Modified cold storage of rat livers with self-made HYD solution

目的　为探讨改进大鼠肝脏低温保存方法对鼠肝的影响 ,将切取后的鼠肝灌入一定量的自制保存液 (HYD液 )后 ,结扎进出肝脏的各血管。方法　应用大鼠离体肝灌注模型 ,对传统保存法 (对照组 )和改进保存法 (实验组 :2 0 %组、30 %组和 4 0 %组 )肝脏微循环指标 (门静脉灌注压、流出液内皮素 1、台盼蓝分布时间及组织学改变 )、流出液有关酶指标水平及分泌胆汁量进行了观察。结果　 2 0 %组、30 %组及 4 0 %组肝脏微循环指标及分泌胆汁量明显优于对照组 (P <0 0 5) ,30 %组肝脏有关酶指标水平明显低于对照组 (P <0 0 5) ,其中 30 %组保存肝脏效果最佳。结论　改进法保存鼠肝的各项指标明显优于传统法 ,故提示本法行之有效 ,值得临床工作借鉴。     

一氧化氮促释放剂在保护大鼠减体积肝移植后缺血再灌注损伤中的作用

目的:探讨一氧化氮促释放剂(FK409)在保护减体积肝移植大鼠移植肝脏缺血再灌注损伤中的作用。方法:将56对(112只)SD大鼠分为两组,分别为FK409处理组和对照组。其中各组的8对大鼠用于观察存活率,20对用于观察移植后血中肝脏天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)、总胆红素、NO水平及移植物内早期生长反应因子-1(EGR-1)蛋白水平的变化。结果:FK409处理组的1周存活率、血清NO水平均高于对照组(P<0.05),ALT、AST及肝组织中的EGR-1蛋白含量则低于对照组(P<0.05)。结论:一氧化氮促释放剂(FK409)能够通过增加NO的合成,来减轻原位减体积肝移植大鼠的再灌注损伤,改善微循环,提高移植肝脏的功能。     

DBcAMP对大鼠肝脏冷缺血再灌注损伤的保护作用

目的:探讨DBcAMP对大鼠移植肝脏在冷缺血再灌注损伤的保护作用。方法:应用SD大鼠原位肝移植(OLT)动物模型,根据保存液的不同,80只大鼠随机分为3个组:HC-A液对照组(A组,n=30);DBcAMP+HC-A液实验组(B组,n=30);UW液对照组(C组,n=20)。所有组在单纯低温保存8h再行原位肝移植术,测定肝组织ATP含量和肝细胞内游离Ca2+浓度变化情况、血清转氨酶,进行组化染色。结果:B组与A组相比,低温保存8h末ATP含量(μmol/g湿重)较高,分别为0.53±0.10,0.48±0.08组间有显著性差异(P<0.05)。B组与A组相比,门静脉开放6h后肝细胞内游离Ca2+浓度(nmol/L)低,分别为419.30±51.12,472.20±57.93,组间有显著性差异(P<0.05);血清ALT、AST(U/L)均较低,分别为3153±741.79,2208±450.34;3790±1029.82,2527±662.20组间有显著性差异(P<0.01)。PAS染色示门静脉开放后,B组较A组肝组织糖原含量少。HE染色及电镜下观察B组较A损伤明显减轻,但是仍比C组重。结论:DBcAMP对大鼠肝脏冷缺血再灌注损伤具有保护作用。     

自制含氟碳液在机器灌注保存供肝中的应用

目的 探讨机器灌注保存供肝过程中,自制含氟碳肝脏保存液对供肝的保护作用. 方法 雄性Wistar大鼠60只,随机分成3组:A组(n=20)单纯冷藏保存组,B组(n=20)离体肾脏保存液(HCA)灌注保存组,C组(n=20)自制含氟碳液灌注保存组.通过建立低温机器灌注保存供肝的实验模型,灌注12 h后,比较A、B、C三组肝脏保存再灌注后灌注流出液中丙氨酸转氨酶(ALT)、肿瘤坏死因子(TNF-α)的含量及透明质酸(HA)摄取量的变化. 结果 与A组比较,B组和C组各指标均降低.与B组比较,C组指标降低更为显著.ALT、TNF-α含量与HA摄取量在各组之间比较差异有统计学意义(均P<0.05). 结论 自制含氟碳肝脏保存液对供肝具有比单纯冷藏和单纯HCA液灌注保存更好的效果.     

Cold preservation of pig liver grafts with warm ischemia and pentoxifylline-UW solution

BACKGROUND: We undertook this study to investigate the safe time limits of cold preservation in UW solution of liver grafts subjected to warm ischemia (WI) for 20 min and the changes of the limits when pentoxifylline is added to UW solution. METHODS: The safe time limit was studied in a simple porcine orthotopic liver transplantation (LTx) model. In donors, livers were subjected to 20 min of WI and subsequent 12-h (group 1, n = 5), 16-h (group 2, n = 5), and 20-h (group 3, n = 3) cold preservation in UW solution, respectively. After the safe time limits were clear, another group (group 4, n = 5) was built to test whether or not the limits can be changed when pentoxifylline is added to UW solution in an unsafe time limit group. RESULTS: All five animals in group 1 survived up to 7 days of the survey endpoint. In group 2, only one animal survived up to the same survey endpoint and all animals in group 3 died within 12 h. The 1-week survival rate of group 1 was significantly higher than the other two groups. Group 1 had a lower level of alanine aminotransferase (ALT) or aspartase aminotransferase (AST) after LTx, less pathological damage, higher concentration of adenosine triphosphate (ATP) and higher microcirculation blood flux in the grafted liver tissue at 1 h after reperfusion than the other two groups. The results primarily showed that 12-h cold preservation was safe, 16 h was unsafe, and 20 h was highly unsafe. But when pentoxifylline was added to UW solution in cold preservation (16-h group, group 4), in contrast to group 2, the incidence of liver tissue necrosis and primary graft nonfunction was significantly lower in group 4 than in group 2. The 1-week survival rate of the pigs was 100% in the former and 20% in latter group. Levels of ALT and AST in recipients' artery blood, malondialdehyde and TNF-alpha concentration in grafted liver tissue, resistance of portal vein and hepatic artery after preservation in group 4 were significantly reduced, whereas microcirculation blood flux of the grafted liver, superoxide dismutase concentration and ATP concentration in grafted liver tissue were significantly elevated. CONCLUSIONS: The safe time limit of cold preservation in UW solution of liver grafts subjected to WI for 20 min was about 12 h and the limits can be prolonged to 16 h when pentoxifylline is added to UW solution. Many mechanisms were involved.     

Effect of fusion protein TAT and heme oxygenase-1 on liver sinusoidal endothelial cells apoptosis during preservation injury

Background Proteins or peptides can be directly transferred into cells when covalently linked to protein transduction domains （PTDs）. TAT is one of the most widely studied PTDs. The effect of fusion protein TAT and heme oxygenase-1 （HO-1） on liver sinusoidal endothelial cells （SECs） apoptosis during cold storage is unknown. The present study aimed to determine whether fusion protein TAT-HO-1 would transduce efficiently into liver during cold storage, and, if so, to determine whether TAT-HO-1 would attenuate SECs apoptosis during preservation injury in rat. Methods Livers of Sprague-Dawley rats were harvested and randomly assigned to group 1 （HTK solution） and group 2 （HTK solution containing TAT-HO-1 fusion protein） according to the type of the preservation solution. The transduction efficiency of TAT-HO-1 was examined and the impairment of SECs was assessed during the period of cold storage followed by 1 hour of reperfusion. Results TAT-HO-1 can transduce efficiently into liver during cold storage. A significantly lower apoptotic index of SECs was observed in group 2, at 6, 12 and 18 hours of cold storage after 1 hour reperfusion, when compared with group 1. TAT-HO-1 reduced HA and ET levels in liver at each time point. Both Bcl-2 and Bax protein were expressed in hepatocytes and SECs at the periphery of the sinusoidal space. Moreover, higher Bcl-2 expression and lower Bax expression were observed in group 2. Conclusions TAT-HO-1 can transduce efficiently into rat livers and shows a protective effect on SECs by attenuating apoptosis during cold ischemia/reperfusion injury. Protein transduction will be a novel therapeutic strategy to reduce the risk of preservation injury in liver transplantation.     

EXPERIMENTAL STUDY ON PRESERVATION OF RAT FATTY LIVER

Objective. To investigate the effects or cold preservation on rat ratty liver. Methods. We observed the changes of portal perfusion pressure, endothelin-1, enzymes relesase in the effluent and mortality of sinusoid lining cell after 0h, 6h, or 12h preservation respectively and a subsequent 30 min reperfusion in rat fatty liver groups and control groups by using isolated perfused rat liver model.And we compared fatty liver groups with control groups by these indices. Results. There was no obvious difference between mildly fatty liver group and control group after long time (12h)preservation, between moderately fatty liver group and control group after short time( 6h)preservation, between severely ratty liver group and control group without preservation(0h), while preservation reperfusion injury was more severe in moderately fatty liver group than in control group after long time (12h) preservation and in severely ratty liver group than in control group after short time (6h)preservation. Conclusions. The authors suggested that a mildly ratty liver donor could be used in the same way as nonfatty liver and a moderately fatty liver donor could he used depending on the time of preservation and the balance of the emergent needs of recipient and donor organ supply, while severely fatty liver doncr should be discarded without hesitation.     

上海多器官保存液保存离体大鼠肝脏的实验研究

目的:观察上海多器官保存液(Shanghai-mutil-organ solution,SMO液)对离体大鼠肝脏的保存效果,探讨应用SMO液保存离体供肝的可行性.方法:SD大鼠随机分为SMO液、UW液和HTK液保存组,建立离体肝脏单纯低温保存模型,保存液保存8、16、24、36 h分析肝脏组织能量代谢情况,观察肝组织形态学改变和肝脏细胞凋亡情况.结果:保存16、24、36 h,SMO液组肝组织三磷酸腺苷(ATP)、磷酸腺苷总量(TAN)及Atkinson能荷(AEC)均明显高于同时点HTK液组 (P＜0.05),与同时点UW液组无显著差异;形态学检查见SMO液组组织损伤较同时点HTK液组轻,除细胞肿胀较同时间点UW液组明显外,其余表现基本一致.保存24、36 h,SMO液组凋亡指数明显低于HTK液组(P＜0.05),而与UW液组无明显差异.结论:SMO液对大鼠离体肝脏的保存效果总体上与UW液相当,优于HTK液,仅在防止细胞水肿方面较UW液稍差.     

维拉帕米对大鼠肝脏低温保存再灌注损伤的防护机制

目的 在自制的器官保存液XJ－1液已取得较好结果的基础上,探讨维拉帕米对大鼠肝脏低温保存再灌注损伤的防护作用及其机制。方法 XJ－1液与XJ－1＋维拉帕米液保存大鼠肝脏18h后施行肝脏移植,检测术后血清转氨酶水平、肝组织钙离子及丙二醛含量等指标,观察大鼠术后1wk存活率及移植肝脏的组织病理学改变。结果 XJ－1＋维液保存的大鼠肝脏移植后4h,血清ALT,AST水平、肝组织钙离子含量、丙二醛含量均显低于XJ－1液组（P＜0．01,P＜0．05）,光镜与电镜下肝组织形态学改变较XJ－1液组好,同时术后生存状况及1wk存活率也优于XJ－1液（71％vs 57%)。结论 维拉帕米能够减轻肝脏体外保存再灌注损伤,明显增强了XJ－1液对大鼠肝脏的保存效果,原因在于其阻滞钙通道的钙离子拮抗作用。     

自制供肝保存液对大鼠肝分泌细胞间黏附分子-1和一氧化氮影响的比较研究

目的通过与UW液及HC-A液比较,检验自制供肝保存液保存肝脏效果,探讨自制保存液对大鼠肝分泌细胞间黏附分子(ICAM)-1及一氧化氮(NO)的影响。方法无菌条件配制自制供肝保存液;建立大鼠肝脏非循环离体灌注模型(IPRL);根据保存液的不同将大鼠随机分组,检测不同时间点肝灌注流出液中转氨酶含量及肝组织中ICAM-1和NO含量,观察肝组织形态学变化。结果对于丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST),自制液组与UW液组相近,但明显低于HC-A液组(P<0.05);对于ICAM-1和NO,自制液组与UW液组同步升高,两者差异无统计学意义(P>0.05),但与HC-A液比较差异有统计学意义(P<0.05)。同时间点比较自制液组ICAM-1含量较UW液组为高,NO则较低。形态学变化自制液组稍重于UW液组,轻于HC-A组。结论从生化指标和肝脏形态学变化反映出,自制供肝保存液已与世界先进的UW液保存效果相当,明显优于HC-A液。ICAM-1仅在肝细胞损伤时才有表达,其变化程度与酶学指标改变相平行。NO在自制液组较低,反映了自制供肝保存液在维持细胞膜稳定和抑制自由基产生方面具有优势。ICAM-1和NO变化反应肝组织损伤程度,可以做为评价保存液优劣的指标。     

药物H、Y、D对大鼠肝脏低温保存损伤的保护作用

目的 研究药物Ｈ、Ｙ、Ｄ对大鼠肝脏保存损伤的保护作用。方法 采用大鼠肝脏离体非循环灌注模型,观察乳酸林格液（ＬＲ液）中加入不同剂量药物Ｈ、Ｙ、Ｄ后保存大鼠脏脏１２ｈ的效果。结果 鼠肝经加入药物Ｈ、Ｙ、Ｄ的ＬＲ液温保存后,其肝组织ＡＴＰ、ＴＡＮ含量、ＡＥＣ及分泌胆汁量明显高于对照组（Ｐ〈０．０５）,且ＡＴＰ、分泌胆汁量与ＬＲ液内Ｈ、Ｙ、Ｄ剂量在一定范围内呈正相关。药物Ｈ、Ｙ、Ｄ具有抗．ＯＨ的作用,可     

血管紧张素转换酶2在大鼠移植肝保存性损伤中的表达及意义

目的:观察血管紧张素转换酶2(ACE-2)在大鼠移植肝中的表达及其与移植后保存性损伤(PI)的相关性,初步探讨局部肾素-血管紧张素系统(RAS)在肝移植PI中的可能作用。方法:根据供体肝冷保存时间将移植大鼠分为冷保存(CP)组及未进行冷保存(NCP)组,未进行肝移植的假手术组作对照;组织学检查观察肝脏保存性损伤程度,实时定量RT-PCR、蛋白质印迹、免疫组化法分别检测术后1、24、48 h肝组织ACE-2的mRNA、蛋白水平表达及其组织定位,哌莫硝唑免疫组化染色检测组织缺氧程度。结果:肝移植后存在不同程度组织损伤,CP组更明显;与假手术组相比,肝移植组术后ACE-2 mRNA及蛋白水平均显著升高(P<0.01或P<0.05),且CP组明显高于NCP组(P<0.05);免疫组化染色显示移植组在肝静脉周围呈阳性表达,染色阳性程度与ACE-2 mRNA表达明显正相关(r=0.78,P<0.001)。结论: ACE-2表达与冷保存导致的移植肝保存性损伤的组织缺氧密切相关,局部RAS可能参与了移植肝保存性损伤。     

低温机器灌注保存供肝新方法探索

目的：通过建立低温机器灌注保存供肝8h的实验模型，探讨灌注过程中自制含氟碳肝脏保存液对供肝的保护作用。方法：雄性Wistar大鼠60只，随机分成3组，单纯冷藏组、HC—A液组、含氟碳液组。经实验处理后，通过比较三组肝脏保存再灌注后灌注流出液中ALT、TNF-α的含量及HA摄取量。结果：单纯冷藏组与HC—A液组各指标比较有明显差异（P〈0．05），HC—A液组与含氟碳液组比较有明显差异（P〈0．05）。结论：在低温机器灌注保存供肝8h条件下，自制含氟碳液对供肝具有比单纯冷藏和单纯HC—A液灌注保存更好的效果。     

Comparative Study on the Effects of Self-made Liver Preservation Solution on Secretion of ICAM-1 and NO in Rats

In order to study the effect of self-made liver preservation solution on liver preservation by comparing with UW solution and HC-A solution, the self-made liver preservation solution （SM） and perfusion solution were prepared under the aseptic conditions. The isolated non-circulated perfusion rat liver model was established. According to the different preservation solutions, the rats were randomly divided into UW group, SM group and HC-A group. The three groups were divided into 6 subgroups according to the preservation duration （n=6 in each group）. The transferase in liver perfusion solution and intercellular adhesion molecule-1 （ICAM-1） and nitric oxide （NO） in liver tissues were determined at 2, 8 and 24 h respectively. The results showed that the levels of alanine aminotransferase （ALT） and aspartate aminotransferase （AST） had no significant difference between SM group and UW group, but significantly lower than in HC-A group. The levels of ICAM-1 and NO were increased simultaneously in SM group and UW group （P〉0.05）, but there was significant difference as compared with HC-A group （P〈0.05）. At the same time point, the level of ICAM-1 was higher in SM group than in UW group, but NO was lower. The preservation effect of SM solution is the same as UW solution, but better than HC-A solution.     

Comparative Study on the Effects of Serf-made Liver Preservation Solution on Secretion of ICAM-1 and NO in Rats

In order to study the effect of self-made liver preservation solution on liver preservation by comparing with UW solution and HC-A solution, the self-made liver preservation solution (SM) and perfusion solution were prepared under the aseptic conditions. The isolated non-circulated perfu-sion rat liver model was established. According to the different preservation solutions, the rats were randomly divided into UW group, SM group and HC-A group. The three groups were divided into 6subgroups according to the preservation duration (n=6 in each group). The transferase in liver perfu-sion solution and intercellular adhesion molecule-1 (ICAM-1) and nitric oxide (NO) in liver tissues were determined at 2, 8 and 24 h respectively. The results showed that the levels of alanine aml- notransferase (ALT) and aspartate aminotransferase (AST) had no significant difference between SM group and UW group, but significantly lower than in HC-A group. The levels of ICAM-1 and NO were increased simultaneously in SM group and UW group (P＞0.05), but there was significant dif-ference as compared with HC-A group (P＜0.05). At the same time point, the level of ICAM-1 was higher in SM group than in UW group, but NO was lower. The preservation effect of SM solution is the same as UW solution, but better than HC-A solution.     

冷保存和再灌注时肝内自由基生成的初步观察

目的 观察移植肝在冷保存与再灌注早期是否有氧自由基与一氧化氮（ＮＯ）产生，并探讨可能的发生机制。方法 采用猪异体原位肝移植模型，分别测定移植肝冷保存后与再灌注早期肝灌洗流出液／血中脂质过氧化物丙二醛（ＭＤＡ）与ＮＯ浓度。并观察肝再灌注时，出肝血中白细胞计数的变化。结果 冷保存后肝灌洗流出液中ＭＤＡ与ＮＯ浓度有明显增高。门静脉血经肝再灌注后ＭＤＡ含量较入肝前显著增加，粒细胞显著减少，但ＮＯ浓度无明显     

苦参碱防治肝血窦内皮细胞冷缺血-再灌注损伤

目的：探讨苦参碱对大鼠原位肝移植中肝血窦内皮细胞冷缺血-再灌注损伤的保护作用及其机制。方法：应用大鼠原位肝移植模型，供肝保存5h，168只大鼠随机分为对照组、小剂量治疗组、大剂量治疗组和假手术组，分别检测移植术后1、2、4、24h血透明质酸(HA)、一氧化氮(NO)、肿瘤坏死因子α(TNF-α)及肝组织丙二醛(MDA)、超氧化物歧化酶(SOD)、细胞间黏附分子-1(ICAM-1)的含量，并观察肝血窦内皮细胞形态学的改变。结果：与对照组比较，治疗组术后各时间点的HA和ICAM-1表达均显著减少，NO含量增加，TNF—α、MDA、SOD水平均明显降低，肝血窦内皮细胞形态出现改善。结论：苦参碱对大鼠原位肝移植冷缺血-再灌注中肝血窦内皮细胞损伤具有保护作用。     

华西Ⅲ号液保存鼠肾的实验研究

利用大鼠异体肾移植模型，以ＵＷ液为对照，研究华西Ⅲ号液对鼠肾的保存效果。实验采用封闭群ＳＤ大鼠，体重２００￣２８０ｇ，供受体同性，随机分为对照组（ＵＷ液组）和实验组（ＨＸ－Ⅲ液组）２组。各组又根据保存４８及７２小时分为两个亚组，每个亚组各１０只大鼠。原位灌注切取供体大鼠左肾后低温保存４８、７２小时行异体肾移植。结果：ＵＷ液保存鼠肾４８、７２小时移植后的存活率分别为１００％和９０％；ＨＸ－Ⅲ液保存鼠     

改良心肌保存液热缺血冷保存对猪心保护的作用

目的 研究改良心肌保存液在心脏移植术中对热缺血冷保存猪供心的心肌保存效果.方法 16对供猪随机分为2组,建立猪同种异体原位心脏移植模型.A组以St Thomas 液、B组以自制液作为停搏液、灌注冲洗液和保存液.供心心脏停搏热缺血5 min后,以停搏液灌注,切取心脏,4 ℃灌注冲洗并低温浸泡保存4 h,施行同种异体原位心脏移植.观察移植后心脏复搏的一般情况及血清肌酸激酶同工酶(CK-MB)变化,常规苏木精-伊红、PAS染色,光镜观察心肌组织.结果 浸泡保存后B组心肌含水量低于A组,复灌后CK-MB含量也低于A组(t=2.89～3.38,P<0.05).B组PAS糖原染色评分高于A组(T=24,P<0.01).结论 改良的新型心肌保存液能较好恢复热缺血冷保存的猪供心心脏功能,减轻复灌后的心肌再灌注损伤,减少保存期间细胞内糖原的消耗,其对心肌的保护明显优于St Thomas液.     

稳定大鼠肝移植模型的规范及移植肝灌注方式比较

目的总结建立大鼠原位肝移植稳定模型的规范和提高成功率的措施,比较经门静脉灌注和经腹主动脉灌注对移植肝功能的影响。方法手术显微镜下,用改良的二袖套法进行100例大鼠原位肝移植,并依据灌注方式分组:经门静脉灌注和经腹主动脉灌注组(n=50)。检测术后肝功能,观察组织病理学改变、术后生存率以及手术并发症。结果两组组织病理学表现相同。手术成功率分别为98%(49/50)和96%(48/50),3月存活率分别为93.5%(29/31)和93.3%(28/30),术后肝功能、手术成功率和3月存活率差异无显著性(P>0.05)。结论供肝经门静脉灌注和经腹主动脉灌注均可采用,应根据研究目的选择适当肝移植模型。熟练的显微外科技术、规范细致的手术操作、缩短无肝期时间是提高成功率、减少术后并发症的关键。