18 +/- 4 vs. 45 +/- 6 mU/mL (P < .01); ALT, 1.5 +/- .07 vs. 6 +/- 0.5 mU/mL (P < .01); and LDH, 82 +/- 2 vs. 135 +/- 29 mU/mL (P < .05). No difference was observed between bile production between MP and cold storage. High levels of biliary GGT and LDH were found in cold preserved livers. ATP levels were higher in livers preserved with MP compared with those preserved by cold storage. CONCLUSIONS: MP at 20 degrees C resulted in a better quality of liver preservation, improving hepatocyte survival, compared with conventional cold storage. This may provide a new method for successful utilization of marginal livers, in particular fatty livers.     

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.     

SX-1液对肝脏保存效果的实验研究

目的 应用改良后的Kamada二袖套法大鼠原位肝移植模型,检验SX-1液、HC-A液和UW液对肝脏保存的效果.方法 在无菌条件下配制肝脏保存液.建立大鼠原位肝移植模型.使用SX-1液、UW液和HC-A液保存大鼠肝脏2、8、24 h后行大鼠原位肝移植,于移植后6 h比较各项肝脏功能.结果 对于ALT、AST,SX-1液组(2、8、24 h)与UW液组同步升高,分析无统计学意义(P＞0.05),与HC-A液组相比,差异有统计学意义(P＜0.05).对于LDH,SX-1液组(2 h、8 h、24 h)与HC-A液组同步升高,差异无统计学意义(P＞0.05),与UW液组相比,差异有统计学意义(P＜0.05).对于分泌胆汁的肝脏个数,各组别与分泌胆汁的肝脏个数无差别(P＞0.05).本组内各时间点分泌胆汁个数有差别(P＜0.05).随肝脏保存时间增长,分泌胆汁的肝脏个数减少.结论 经大鼠原位肝移植模型证实,SX-1液在肝脏酶学方面与UW液作用相当,超过HC-A液水平.肝移植后6 h肝脏分泌胆汁的个数方面,SX-1液与HC-A液、UW液间无明显差别.     

他克莫司在大鼠肝脏保存中的作用

目的了解他克莫司对肝脏缺血再灌注损伤的保护作用。方法在供肝保存期间向保存液中加入他克莫司,检测保存12 h后的肝脏能量物质负荷、肝移植术后受体大鼠血清中的肝酶浓度以及胆汁分泌情况。结果保存液中加入他克莫司后,肝组织在保存期间的能量物质消耗明显小于对照组,移植术后受体大鼠血清中ALT和LDH含量明显较对照组为低,胆汁分泌量则明显高于对照组,差异均具有显著性(P0.05)。结论他克莫司用于肝脏保存能够有效地减轻缺血再灌注损伤,提高肝细胞能荷,改善移植术后的肝脏功能。     

Efficacy of polyethylene glycols in University of Wisconsin preservation solutions: a study of isolated perfused rat liver

Recent reports argue that the performance of University of Wisconsin (UW) solution is limited by the presence of hydroxyethyl starch (HES) as an additive, since HES could be responsible for human red blood cell aggregation. We investigated the effect on rat liver preservation of replacing HES in UW solution by polyethylene glycols (PEG20 and PEG35) at two concentrations. An isolated perfused rat liver model was used. Six groups of preserved livers (n = 7 for each group) were compared to controls (nonpreserved livers, n = 7). The following preservation solutions were assayed: UW without oncotic supply, UW-HES (0.25 mmol/L), UW-PEG20 (0.03 and 0.25 mmol/L), and UW-PEG35 (0.03 and 0.25 mmol/L). After 24-hour cold storage, the livers were perfused for 120 minutes at 37 degrees C with oxygenated Krebs-Henseleit solution. During perfusion, transaminase release, portal and bile flows, and bromosulfophthalein (BSP) clearance were assessed. Results showed that the omission of oncotic supply in UW statistically increased ALT and AST release in perfusate and decreased bile and portal flows. PEG addition in UW solution, especially PEG35 at 0.25 mmol/L, effectively protected the rat liver graft from the onset of hypothermic ischemia/reperfusion damage. In conclusion, data reported here reveal that oncotic supply is essential for liver preservation and that HES can be effectively replaced by PEG in UW solution.     

Fructose 1-6 bisphosphate versus University of Wisconsin solution for rat liver preservation: does FBP prevent early mitochondrial injury?

Background

Fructose 1,6-biphosphate (FBP) has been shown to exert therapeutic effects in models of ischemia-reperfusion in organs other than the liver. This study compared FBP and University of Wisconsin (UW) solution during cold storage and reperfusion, among mitochondria of adult male Wistar rat livers.

Methods

Adult male Wistar rats were assigned to two groups according to the preservation solution used; UW or FBP Aspartate transaminase (AST), alanine transferase (ALT); and lactic dehydrogenase (LDH) were measured in samples of the storage solution obtained at 2, 4 and 6 hours of preservation. After 6 hours of cold storage, we reperfused the liver, taking blood samples to measure AST, ALT, LDH, and throbarbituric acid reactive substances (TBARS). Hepatic fragments were processed for histologic analysis; for determinations of TBARS, catalase, and nitric oxide as well as for mitochondrial evaluation by infrared spectroscopy.

Results

During cold preservation, levels of AST and LDH in the storage solution were lower among the FBP group, but after reperfusion, serum levels of AST, ALT, and LDH were higher in this group, as was catalase activity. TBARS and nitric oxide were comparable between the groups. In the UW group there was a higher amide I/amide II ratio than in the FBP group, suggesting an abnormal protein structure of the mitochondrial membrane. No signs of preservation injury were observed in any liver biopsy, but sinusoidal congestion was present in livers preserved with FBP.

Conclusion

FBP showed a protective effect for preservation during cold storage seeming to protect the mitochondrial membrane although it did not prevent reperfusion injury.     

SX-1液对肝脏保存效果的形态学观察

目的 探讨新研制的肝脏灌注保存液SX-1液对保存肝脏的形态学影响.方法 使用SX-1液、威斯康星大学保存液(UW液)和高渗枸橼酸盐腺嘌呤液(HC-A液)保存大鼠肝脏2 h、8 h、24 h后行大鼠原位肝移植(OLT),于移植后6 h处死大鼠提取肝脏组织在光镜进行形态学观察,比较各组保存液对肝脏的保存效果.另外,取SX-...     

A comparison of histidine-lactobionate and UW solution in 48-hour dog liver preservation.

Many modifications of the UW solution have been reported to yield successful results in rat liver preservation and transplantation. One solution used histidine, in combination with lactobionate (HL-I), and gave superior preservation of the rat liver when compared with the UW solution. In this study we have compared the HL-I solution with 90 mM histidine, HL-II solution with 30 mM histidine, and the UW solution in dog liver preservation and transplantation. Dog livers were preserved for 48 hr in one of the three solutions and transplanted. The peak AST and ALT values were highest in livers preserved in HL-I, intermediate in UW solution, and lowest in HL-II. However, there were no significant differences among survival rates (average 5-7 days per group), posttransplant serum concentration of liver enzymes (AST, ALT, LDH, and alk-phos), clotting factors (PT and PTT), bilirubin, and fibrinogen concentration for each group. Dogs were sacrificed or died within 5-7 days due to rejection in nonimmunosuppressed dogs. Also, rat livers were preserved in the HL-II solution or in a solution in which histidine was replaced by isoleucine (IL-I). Isoleucine is an amino acid with a molecular mass similar to that of histidine, but is not as good a hydrogen ion buffer as histidine at the pH used for liver preservation (7.4). The buffer capacity of the IL-I solution was similar to the UW solution, but about one-half as much as the HL-II solution. Rats receiving a liver preserved for 30 hr in HL-II or IL-I were 100% viable. Rats receiving a liver preserved for 40-44 hr in HL-II or IL-I showed less survival (33% and 25%, respectively). This shows that histidine can be effectively replaced by isoleucine in a preservation solution and gives equivalent preservation results. Thus, the mechanism of improvement of liver preservation with histidine is not due to its action as a hydrogen ion buffer. These studies show that, although the HL solutions are superior for preservation of the rat liver, they are not superior to the UW solution for preservation of the dog liver. However, as others have shown in the rat liver transplant model, a simplified UW solution (HL-II) appears effective in dog liver preservation. The dog liver transplant model remains a more appropriate model for testing new preservation solutions prior to initiation of clinical trials.