Omega-3脂肪酸对大鼠冷保存供肝肝移植后肝损伤保护作用的实验研究
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摘要
研究背景
供肝的冷保存缺血再灌注损伤以及由此引发一系列问题诸如移植后早期肝功能不良、原发移植肝无功能、胆道并发症等一直是肝移植研究的热点问题。目前,缺血再灌注损伤的机制仍不十分清楚,一致观点是供肝复流后会激活天然免疫系统并由此引发的局灶性促炎反应。供肝冷保存缺血性损害主要表现为局限性代谢紊乱、实质细胞死亡,并由此引发配体激活、线粒体氧自由基释放。复流后,上述不良因素会在Kupffer细胞、树突状细胞、T细胞、NK细胞和中性粒细胞等多种非实质细胞参与下激活宿主天然免疫系统,进一步加剧炎症反应。外周血中免疫细胞的加入会使这一免疫激活-炎症级联反应持续进展。简而言之,冷保存造成的缺血再灌注损伤是宿主天然免疫系统介导的局限性促炎反应,最终造成肝脏实质细胞的损害。
既往研究证实,omega-3多不饱和脂肪酸(以下简称omega-3脂肪酸)能改善胃肠外营养相关性肝病预后；减少炎症介质产生、降低黏附分子的表达,从而起到直接或间接抗炎的作用；预防脂肪肝小鼠缺血再灌注损伤和微循环衰竭的作用；减轻小鼠化学性肝损伤和梗阻性黄疸肝细胞损伤的作用。新近研究证实,omega-3脂肪酸还具有改善大鼠缺血再灌注损伤,促进肝切除后肝再生的作用。
虽然最近有omega-3脂肪酸能改善肝移植患者术后肝损伤的临床报道,但其确切作用机制还不十分清楚。众所周知,即使在医学迅猛发展的今天,肝移植过程中冷保存缺血再灌注损伤仍不可避免。因而如何缓解冷保存缺血再灌注损伤,对于改善肝移植患者肝功能、提高手术效果具有非常重要的意义。缺血再灌注损伤时宿主天然免疫系统激活,肝脏非实质细胞释放的各种炎症介质对于免疫激活-炎症级联反应的维持非常关键。目前已明确omega-3脂肪酸能在转基因小鼠化学性肝炎模型中起到抗炎作用。因此,我们设想,首先通过Kamada'‘二袖套法”建立基于精准外科理念下的不重建肝动脉的大鼠原位肝移植模型,找出静脉和胆管最合适的重建方法。并在此基础上建立重建肝动脉的冷保存大鼠原位肝移植模型,探究不同冷保存时间对移植术后近期肝功能和微循环的影响,找出动物实验研究合适的冷保存时间长度。最后对重建肝动脉冷保存肝移植大鼠行术后omega-3脂肪酸干预,以明确omega-3脂肪酸是否对冷保存缺血再灌注后的供肝具有保护作用。
第一部分基于精准外科理念的大鼠肝移植模型的建立和评价
目的：基于精准外科理念下,建立不重建肝动脉血供的成熟稳定的同品系大鼠原位肝移植模型。并对肝移植模型进行评价,探索二袖套法肝移植中最佳的肝上腔静脉显微缝合方法,最佳的静脉吻合用袖套管和胆管吻合支架管。
方法：健康成年雄性SD大鼠,体重250-300g。供受体体重相差≤20g。根据不同的显微缝合方法、袖套和支架类型,将移植组大鼠随机分为3组。第1组：新的肝上腔静脉显微缝合方法+单槽袖套+无损伤胆管支架；第2组：新的肝上腔静脉显微缝合方法+单槽袖套+粗制胆管支架；第3组：肝上腔静脉采用传统的双定点连续缝合法+多槽袖套+无损伤胆管支架。第4组为对照组,实施假手术,仅开腹游离肝周韧带和移植相关血管。记录移植组大鼠各个操作步骤时长、一周和一月的生存率。1月后全部大鼠均在乙醚麻醉下处死,测定其血液肝功能生化指标,并详细解剖肝上、肝下腔静脉、门静脉和肝门部胆管,以明确有无血管扭转、血栓形成和胆汁瘤、胆道梗阻等并发症。P<0.05视为有统计学意义。所有的统计数据使用PASW18.0来分析。
结果：新的肝上腔静脉显微缝合方法较传统的双定点连续缝合法速度更快(P<0.05),无术后肝上腔静脉吻合口出血的发生。单槽袖套明显缩短肝下腔静脉和门静脉的重建时间(P<0.05),同时术后血栓也明显减少。新的肝上腔静脉显微缝合方法结合单槽袖套较传统连续缝合与多槽袖套能明显缩短无肝期(P<0.05)。1月后移植组大鼠的肝功能各项指标已恢复正常,与对照组比较无明显差异(P>0.05)。新的胆管支架制作方法能够明显降低大鼠肝移植术后胆道并发症的发生,进而提高了大鼠的一周和一月存活率(P<0.05)
结论：新的肝上腔静脉显微缝合方法结合重新设计制作的单槽袖套较传统缝合方法和袖套能够以较小创伤,更简单、迅速、可靠地吻合肝上、肝下腔静脉和门静脉,显著地缩短无肝期,更符合精准外科“最小侵袭,最大脏器保护”的理念。同时,无损伤胆管支架明显较传统粗制支架减弱了对胆管内壁的损伤。因而显著地降低了移植术后胆道并发症的发生。简言之,基于精准外科理念的大鼠肝移植模型创伤更小,耗时更短,模型更稳定、可靠,并发症更少,能够获得更好的远期生存。
第二部分冷保存对大鼠肝移植术后肝功能和即时微循环的影响
目的：建立成熟稳定、重建肝动脉血供的大鼠冷保存肝移植模型。探讨不同冷保存时间对大鼠肝移植术后肝功能和即时微循环的影响,为进行冷保存肝移植后肝损伤的药物治疗研究奠定基础。
方法：实验动物同第一部分。采用第一部分的手术方法建立恢复肝动脉血供的冷保存大鼠原位肝移植模型。根据不同的冷保存时间,将大鼠随机分为3组：第1组,冷保存12h组：供肝使用4℃HTK液灌注并冷保存12h；第2组,冷保存24h组：供肝使用4℃HTK液灌注并冷保存24h；第3组：正常对照组,实施假手术,仅开腹游离肝周韧带和移植相关血管。记录无肝期和供、受体所用手术时间和术后7天受体的一般情况。移植组供肝复流后3、10、20min、对照组完成游离操作后3min分别使用激光散斑灌注成像法对肝脏表面微循环进行测定。移植组大鼠各取10只受体进行7天生存率的比较,其余受体分别于术后1天(24h)、3天、7天处死取血和肝组织标本,每一时间点取6只受体。对照组6只正常大鼠于术后7天处死取血。移植组和对照组检测血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase, AST)、,白蛋白(albumin, Alb)、碱性磷酸酶(alkaline phosphatase, ALP)和总胆红素(total bilirubin, TB)等肝功能指标。肝组织行HE染色,分别从门管区炎细胞浸润、局灶性坏死、胆管增殖、胆管损伤和间质纤维化等5方面对肝损伤程度进行半定量评分。所有大鼠在处死前均经腹壁行肝脏中叶的超声实时弹性成像。所有数据使用PASW18.0软件进行统计分析,P<0.05视为有统计学意义。
结果：
1.手术时间及术后一般情况：两冷保存移植组大鼠的供、受体手术操作时间无明显差别(P>0.05),无肝期均控制在14min以内。冷保存12h组受体术后恢复较快,麻醉清醒后即刻活动,进饮食水。两组存活受体均未见巩膜、双耳、爪子和尿液的黄染。
2.供肝复流后肝脏表面微循环变化：冷保存肝移植大鼠的肝脏表面微循环流量值明显低于正常对照组(P<0.05)。冷保存12h组肝脏表面微循环流量值明显高于冷保存24h(P<0.05)。
3.生存率：冷保存12h组明显高于冷保存24h组(P<0.05)。
4.血清肝功能指标变化情况：在术后1、3、7天,两冷保存肝移植组大鼠肝功能受损严重,各项指标均明显差于对照组(P<0.05),且同时间点冷保存24h组明显差于冷保存12h组(P<0.05)。随时间延续,两冷保存组ALT、AST呈逐渐下降趋势,ALP则呈先降低后升高,Alb和TB均呈逐渐增高趋势。
5.肝组织HE染色半定量评分：冷保存肝移植术后1天肝损伤已非常明显：肝细胞肿胀,近中央静脉的肝细胞空泡变性严重,肝血窦扩张充血,门管区炎细胞浸润,胆管内皮细胞肿胀、坏死。随时间延续,肝细胞肿胀、肝血窦扩张有所减轻,空泡变逐渐缓解,门管区炎细胞浸润和胆管增生较前明显,局灶性坏死有所减轻,胆管损伤有加重趋势,间质纤维化改变不明显。同时间点,冷保存12组在门管区炎细胞浸润、胆管损伤和局灶性坏死等方面的评分均低于冷保存24h组(P<0.05),冷保存12h组的胆管增殖评分则在术后3天和一周低于冷保存24h组(P<0.05)；两组间在间质纤维化上评分差别不显著(P>0.05)。
6.超声实时弹性成像：以对照组的肝脏弹性模量值为正常参考值,冷保存肝移植组在术后第1天肝脏弹性模量值即高于正常值(P<0.05),但随时间延续,各组的肝脏弹性模量值变化不大(P>0.05)。冷保存24h组肝脏弹性模量值与冷保存12h组差别不明显(P>0.05),但两冷保存组肝脏弹性模量值始终高于正常值(P<0.05)。
结论：
1.供肝冷保存会造成肝移植大鼠肝功能的严重损害,且对于胆管细胞的损伤要重于肝细胞。随着冷保存时间的延长,这种肝功能损害随之加重。
2.供肝冷保存会造成术后早期的肝脏微循环障碍,随着供肝冷保存时间延长,微循环损害加重。激光散斑灌注成像可以作为供肝冷保存缺血损伤严重程度的早期评价指标之一。
3.供肝冷保存12h的大鼠原位肝移植是作为研究冷保存肝移植后肝损伤的比较合适的动物模型。
4.超声实时弹性成像可以作为安全、无创地评价肝脏功能的一个参考指标。
第三部分Omega-3脂肪酸对大鼠冷保存供肝肝移植术后肝功能的影响
目的：明确omega-3旨肪酸对大鼠冷保存肝移植术后肝功能影响并对其作用机制作初步探讨。
方法：实验动物及手术方法同第二部分。根据供肝有无冷保存以及术后不同处理,将大鼠随机分为3组：其中第1组实施正常肝移植,第2、3组实施供肝冷保存12h肝移植。术后1-7天,第1、2组予每天生理盐水12ml/kg灌胃,第3组予每天omega-3鱼油脂肪乳12ml/kg灌胃。每组取10只受体进行4周生存率的比较。分别于术后3天、1周、2周、3周和4周处死受体,每一时间点取6只受体,取血和肝组织标本。血清肝功能指标检测和肝组织HE染色半定量评分方法同第二部分。术后各时间点,免疫组织化学方法检测肝组织中Ki-67的表达,Masson染色方法描述肝组织中纤维组织增生的程度。所有受体处死前经腹壁行肝脏超声实时弹性成像,方法同第二部分。另外,对术后3天、1周、2周标本分别使用酶联免疫吸附试验检测血清、实时荧光定量PCR技术检测肝组织中TNF-alpha, IL-6, IFN-gamma和TGF-betal的表达情况。所有数据使用PASW18.0软件进行统计分析,P<0.05视为有统计学意义。
结果：
1.生存率：正常肝移植组大鼠术后4周生存率高于冷保存组(P<0.05),两冷保存组间生存率无显著差异(P>0.05)
2.血清肝功能指标：术后2周内,第2组术后肝功能指标除Alb外明显差于第1、3组(P<0.05),第3组虽较第2组有所改善但仍未达第1组水平(P<0.05)。各组术后2-3周肝功能指标明显好转,并在3-4周趋于平缓。4周观察期末,第1、3肝功能基本恢复,但第2组肝功能指标仍未达正常值(P<0.05)。
3.肝组织HE染色半定量评分：同时间点,各组局灶性坏死和间质纤维化评分无差别。胆管损伤则第1组最弱,余两组无明显差别。门管区炎细胞浸润第2组最强,其余两组无明显差别。胆管增殖：术后第1、2周,第1组最弱,其余两组无显著差别；术后第3周第2组最强,其余两组无明显差别。
4.肝脏超声实时弹性成像：各组肝脏弹性模量值总体呈逐渐降低趋势,两冷保存组相同时间点无明显差别(P>0.05),但均较正常移植组为高(P<0.05)。
5.血清细胞因子水平：各组大鼠血清中TNF-alph、IFN-gama、IL-6和TGF-betal均呈先升后降趋势,术后1周为峰值。相同时间点,第2组术后血清中TNF-alpha、IFN-gamma、IL-6水平最高(P<0.05),第1、3组间无明显差别(P>0.05)。而同时间点各组间TGF-betal无明显差别(P>0.05)。
6.肝组织细胞因子mRNA表达情况：肝组织中TNF-alpha, IFN-gamma, IL-6和TGF-betal的mRNA表达强度与血清中的变化趋势类似。相同时间点上,TNF-alpha、IFN-gamma和IL-6各细胞因子mRNA的表达,第2组明显高于其余两组(P<0.05),且1、3组间则无明显差别(P＞0.05), TGF-betal则在各组间无明显差别(P>0.05)。
7.肝细胞增殖情况：肝组织Ki-67结果显示,各组术后肝细胞均有不同程度增殖,1周内增殖活跃,其后逐渐减弱。第1、3组间肝细胞增殖程度无明显差别(P>0.05)。前2周内,第2组肝细胞增殖程度明显强于第1、3组(P<0.05)；第3周,第2组肝细胞增殖程度仍强于第3组(P<0.05),第4周各组间差别不显著(P>0.05)。
8.肝组织中胶原纤维的检测：Masson染色评分显示,各组术后肝组织中胶原纤维含量呈先升后降趋势,以第2、3周为高。相同时间点,各组胶原纤维含量无差别(P>0.05)。
结论：
1.与正常移植相比,供肝冷保存12h会显著降低肝移植大鼠术后4周生存率,给予(>mega-3脂肪酸并不能提高其4周生存率。
2.供肝冷保存12h肝移植后给予omega-3脂肪酸能在一定程度上改善术后肝功能生化指标,但不能改变其恢复的进程。
3. Omega-3脂肪酸通过降低肝组织和血清中TNF-alpha、IFN-gamma和IL-6等促炎细胞因子的表达从而起到抑制炎症反应,缓解缺血再灌注损伤,改善肝功能的作用。Omega-3旨肪酸的使用不增加肝脏纤维化的风险,但对于改善冷保存造成的胆管损伤和胆管增殖效果不明显。
Backgroud
Ischemia-reperfusion injury (IRI) caused by graft cryopreservation has triggered a series of problems such as poor early graft function, primary non function and biliary complications. Therefore, IRI has been the hot topics in the study of liver transplantation. Nowdays, the mechanism of IRI is still unclear. The consensus is that the innate immune system will be activated after reperfusion of the graft. And then local proinflammatory response will be appeared. The ischemic damage to the graft mainly show metabolic disorder and parenchyma cell death which lead to ligands activation and mitochondrial release oxygen free radicals. After the reperfusion, the adverse factors above with the involvement of Kupffer cells, NK cells, dendritic cells, T cells, neutrophils and other non parenchymal cells will activate the host's innate immune system, further aggravate the inflammatory response. The involvement of immune cells in peripheral blood can make the immune activation-inflammatory cascade a continued progress. In short, the IRI is a local proinflammatory response which is mediated by host's innate immune system and finnaly lead to damage of the liver parenchymal cells.
It has been reported that omega-3polyunsaturated fatty acids (n-3PUFA) could improve the prognosis of parenteral nutrition correlation of liver disease, to reduce the production of inflammation medium and adhesion molecules to play a role in anti-inflammatory, to prevent IRI and circulation failure in mice with fatty liver, to alleviate chemically induced acute hepatitis and liver cell damage caused by obstructive jaundice in rats and so on. Recently, it has been proved that n-3PUFA could also attenuate IRI and promote hepatic regeneration following partial liver resection in normal or fatty liver.
Although there is report about the liver-protecting effects of n-3PUFA for patients after liver transplantation, the influence of n-3PUFA to the IRI which caused by graft cryopreservation and the exact mechanism is still not very clear. As we know, even with the rapid development of medicine, the IRI of donor liver caused by cryopreservation is still completely unavoidable. Hence, it is clinically meaningful to maximumly reduce IRI and improve liver function for patients after liver transplantation. As mentioned above, the IRI is a local proinflammatory response mediated by host's innate immune system. In this pathophysiological process, the parenchymal cells release a variety of inflammatory mediators such as TNF alpha, IL-6, IL-1beta and IFN-gamma after the activation of host's inane immune. This is the key to maintain the inflammatory cascade. It has been proved that omega-3fatty acids could play an anti-inflammatory effect in mouse with chemical-induced hepatitis. In this study, firstly we established an orthotopic liver transplantation model in rats without the reconstruction of hepatic artery. This modal was performed according to Kamada's two cuff method under the concept of precision surgery. We found the best method for reconstruction of veins and bile duct for ROLT. Secondly, we established an cryopreserved orthotopic liver transplantation model in rats and explored the effect of graft cryopreservation to liver function and microcirculation. In the meantime, we found out the exact length of cropreservation time which was suitable for animal experimental study. At last, we treated the rats wihich subjected to cryopreserved liver transplantation with n-3PUFA. And then the treatment effect of n-3PUFA to the rats was investigated. We try to prove that n-3PUFA could inhibit liver inflammation, improve liver function and further increase the long-term survival of the recipients.
Part I Establishment and Evaluation of Liver Transplantation Model in Rats Under the Concept of Precision Surgery
OBJECTIVE:Establishment of rat orthotopic liver transplantation (ROLT) model under the concept of precision surgery. The hepatic artery was not reconstructed. The modal was evaluated for exploring the best microsuture method for reconstruction of suprahepatic vena cava (SHVC), the best cuff and stent for reconstruction of veins and bile ducts.
METHODS:
Male SD rats weighing250to300g were used as donors and recipients. The weight difference was equal or lesser than20g. According to different microsuture methods for the SHVC and different types of cuffs and stents, three ROLT groups were created to compare the operation times and prognoses. Group1:new microsuture method for SHVC+single-groove cuff+blade-cut stents. Group2:new microsuture method for SHVC+single-groove cuff+scissors-cut stents. Group3: conventional continuous suture method for SHVC+multi-groove cuff+blade-cut stents. Sham operations were performed as controls in group4. The time expenditures with each step were compared among the transplantation groups. The one week-and one month-survival rates of the transplantation groups were recorded and compared. All of the animals that survived one month were sacrificed and the biochemical parameters were tested. The SHVC, infrahepatic vena cava (IHVC), portal vein (PV) and hilar bile duct was detailedly dissected to make sure if there are torsion of the vessels, thrombosis and biliary complications. P＜0.05was considered to indicate statistical significance. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA).
RESULTS:
Our new microsuture method was faster than the conventional continuous suture method for SHVC anastomosis (P＜0.05). There was no bleeding of the anastomotic stoma of the SHVC. The use of a single-groove cuff for reconstruction of the portal vein and the infrahepatic vena cava shortened the anastomotic time (P＜0.05) and significantly reduce the postoperative thrombosis. The new microsuture method companied with the single-groove cuff shortened the anhepatic time when compared to the conventional continuous suture method with the multi-groove cuff (P＜0.05). The biochemical parameters reached to normal level at one month after the transplantation. The The use of blade-cut stents resulted in fewer biliary complications and better survival over the short and long terms (P＜0.05).
CONCLUSIONS:
Our new microsuture method and the single-groove cuffs proved to be a precise method for venous reconstruction which shortened the anhepatic time and the anastomotic time significantly. These new methods are more in line with the concept "minimizing surgical invasiveness and maximizing organ protection" of precision surgery. The blade-cut stents apparently decreased the incidence of biliary complications by reducing the injury to the inner wall of the bile duct. In summary, with this precise microsuture method and delicate cuffs and stents, excellent long-term survival can be achieved easily and stably for ROLT.
Part II The Effect of Graft Cropreservation to Postoperative Liver Function and Instant Microcirculation For Rats Subjected to Liver Transplantation
OBJECTIVE:To establish a mature and stable cryopreserved liver transplantation modal in rats with the reconstruction of hepatic artery. To explore the effect of different cryopreservation time to postoperative liver function and instant microcirculation. To lay the foundation for drug treatment of damage caused by cryopreserved liver transplantation.
METHODS:
Animals were similar to Part Ⅰ. To establish cryopreserved rat liver transplantation modal with the reconstruction of hepatic artery. The methods mention in Part Ⅰ was applied. According to different cryopreservation time, three ROLT groups were created. In group1, donor liver was perfused and preserved for12h by4℃HTK solution (P12h group) and in group2for24h by4℃HTK solution (P24h group). In group3, sham operations were performed as controls. Anhepatic time, the time used for donor and recipient and general conditions on the7th day after the operation were recorded. For the recipients, the laser speckle perfusion images were performed3,10and20min after the reperfusion of the donor liver. Ten recipients were used for comparison of7day-survival rate. Blood and liver specimens were gained on postoperative day1(24h), day3and day7(POD1,3,7) when the recipients were sacrificed. There were six recipients for each point. The six recipients in the control group were also sacrificed on postoperative day7and blood specimens were collected. Biochemical parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), alkaline phosphatase (ALP) and total bilirubin (TB) were measured for all the blood specimens. Hematoxylin-eosin staining was done for the liver tissue specimens. The liver tissue were semi-quantitatively scored from the following items:portal inflammation, focal necrosis, ductular proliferation, bile duct damage and septal fibrosis. The real-time elastography was performed transabdominally for all the survivors before they were sacrificed. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.
RESULTS:
1. Time expenditure and general conditions. There were no significant difference of the time used in donors and recipients between P12h and P24h groups. The anhepatic time were controlled within14min. The animals in P12h group recovered faster after the operation. There were no icteric sclera, yellow claws and urine for all the survivors in the P12h and P24h groups.
2. The microcirculation changes of the graft after the reperfusion. The flux value of the graft liver in the cryopreserved groups were significantly lower than the control. While the flux value in the P12h group was higher than that in the P24h group (P＜0.05).
3. The survival rate in the P12h group was significantly higher than that in the P24h group (P＜0.05).
4. The biochemical parameters of the serum. The liver function in the cryopreservation groups were heavily damaged when compared with the control group. On POD1,3and7, all the parameters were all worse than that in the control group obviously (P＜0.05). In the cryopreservation groups, ALT and AST decreased over time. While ALP increased at first and then decreased. The Alb and TB value increased over time.
5. The semi-quantitative scores of the liver tissue. The liver damage was very obvious even on POD1for the recipients in P12h and P24h groups. Following changes were found in HE staining:Swelling and vacuoles degeneration of the liver cells, hepatic sinusoid congestion, inflammation cells infiltration of the portal tract, bile duct endothelial cells swollen and necrosis. As an extension of time, swelling and vacuoles degeneration of the liver cells and hepatic sinusoid congestion alleviated. Portal inflammation and ductular proliferation were obvious than POD1. Focal necrosis alleviated but bile duct damage aggravated over time. The septal fibrosis was not obvious. At the same points, the P12h group has lower scores than the P24h group with respect to portal inflammation, ductular proliferation, focal necrosis and bile duct damage (P＜0.05). But there were no significant difference of the scores for septal fibrosis (P＞0.05).
6. The real-time elastography of recipients'liver. The mean elastic ratio of the liver in the control group was setted as normal value. The elastic ratios in the cryopreservation groups were higher than normal value just on POD1and didn't change a lot over time. The elastic ratios of liver in the P12h group were similar to that in the P24h group at the same point (P＞0.05). However, the elastic ratios of the cryopreservation groups were always higher than normal value during the observation time (P＜0.05).
CONCLUSIONS:
1. Cryopreservation of the donor liver caused serious liver function damage. The damage of bile duct cells were heavier than liver cells. The longer the donor liver was cryopreserved, the heavier the liver was impaired.
2. Cryopreservation of the donor liver could cause early postoperative damage to hepatic microcirculation. The longer the donor liver was cryopreserved, the heavier the microcirculation was impaired. Laser speckle perfusion imaging can be used as an index for evaluating of early ischemic injury caused by the cryopreservation of the donor liver
3. Twelve hours cryopreservation of donor liver was suitable for study of liver impairment after cryopreserved liver transplantation.
4. The real-time elastography was a safe and noninvasive method for evaluation of liver function.
Part Ⅲ The Effects of Omega-3Fatty Acids to Postoperative Liver Function for Rats Subjected to Cryopreserved Liver Transplantation
OBJECTIVE:To investigate the effects of omega-3fatty acids to postoperative liver function for rats subjected to cryopreserved liver transplantation and preliminarily discuss the mechanism.
METHODS:
According to different time of graft preservation and postoperative administration, rats were randomly divided into three transplantation groups. Our new methods for ROLT which was based on precision liver surgery was used here. In group1, normal liver transplantation was performed. In group2and3, donor liver was perfused and preserved for12h by4℃HTK solution and then transplanted. During postoperative day (POD)1to7, the recipients in group land2received saline solution (12ml/kg/day, via orogastric gavage) while recipients in group3received omega-3fish oil fat emulsion (12ml/kg/day, via orogastric gavage). Ten recipients were used in each group for comparison of four week survival rate. On POD3,7,14,21and28, all survivors in each group were sacrificed, blood and liver specimens were collected. There were six recipients for each point. Biochemical parameters and semi-quantitative score of the hematoxylin-eosin staining of liver tissue were similar to that in Part Ⅰ. At various points after the operation, Ki-67level of the liver tissue was measured by immunohistochemical analysis. Meanwhile, Masson staining was used to describe the degree of fibrosis in liver tissue. The real-time elastography was performed transabdominally for all the survivors just as Part Ⅱ. In addition, on POD3,14and21, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal in the serum was measured by ELISA kit. The expression of mRNA of the same cytokines in the live tissue was measured by real time quantitative PCR in the same point. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.
RESULTS:
1. Suvival rate. The normal liver transplantation group was higher than that in the cryopreserved group (P＜0.05). There were no significant difference between group2and3which cryopreserved liver transplantation were performed (P＞0.05).
2. The biochemical parameters of the serum. The biochemical parameters except Alb in group2was significantly worse than that in group1and3(P＜0.05). The postoperative liver function in group3was improved but still worse than that in group1(P＜0.05). The biochemical parameters in each group improved during POD14-POD21and flatten out during POD21-POD28. At the end of POD28, the animals in group1and3got nearly normal liver function but the biochemical parameters in group2were still not better than normal value.
3. The semi-quantitative scores of the liver tissue. At the same time point, there were no difference for septal fibrosis and focal necrosis among the three groups. For the bile duct damage, group1was the lightest and there were no difference between the other two groups. For portal inflammation, group2was the worst and there were no difference between the other two groups. For ductular proliferation, on POD7and POD14, group1was the lightest and there were no difference between the other two groups. On POD21, group2was the worst and there were no difference between the other two groups.
4. The real-time elastography of liver. The elastic ratio of the liver decreased after the operation. There was no difference between group2and3at the same time point(P＞0.05). But the elastic ratio of group2and3were higher than that in group1(P＜0.05).
5. The levels of cytokines in the serum. On POD3,7and14, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal increased first and then decreased. The levels of cytokines were the highest on POD7. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.
6. The expression of mRNA of the cytokines in the live tissue. The changes of the mRNA of the cytokines in liver tissue was similar to that in the serum.The expression of mRNA of the cytokines were also first increased and then decreased. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.
7. The proliferation of liver cells. The scores of Ki-67levels in the liver tissue showed that the liver cells proliferated most obviously during one week among the three groups after the operation. Then the proliferatation gradually weakened. There were no difference of the scores of Ki-67between group land3(P＞0.05). During the first two weeks, the scores of Ki-67in group2were higher than that in group1and3(P＜0.05). On POD21, the scores of Ki-67in group2were still higher than that in group3(P＜0.05). And there were no difference among the three groups on POD28.
8. Detection of collagen fibers in the liver tissue. The scores of Masson staining showed that the content of collagen fibers in the liver tissue increased first and then decreased. The scores on POD14and POD21were more higher than other time point. But there were no difference between the three groups at the same time points.
CONCLUSIONS:
1. When compared with normal liver transportation,12hours cryopreservation of the donor liver decreased the four week survival rate of the recipients whether or not usage of omega-3fatty acids.
2. Omega-3fatty acids could improve the postoperative liver function in some extent for rats subjected to cryopreservation transplantation. However, it couldn't change the restore speed of the liver function.
3. Omega-3fatty acids probably play aniti-inflammatory role through the inhibition of the synthesis of inflammatory cytokines such as TNF-alpha, IL-6and IFN-gamma in the liver tissue. Then the ischemic reperfusion injury alleviated and the liver function got better. Omega-3fatty acids didn't increase the risk of fibrosis of the liver. But it couldn't improve the ductular proliferation and bile duct damage caused by cryopreservation of the donor liver, either.
供肝的冷保存缺血再灌注损伤以及由此引发一系列问题诸如移植后早期肝功能不良、原发移植肝无功能、胆道并发症等一直是肝移植研究的热点问题。目前,缺血再灌注损伤的机制仍不十分清楚,一致观点是供肝复流后会激活天然免疫系统并由此引发的局灶性促炎反应。供肝冷保存缺血性损害主要表现为局限性代谢紊乱、实质细胞死亡,并由此引发配体激活、线粒体氧自由基释放。复流后,上述不良因素会在Kupffer细胞、树突状细胞、T细胞、NK细胞和中性粒细胞等多种非实质细胞参与下激活宿主天然免疫系统,进一步加剧炎症反应。外周血中免疫细胞的加入会使这一免疫激活-炎症级联反应持续进展。简而言之,冷保存造成的缺血再灌注损伤是宿主天然免疫系统介导的局限性促炎反应,最终造成肝脏实质细胞的损害。
既往研究证实,omega-3多不饱和脂肪酸(以下简称omega-3脂肪酸)能改善胃肠外营养相关性肝病预后；减少炎症介质产生、降低黏附分子的表达,从而起到直接或间接抗炎的作用；预防脂肪肝小鼠缺血再灌注损伤和微循环衰竭的作用；减轻小鼠化学性肝损伤和梗阻性黄疸肝细胞损伤的作用。新近研究证实,omega-3脂肪酸还具有改善大鼠缺血再灌注损伤,促进肝切除后肝再生的作用。
虽然最近有omega-3脂肪酸能改善肝移植患者术后肝损伤的临床报道,但其确切作用机制还不十分清楚。众所周知,即使在医学迅猛发展的今天,肝移植过程中冷保存缺血再灌注损伤仍不可避免。因而如何缓解冷保存缺血再灌注损伤,对于改善肝移植患者肝功能、提高手术效果具有非常重要的意义。缺血再灌注损伤时宿主天然免疫系统激活,肝脏非实质细胞释放的各种炎症介质对于免疫激活-炎症级联反应的维持非常关键。目前已明确omega-3脂肪酸能在转基因小鼠化学性肝炎模型中起到抗炎作用。因此,我们设想,首先通过Kamada'‘二袖套法”建立基于精准外科理念下的不重建肝动脉的大鼠原位肝移植模型,找出静脉和胆管最合适的重建方法。并在此基础上建立重建肝动脉的冷保存大鼠原位肝移植模型,探究不同冷保存时间对移植术后近期肝功能和微循环的影响,找出动物实验研究合适的冷保存时间长度。最后对重建肝动脉冷保存肝移植大鼠行术后omega-3脂肪酸干预,以明确omega-3脂肪酸是否对冷保存缺血再灌注后的供肝具有保护作用。
第一部分基于精准外科理念的大鼠肝移植模型的建立和评价
目的：基于精准外科理念下,建立不重建肝动脉血供的成熟稳定的同品系大鼠原位肝移植模型。并对肝移植模型进行评价,探索二袖套法肝移植中最佳的肝上腔静脉显微缝合方法,最佳的静脉吻合用袖套管和胆管吻合支架管。
方法：健康成年雄性SD大鼠,体重250-300g。供受体体重相差≤20g。根据不同的显微缝合方法、袖套和支架类型,将移植组大鼠随机分为3组。第1组：新的肝上腔静脉显微缝合方法+单槽袖套+无损伤胆管支架；第2组：新的肝上腔静脉显微缝合方法+单槽袖套+粗制胆管支架；第3组：肝上腔静脉采用传统的双定点连续缝合法+多槽袖套+无损伤胆管支架。第4组为对照组,实施假手术,仅开腹游离肝周韧带和移植相关血管。记录移植组大鼠各个操作步骤时长、一周和一月的生存率。1月后全部大鼠均在乙醚麻醉下处死,测定其血液肝功能生化指标,并详细解剖肝上、肝下腔静脉、门静脉和肝门部胆管,以明确有无血管扭转、血栓形成和胆汁瘤、胆道梗阻等并发症。P<0.05视为有统计学意义。所有的统计数据使用PASW18.0来分析。
结果：新的肝上腔静脉显微缝合方法较传统的双定点连续缝合法速度更快(P<0.05),无术后肝上腔静脉吻合口出血的发生。单槽袖套明显缩短肝下腔静脉和门静脉的重建时间(P<0.05),同时术后血栓也明显减少。新的肝上腔静脉显微缝合方法结合单槽袖套较传统连续缝合与多槽袖套能明显缩短无肝期(P<0.05)。1月后移植组大鼠的肝功能各项指标已恢复正常,与对照组比较无明显差异(P>0.05)。新的胆管支架制作方法能够明显降低大鼠肝移植术后胆道并发症的发生,进而提高了大鼠的一周和一月存活率(P<0.05)
结论：新的肝上腔静脉显微缝合方法结合重新设计制作的单槽袖套较传统缝合方法和袖套能够以较小创伤,更简单、迅速、可靠地吻合肝上、肝下腔静脉和门静脉,显著地缩短无肝期,更符合精准外科“最小侵袭,最大脏器保护”的理念。同时,无损伤胆管支架明显较传统粗制支架减弱了对胆管内壁的损伤。因而显著地降低了移植术后胆道并发症的发生。简言之,基于精准外科理念的大鼠肝移植模型创伤更小,耗时更短,模型更稳定、可靠,并发症更少,能够获得更好的远期生存。
第二部分冷保存对大鼠肝移植术后肝功能和即时微循环的影响
目的：建立成熟稳定、重建肝动脉血供的大鼠冷保存肝移植模型。探讨不同冷保存时间对大鼠肝移植术后肝功能和即时微循环的影响,为进行冷保存肝移植后肝损伤的药物治疗研究奠定基础。
方法：实验动物同第一部分。采用第一部分的手术方法建立恢复肝动脉血供的冷保存大鼠原位肝移植模型。根据不同的冷保存时间,将大鼠随机分为3组：第1组,冷保存12h组：供肝使用4℃HTK液灌注并冷保存12h；第2组,冷保存24h组：供肝使用4℃HTK液灌注并冷保存24h；第3组：正常对照组,实施假手术,仅开腹游离肝周韧带和移植相关血管。记录无肝期和供、受体所用手术时间和术后7天受体的一般情况。移植组供肝复流后3、10、20min、对照组完成游离操作后3min分别使用激光散斑灌注成像法对肝脏表面微循环进行测定。移植组大鼠各取10只受体进行7天生存率的比较,其余受体分别于术后1天(24h)、3天、7天处死取血和肝组织标本,每一时间点取6只受体。对照组6只正常大鼠于术后7天处死取血。移植组和对照组检测血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase, AST)、,白蛋白(albumin, Alb)、碱性磷酸酶(alkaline phosphatase, ALP)和总胆红素(total bilirubin, TB)等肝功能指标。肝组织行HE染色,分别从门管区炎细胞浸润、局灶性坏死、胆管增殖、胆管损伤和间质纤维化等5方面对肝损伤程度进行半定量评分。所有大鼠在处死前均经腹壁行肝脏中叶的超声实时弹性成像。所有数据使用PASW18.0软件进行统计分析,P<0.05视为有统计学意义。
结果：
1.手术时间及术后一般情况：两冷保存移植组大鼠的供、受体手术操作时间无明显差别(P>0.05),无肝期均控制在14min以内。冷保存12h组受体术后恢复较快,麻醉清醒后即刻活动,进饮食水。两组存活受体均未见巩膜、双耳、爪子和尿液的黄染。
2.供肝复流后肝脏表面微循环变化：冷保存肝移植大鼠的肝脏表面微循环流量值明显低于正常对照组(P<0.05)。冷保存12h组肝脏表面微循环流量值明显高于冷保存24h(P<0.05)。
3.生存率：冷保存12h组明显高于冷保存24h组(P<0.05)。
4.血清肝功能指标变化情况：在术后1、3、7天,两冷保存肝移植组大鼠肝功能受损严重,各项指标均明显差于对照组(P<0.05),且同时间点冷保存24h组明显差于冷保存12h组(P<0.05)。随时间延续,两冷保存组ALT、AST呈逐渐下降趋势,ALP则呈先降低后升高,Alb和TB均呈逐渐增高趋势。
5.肝组织HE染色半定量评分：冷保存肝移植术后1天肝损伤已非常明显：肝细胞肿胀,近中央静脉的肝细胞空泡变性严重,肝血窦扩张充血,门管区炎细胞浸润,胆管内皮细胞肿胀、坏死。随时间延续,肝细胞肿胀、肝血窦扩张有所减轻,空泡变逐渐缓解,门管区炎细胞浸润和胆管增生较前明显,局灶性坏死有所减轻,胆管损伤有加重趋势,间质纤维化改变不明显。同时间点,冷保存12组在门管区炎细胞浸润、胆管损伤和局灶性坏死等方面的评分均低于冷保存24h组(P<0.05),冷保存12h组的胆管增殖评分则在术后3天和一周低于冷保存24h组(P<0.05)；两组间在间质纤维化上评分差别不显著(P>0.05)。
6.超声实时弹性成像：以对照组的肝脏弹性模量值为正常参考值,冷保存肝移植组在术后第1天肝脏弹性模量值即高于正常值(P<0.05),但随时间延续,各组的肝脏弹性模量值变化不大(P>0.05)。冷保存24h组肝脏弹性模量值与冷保存12h组差别不明显(P>0.05),但两冷保存组肝脏弹性模量值始终高于正常值(P<0.05)。
结论：
1.供肝冷保存会造成肝移植大鼠肝功能的严重损害,且对于胆管细胞的损伤要重于肝细胞。随着冷保存时间的延长,这种肝功能损害随之加重。
2.供肝冷保存会造成术后早期的肝脏微循环障碍,随着供肝冷保存时间延长,微循环损害加重。激光散斑灌注成像可以作为供肝冷保存缺血损伤严重程度的早期评价指标之一。
3.供肝冷保存12h的大鼠原位肝移植是作为研究冷保存肝移植后肝损伤的比较合适的动物模型。
4.超声实时弹性成像可以作为安全、无创地评价肝脏功能的一个参考指标。
第三部分Omega-3脂肪酸对大鼠冷保存供肝肝移植术后肝功能的影响
目的：明确omega-3旨肪酸对大鼠冷保存肝移植术后肝功能影响并对其作用机制作初步探讨。
方法：实验动物及手术方法同第二部分。根据供肝有无冷保存以及术后不同处理,将大鼠随机分为3组：其中第1组实施正常肝移植,第2、3组实施供肝冷保存12h肝移植。术后1-7天,第1、2组予每天生理盐水12ml/kg灌胃,第3组予每天omega-3鱼油脂肪乳12ml/kg灌胃。每组取10只受体进行4周生存率的比较。分别于术后3天、1周、2周、3周和4周处死受体,每一时间点取6只受体,取血和肝组织标本。血清肝功能指标检测和肝组织HE染色半定量评分方法同第二部分。术后各时间点,免疫组织化学方法检测肝组织中Ki-67的表达,Masson染色方法描述肝组织中纤维组织增生的程度。所有受体处死前经腹壁行肝脏超声实时弹性成像,方法同第二部分。另外,对术后3天、1周、2周标本分别使用酶联免疫吸附试验检测血清、实时荧光定量PCR技术检测肝组织中TNF-alpha, IL-6, IFN-gamma和TGF-betal的表达情况。所有数据使用PASW18.0软件进行统计分析,P<0.05视为有统计学意义。
结果：
1.生存率：正常肝移植组大鼠术后4周生存率高于冷保存组(P<0.05),两冷保存组间生存率无显著差异(P>0.05)
2.血清肝功能指标：术后2周内,第2组术后肝功能指标除Alb外明显差于第1、3组(P<0.05),第3组虽较第2组有所改善但仍未达第1组水平(P<0.05)。各组术后2-3周肝功能指标明显好转,并在3-4周趋于平缓。4周观察期末,第1、3肝功能基本恢复,但第2组肝功能指标仍未达正常值(P<0.05)。
3.肝组织HE染色半定量评分：同时间点,各组局灶性坏死和间质纤维化评分无差别。胆管损伤则第1组最弱,余两组无明显差别。门管区炎细胞浸润第2组最强,其余两组无明显差别。胆管增殖：术后第1、2周,第1组最弱,其余两组无显著差别；术后第3周第2组最强,其余两组无明显差别。
4.肝脏超声实时弹性成像：各组肝脏弹性模量值总体呈逐渐降低趋势,两冷保存组相同时间点无明显差别(P>0.05),但均较正常移植组为高(P<0.05)。
5.血清细胞因子水平：各组大鼠血清中TNF-alph、IFN-gama、IL-6和TGF-betal均呈先升后降趋势,术后1周为峰值。相同时间点,第2组术后血清中TNF-alpha、IFN-gamma、IL-6水平最高(P<0.05),第1、3组间无明显差别(P>0.05)。而同时间点各组间TGF-betal无明显差别(P>0.05)。
6.肝组织细胞因子mRNA表达情况：肝组织中TNF-alpha, IFN-gamma, IL-6和TGF-betal的mRNA表达强度与血清中的变化趋势类似。相同时间点上,TNF-alpha、IFN-gamma和IL-6各细胞因子mRNA的表达,第2组明显高于其余两组(P<0.05),且1、3组间则无明显差别(P＞0.05), TGF-betal则在各组间无明显差别(P>0.05)。
7.肝细胞增殖情况：肝组织Ki-67结果显示,各组术后肝细胞均有不同程度增殖,1周内增殖活跃,其后逐渐减弱。第1、3组间肝细胞增殖程度无明显差别(P>0.05)。前2周内,第2组肝细胞增殖程度明显强于第1、3组(P<0.05)；第3周,第2组肝细胞增殖程度仍强于第3组(P<0.05),第4周各组间差别不显著(P>0.05)。
8.肝组织中胶原纤维的检测：Masson染色评分显示,各组术后肝组织中胶原纤维含量呈先升后降趋势,以第2、3周为高。相同时间点,各组胶原纤维含量无差别(P>0.05)。
结论：
1.与正常移植相比,供肝冷保存12h会显著降低肝移植大鼠术后4周生存率,给予(>mega-3脂肪酸并不能提高其4周生存率。
2.供肝冷保存12h肝移植后给予omega-3脂肪酸能在一定程度上改善术后肝功能生化指标,但不能改变其恢复的进程。
3. Omega-3脂肪酸通过降低肝组织和血清中TNF-alpha、IFN-gamma和IL-6等促炎细胞因子的表达从而起到抑制炎症反应,缓解缺血再灌注损伤,改善肝功能的作用。Omega-3旨肪酸的使用不增加肝脏纤维化的风险,但对于改善冷保存造成的胆管损伤和胆管增殖效果不明显。
Backgroud
Ischemia-reperfusion injury (IRI) caused by graft cryopreservation has triggered a series of problems such as poor early graft function, primary non function and biliary complications. Therefore, IRI has been the hot topics in the study of liver transplantation. Nowdays, the mechanism of IRI is still unclear. The consensus is that the innate immune system will be activated after reperfusion of the graft. And then local proinflammatory response will be appeared. The ischemic damage to the graft mainly show metabolic disorder and parenchyma cell death which lead to ligands activation and mitochondrial release oxygen free radicals. After the reperfusion, the adverse factors above with the involvement of Kupffer cells, NK cells, dendritic cells, T cells, neutrophils and other non parenchymal cells will activate the host's innate immune system, further aggravate the inflammatory response. The involvement of immune cells in peripheral blood can make the immune activation-inflammatory cascade a continued progress. In short, the IRI is a local proinflammatory response which is mediated by host's innate immune system and finnaly lead to damage of the liver parenchymal cells.
It has been reported that omega-3polyunsaturated fatty acids (n-3PUFA) could improve the prognosis of parenteral nutrition correlation of liver disease, to reduce the production of inflammation medium and adhesion molecules to play a role in anti-inflammatory, to prevent IRI and circulation failure in mice with fatty liver, to alleviate chemically induced acute hepatitis and liver cell damage caused by obstructive jaundice in rats and so on. Recently, it has been proved that n-3PUFA could also attenuate IRI and promote hepatic regeneration following partial liver resection in normal or fatty liver.
Although there is report about the liver-protecting effects of n-3PUFA for patients after liver transplantation, the influence of n-3PUFA to the IRI which caused by graft cryopreservation and the exact mechanism is still not very clear. As we know, even with the rapid development of medicine, the IRI of donor liver caused by cryopreservation is still completely unavoidable. Hence, it is clinically meaningful to maximumly reduce IRI and improve liver function for patients after liver transplantation. As mentioned above, the IRI is a local proinflammatory response mediated by host's innate immune system. In this pathophysiological process, the parenchymal cells release a variety of inflammatory mediators such as TNF alpha, IL-6, IL-1beta and IFN-gamma after the activation of host's inane immune. This is the key to maintain the inflammatory cascade. It has been proved that omega-3fatty acids could play an anti-inflammatory effect in mouse with chemical-induced hepatitis. In this study, firstly we established an orthotopic liver transplantation model in rats without the reconstruction of hepatic artery. This modal was performed according to Kamada's two cuff method under the concept of precision surgery. We found the best method for reconstruction of veins and bile duct for ROLT. Secondly, we established an cryopreserved orthotopic liver transplantation model in rats and explored the effect of graft cryopreservation to liver function and microcirculation. In the meantime, we found out the exact length of cropreservation time which was suitable for animal experimental study. At last, we treated the rats wihich subjected to cryopreserved liver transplantation with n-3PUFA. And then the treatment effect of n-3PUFA to the rats was investigated. We try to prove that n-3PUFA could inhibit liver inflammation, improve liver function and further increase the long-term survival of the recipients.
Part I Establishment and Evaluation of Liver Transplantation Model in Rats Under the Concept of Precision Surgery
OBJECTIVE:Establishment of rat orthotopic liver transplantation (ROLT) model under the concept of precision surgery. The hepatic artery was not reconstructed. The modal was evaluated for exploring the best microsuture method for reconstruction of suprahepatic vena cava (SHVC), the best cuff and stent for reconstruction of veins and bile ducts.
METHODS:
Male SD rats weighing250to300g were used as donors and recipients. The weight difference was equal or lesser than20g. According to different microsuture methods for the SHVC and different types of cuffs and stents, three ROLT groups were created to compare the operation times and prognoses. Group1:new microsuture method for SHVC+single-groove cuff+blade-cut stents. Group2:new microsuture method for SHVC+single-groove cuff+scissors-cut stents. Group3: conventional continuous suture method for SHVC+multi-groove cuff+blade-cut stents. Sham operations were performed as controls in group4. The time expenditures with each step were compared among the transplantation groups. The one week-and one month-survival rates of the transplantation groups were recorded and compared. All of the animals that survived one month were sacrificed and the biochemical parameters were tested. The SHVC, infrahepatic vena cava (IHVC), portal vein (PV) and hilar bile duct was detailedly dissected to make sure if there are torsion of the vessels, thrombosis and biliary complications. P＜0.05was considered to indicate statistical significance. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA).
RESULTS:
Our new microsuture method was faster than the conventional continuous suture method for SHVC anastomosis (P＜0.05). There was no bleeding of the anastomotic stoma of the SHVC. The use of a single-groove cuff for reconstruction of the portal vein and the infrahepatic vena cava shortened the anastomotic time (P＜0.05) and significantly reduce the postoperative thrombosis. The new microsuture method companied with the single-groove cuff shortened the anhepatic time when compared to the conventional continuous suture method with the multi-groove cuff (P＜0.05). The biochemical parameters reached to normal level at one month after the transplantation. The The use of blade-cut stents resulted in fewer biliary complications and better survival over the short and long terms (P＜0.05).
CONCLUSIONS:
Our new microsuture method and the single-groove cuffs proved to be a precise method for venous reconstruction which shortened the anhepatic time and the anastomotic time significantly. These new methods are more in line with the concept "minimizing surgical invasiveness and maximizing organ protection" of precision surgery. The blade-cut stents apparently decreased the incidence of biliary complications by reducing the injury to the inner wall of the bile duct. In summary, with this precise microsuture method and delicate cuffs and stents, excellent long-term survival can be achieved easily and stably for ROLT.
Part II The Effect of Graft Cropreservation to Postoperative Liver Function and Instant Microcirculation For Rats Subjected to Liver Transplantation
OBJECTIVE:To establish a mature and stable cryopreserved liver transplantation modal in rats with the reconstruction of hepatic artery. To explore the effect of different cryopreservation time to postoperative liver function and instant microcirculation. To lay the foundation for drug treatment of damage caused by cryopreserved liver transplantation.
METHODS:
Animals were similar to Part Ⅰ. To establish cryopreserved rat liver transplantation modal with the reconstruction of hepatic artery. The methods mention in Part Ⅰ was applied. According to different cryopreservation time, three ROLT groups were created. In group1, donor liver was perfused and preserved for12h by4℃HTK solution (P12h group) and in group2for24h by4℃HTK solution (P24h group). In group3, sham operations were performed as controls. Anhepatic time, the time used for donor and recipient and general conditions on the7th day after the operation were recorded. For the recipients, the laser speckle perfusion images were performed3,10and20min after the reperfusion of the donor liver. Ten recipients were used for comparison of7day-survival rate. Blood and liver specimens were gained on postoperative day1(24h), day3and day7(POD1,3,7) when the recipients were sacrificed. There were six recipients for each point. The six recipients in the control group were also sacrificed on postoperative day7and blood specimens were collected. Biochemical parameters included alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (Alb), alkaline phosphatase (ALP) and total bilirubin (TB) were measured for all the blood specimens. Hematoxylin-eosin staining was done for the liver tissue specimens. The liver tissue were semi-quantitatively scored from the following items:portal inflammation, focal necrosis, ductular proliferation, bile duct damage and septal fibrosis. The real-time elastography was performed transabdominally for all the survivors before they were sacrificed. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.
RESULTS:
1. Time expenditure and general conditions. There were no significant difference of the time used in donors and recipients between P12h and P24h groups. The anhepatic time were controlled within14min. The animals in P12h group recovered faster after the operation. There were no icteric sclera, yellow claws and urine for all the survivors in the P12h and P24h groups.
2. The microcirculation changes of the graft after the reperfusion. The flux value of the graft liver in the cryopreserved groups were significantly lower than the control. While the flux value in the P12h group was higher than that in the P24h group (P＜0.05).
3. The survival rate in the P12h group was significantly higher than that in the P24h group (P＜0.05).
4. The biochemical parameters of the serum. The liver function in the cryopreservation groups were heavily damaged when compared with the control group. On POD1,3and7, all the parameters were all worse than that in the control group obviously (P＜0.05). In the cryopreservation groups, ALT and AST decreased over time. While ALP increased at first and then decreased. The Alb and TB value increased over time.
5. The semi-quantitative scores of the liver tissue. The liver damage was very obvious even on POD1for the recipients in P12h and P24h groups. Following changes were found in HE staining:Swelling and vacuoles degeneration of the liver cells, hepatic sinusoid congestion, inflammation cells infiltration of the portal tract, bile duct endothelial cells swollen and necrosis. As an extension of time, swelling and vacuoles degeneration of the liver cells and hepatic sinusoid congestion alleviated. Portal inflammation and ductular proliferation were obvious than POD1. Focal necrosis alleviated but bile duct damage aggravated over time. The septal fibrosis was not obvious. At the same points, the P12h group has lower scores than the P24h group with respect to portal inflammation, ductular proliferation, focal necrosis and bile duct damage (P＜0.05). But there were no significant difference of the scores for septal fibrosis (P＞0.05).
6. The real-time elastography of recipients'liver. The mean elastic ratio of the liver in the control group was setted as normal value. The elastic ratios in the cryopreservation groups were higher than normal value just on POD1and didn't change a lot over time. The elastic ratios of liver in the P12h group were similar to that in the P24h group at the same point (P＞0.05). However, the elastic ratios of the cryopreservation groups were always higher than normal value during the observation time (P＜0.05).
CONCLUSIONS:
1. Cryopreservation of the donor liver caused serious liver function damage. The damage of bile duct cells were heavier than liver cells. The longer the donor liver was cryopreserved, the heavier the liver was impaired.
2. Cryopreservation of the donor liver could cause early postoperative damage to hepatic microcirculation. The longer the donor liver was cryopreserved, the heavier the microcirculation was impaired. Laser speckle perfusion imaging can be used as an index for evaluating of early ischemic injury caused by the cryopreservation of the donor liver
3. Twelve hours cryopreservation of donor liver was suitable for study of liver impairment after cryopreserved liver transplantation.
4. The real-time elastography was a safe and noninvasive method for evaluation of liver function.
Part Ⅲ The Effects of Omega-3Fatty Acids to Postoperative Liver Function for Rats Subjected to Cryopreserved Liver Transplantation
OBJECTIVE:To investigate the effects of omega-3fatty acids to postoperative liver function for rats subjected to cryopreserved liver transplantation and preliminarily discuss the mechanism.
METHODS:
According to different time of graft preservation and postoperative administration, rats were randomly divided into three transplantation groups. Our new methods for ROLT which was based on precision liver surgery was used here. In group1, normal liver transplantation was performed. In group2and3, donor liver was perfused and preserved for12h by4℃HTK solution and then transplanted. During postoperative day (POD)1to7, the recipients in group land2received saline solution (12ml/kg/day, via orogastric gavage) while recipients in group3received omega-3fish oil fat emulsion (12ml/kg/day, via orogastric gavage). Ten recipients were used in each group for comparison of four week survival rate. On POD3,7,14,21and28, all survivors in each group were sacrificed, blood and liver specimens were collected. There were six recipients for each point. Biochemical parameters and semi-quantitative score of the hematoxylin-eosin staining of liver tissue were similar to that in Part Ⅰ. At various points after the operation, Ki-67level of the liver tissue was measured by immunohistochemical analysis. Meanwhile, Masson staining was used to describe the degree of fibrosis in liver tissue. The real-time elastography was performed transabdominally for all the survivors just as Part Ⅱ. In addition, on POD3,14and21, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal in the serum was measured by ELISA kit. The expression of mRNA of the same cytokines in the live tissue was measured by real time quantitative PCR in the same point. All of the analyses were performed using SPSS software, version18(SPSS, Inc., Chicago, IL, USA). P＜0.05was considered to indicate statistical significance.
RESULTS:
1. Suvival rate. The normal liver transplantation group was higher than that in the cryopreserved group (P＜0.05). There were no significant difference between group2and3which cryopreserved liver transplantation were performed (P＞0.05).
2. The biochemical parameters of the serum. The biochemical parameters except Alb in group2was significantly worse than that in group1and3(P＜0.05). The postoperative liver function in group3was improved but still worse than that in group1(P＜0.05). The biochemical parameters in each group improved during POD14-POD21and flatten out during POD21-POD28. At the end of POD28, the animals in group1and3got nearly normal liver function but the biochemical parameters in group2were still not better than normal value.
3. The semi-quantitative scores of the liver tissue. At the same time point, there were no difference for septal fibrosis and focal necrosis among the three groups. For the bile duct damage, group1was the lightest and there were no difference between the other two groups. For portal inflammation, group2was the worst and there were no difference between the other two groups. For ductular proliferation, on POD7and POD14, group1was the lightest and there were no difference between the other two groups. On POD21, group2was the worst and there were no difference between the other two groups.
4. The real-time elastography of liver. The elastic ratio of the liver decreased after the operation. There was no difference between group2and3at the same time point(P＞0.05). But the elastic ratio of group2and3were higher than that in group1(P＜0.05).
5. The levels of cytokines in the serum. On POD3,7and14, the levels of TNF-alpha, IL-6, IFN-gamma and TGF-betal increased first and then decreased. The levels of cytokines were the highest on POD7. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.
6. The expression of mRNA of the cytokines in the live tissue. The changes of the mRNA of the cytokines in liver tissue was similar to that in the serum.The expression of mRNA of the cytokines were also first increased and then decreased. At the same time point, the levels of TNF-alpha, IL-6and IFN-gamma in group2was the highest and there was no difference between the other two groups. The TGF-betal showed no difference among the three groups.
7. The proliferation of liver cells. The scores of Ki-67levels in the liver tissue showed that the liver cells proliferated most obviously during one week among the three groups after the operation. Then the proliferatation gradually weakened. There were no difference of the scores of Ki-67between group land3(P＞0.05). During the first two weeks, the scores of Ki-67in group2were higher than that in group1and3(P＜0.05). On POD21, the scores of Ki-67in group2were still higher than that in group3(P＜0.05). And there were no difference among the three groups on POD28.
8. Detection of collagen fibers in the liver tissue. The scores of Masson staining showed that the content of collagen fibers in the liver tissue increased first and then decreased. The scores on POD14and POD21were more higher than other time point. But there were no difference between the three groups at the same time points.
CONCLUSIONS:
1. When compared with normal liver transportation,12hours cryopreservation of the donor liver decreased the four week survival rate of the recipients whether or not usage of omega-3fatty acids.
2. Omega-3fatty acids could improve the postoperative liver function in some extent for rats subjected to cryopreservation transplantation. However, it couldn't change the restore speed of the liver function.
3. Omega-3fatty acids probably play aniti-inflammatory role through the inhibition of the synthesis of inflammatory cytokines such as TNF-alpha, IL-6and IFN-gamma in the liver tissue. Then the ischemic reperfusion injury alleviated and the liver function got better. Omega-3fatty acids didn't increase the risk of fibrosis of the liver. But it couldn't improve the ductular proliferation and bile duct damage caused by cryopreservation of the donor liver, either.
引文
1. Wertheim JA, Petrowsky H, Saab S, Kupiec-Weglinski JW, Busuttil RW. Major challenges limiting liver transplantation in the United States. Am J Transplant.2011;11(9):1773-1784.
2. Adam R, Hoti E. Liver transplantation:the current situation. Semin Liver Dis. 2009;29(1):3-18.
3. Merion RM. Current Status and Future of Liver Transplantation. Seminars in Liver Disease.2010;30(4):411-421.
4. Lentsch AB, Kato A, Yoshidome H, McMasters KM, Edwards MJ. Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury. Hepatology.2000;32(2):169-173.
5. Zhai Y, Busuttil RW, Kupiec-Weglinski JW. Liver ischemia and reperfusion injury:new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation. Am J Transplant.2011;11(8):1563-1569.
6. Zhai Y, Petrowsky H, Hong JC, Busuttil RW, Kupiec-Weglinski JW. Ischemia-reperfusion injury, in liver transplantation-from bench to bedside. Nat Rev Gastro Hepat.2013;10(2):79-89.
7. Dyerberg J, Bang HO, Stoffersen E, Moncada S, Vane JR. Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? Lancet. 1978;2(8081):117-119.
8. Daviglus ML, Stamler J, Orencia AJ, et al. Fish consumption and the 30-year risk of fatal myocardial infarction. The New England journal of medicine. 1997;336(15):1046-1053.
9. Hu FB, Bronner L, Willett WC, et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA.2002;287(14):1815-1821.
10. Krauss RM, Eckel RH, Howard B, et al. AHA Dietary Guidelines:revision 2000:A statement for healthcare professionals from the Nutrition Committee of the American Heart Association. Circulation.2000;102(18):2284-2299.
11. Yokoyama M, Origasa H, Matsuzaki M, et al. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS):a randomised open-label, blinded endpoint analysis. Lancet. 2007;369(9567):1090-1098.
12. Spadaro L, Magliocco O, Spampinato D, et al. Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease. Dig Liver Dis. 2008;40(3):194-199.
13. de Meijer VE, Gura KM, Le HD, Meisel JA, Puder M. Fish oil-based lipid emulsions prevent and reverse parenteral nutrition-associated liver disease:the Boston experience. JP EN J Par enter Enteral Nutr.2009;33(5):541-547.
14. Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury. Ann Surg. 2009;250(3):395-402.
15. Gura KM, Duggan CP, Collier SB, et al. Reversal of parenteral nutrition-associated liver disease in two infants with short bowel syndrome using parenteral fish oil:implications for future management. Pediatrics. 2006;118(1):e197-201.
16. Fallon EM, Le HD, Puder M. Prevention of parenteral nutrition-associated liver disease:role of omega-3 fish oil. Curr Opin Organ Transplant. 2010;15(3):334-340.
17. Calder PC. N-3 polyunsaturated fatty acids and inflammation:from molecular biology to the clinic. Lipids.2003;38(4):343-352.
18. Schmocker C, Weylandt KH, Kahlke L, et al. Omega-3 fatty acids alleviate chemically induced acute hepatitis by suppression of cytokines. Hepatology. 2007;45(4):864-869.
19. Hao W, Wong OY, Liu X, Lee P, Chen Y, Wong KK. omega-3 fatty acids suppress inflammatory cytokine production by macrophages and hepatocytes. JPediatr Surg.2010;45(12):2412-2418.
20. Clavien PA, El-Badry AM, Moritz W, Contaldo C, Tian YH, Graf R. Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega-3 fatty acids. Hepatology.2007;45(4):855-863.
21. Lee S, Kim S, Le HD, et al. Reduction of hepatocellular injury after common bile duct ligation using omega-3 fatty acids. J Pediatr Surg. 2008;43(11):2010-2015.
22. Gonzalez-Periz A, Planaguma A, Gronert K, et al. Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA. Faseb J.2006;20(14):2537-2539.
23. Song BJ, Moon KH, Olsson NU, Salem N, Jr. Prevention of alcoholic fatty liver and mitochondrial dysfunction in the rat by long-chain polyunsaturated fatty acids. J Hepatol. 2008;49(2):262-273.
24. Coelho AM, Machado MC, Takahashi HK, et al. Ischemic preconditioning-like effect of polyunsaturated fatty acid-rich diet on hepatic ischemia/reperfusion injury. J Gastrointest Surg.2011;15(10):1679-1688.
25. Marsman HA, Heger M, Kloek JJ, Nienhuis SL, ten Kate FJ, van Gulik TM. Omega-3 fatty acids reduce hepatic steatosis and consequently attenuate ischemia-reperfusion injury following partial hepatectomy in rats. Dig Liver Dis.2011;43(12):984-990.
26. Qiu YD, Wang S, Yang Y, Yan XP. Omega-3 polyunsaturated fatty acids promote liver regeneration after 90% hepatectomy in rats. World J Gastroenterol.2012;18(25):3288-3295.
27. Marsman HA, de Graaf W, Heger M, et al. Hepatic regeneration and functional recovery following partial liver resection in an experimental model of hepatic steatosis treated with omega-3 fatty acids. Br J Surg. 2013;100(5):674-683.
28. Chen CC, Ho CY, Chaung HC, et al. Fish omega-3 fatty acids induce liver fibrosis in the treatment of bile duct-ligated rats. Dig Dis Sci. 2013;58(2):440-447.
29. Zhu XH, Wu YF, Qiu YD, Jiang CP, Ding YT. Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation. World J Gastroenterol. 2012;18(42):6141-6147.
30. Zhu X, Wu Y, Qiu Y, Jiang C, Ding Y. Effects of omega-3 fish oil lipid emulsion combined with parenteral nutrition on patients undergoing liver transplantation. JPEN J Par enter Enteral Nutr.2013;37(1):68-74.
31. Lee S, Charters AC, Chandler JG, Orloff MJ. A technique for orthotopic liver transplantation in the rat. Transplantation.1973;16(6):664-669.
32. Lee S, Charters AC,3rd, Orloff MJ. Simplified technic for orthotopic liver transplantation in the rat. Am J Surg.1975; 130(1):38-40.
33. Kamada N, Calne RY. Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation. 1979;28(1):47-50.
34. Zimmermann FA, Butcher GW, Davies HS, Brons G, Kamada N, Turel O. Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic responses to fully allogeneic liver grafts. Transplant Proc. 1979;11(1):571-577.
35. Miyata M, Fischer JH, Fuhs M, Isselhard W, Kasai Y. A simple method for orthotopic liver transplantation in the rat. Cuff technique for three vascular anastomoses. Transplantation.1980;30(5):335-338.
36. Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery.1983;93(1 Pt 1):64-69.
37. Settaf A, Gugenheim J, Houssin D, Bismuth H. Cuff technique for orthotopic liver transplantation in the rat. A simplified method for the suprahepatic vena cava anastomosis. Transplantation.1986;42(3):330-331.
38. Tsuchimoto S, Kusumoto K, Nakajima Y, et al. Orthotopic liver transplantation in the rat. A simplified technique using the cuff method for suprahepatic vena cava anastomosis. Transplantation.1988;45(6):1153-1155.
39. Knoop M, Hutchinson IV. Suprahepatic vena cava cuffs in rat liver transplantation--a simplified method? Transplantation.1989;47(3):576.
40. Xu HS, Rosenlof LK, Selby JB, Jones RS. A simple method for bile duct anastomosis and interval bile collection in the liver-transplanted rat. J Surg Res.1992;53(5):520-523.
41. Kashfi A, Mehrabi A, Pahlavan P, et al. A review of various techniques of orthotopic liver transplantation in the rat. Transpl P.2005;37(1):185-188.
42. Harihara Y, Sanjo K, Idezuki Y. A modified cuff technique for suprahepatic vena cava anastomosis in rat liver transplantation. Transplantation. 1992;53(3):707-709.
43. Tan F, Chen Z, Zhao Y, Liang T, Li J, Wei J. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation. Microsurgery.2005;25(7):556-560.
44. Oldani G, Maestri M, Gaspari A, et al. A Novel Technique for Rat Liver Transplantation Using Quick Linker System:A Preliminary Result. J Surg Res. 2008;149(2):303-309.
45. Ariyakhagorn V, Schmitz V, Olschewski P, et al. Improvement of Microsurgical Techniques in Orthotopic Rat Liver Transplantation. J Surg Res. 2009;153(2):332-339.
46. Delriviere L, Gibbs P, Kobayashi E, Goto S, Kamada N, Gianello P. Detailed modified technique for safer harvesting and preparation of liver graft in the rat. Microsurgery.1996;17(12):690-696.
47. Delriviere L, Gibbs P, Kobayashi E, Goto S, Kamada N, Gianello P. Technical details for safer venous and biliary anastomoses for liver transplantation in the rat. Microsurgery.1998; 18(1):12-18.
48. Hori T, Nguyen JH, Zhao XD, et al. Comprehensive and innovative techniques for liver transplantation in rats:A surgical guide. World J Gastroentero. 2010;16(25):3120-3132.
49.董家鸿,黄志强.精准肝切除——21世纪肝脏外科新理念.中华外群杂志.2009；47(21)：1601-1605.
50. Dong J, Yang S, Zeng J, et al. Precision in liver surgery. Semin Liver Dis. 2013;33(3):189-203.
51. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving Bioscience Research Reporting:The ARRIVE Guidelines for Reporting Animal Research. Plos Biology.2010;8(6).
52.李文滨,李国林,褚忠华,et al单人裸眼建立大鼠原位肝移植模型.中华实验外科杂志.2011；28(11)：1994-1996.
53. Holzen JP, Palmes D, Langer M, Spiegel HU. Microsurgical training curriculum for learning kidney and liver transplantation in the rat. Microsurgery.2005;25(8):614-623.
54. Ozkan O, Ozgentas HE. Open guide suture technique for safe micro vascular anastomosis. Ann Plast Surg.2005;55(3):289-291.
55.宇汝胜.大鼠原位肝移植模型的手术操作技巧探讨.肝胆胰外科杂志.2008；20(1)：7-9.
56.吕和平,薛涛,张启瑜,廖毅,施红旗.单人直视下大鼠减体积肝移植模型的技术改进.中华实验外科杂志.2011；28(2)：208.
57. Sanchez-Patan F, Blanco R, Aller MA, Anchuelo R, Roman FS, Arias J. End-to-side portacaval shunt:a simplified technique. J Invest Surg. 2007;20(2):135-138.
58. Marni A, Ferrero ME, Forti D. End-to-side anastomosis in heterotopic rat organ transplantation. Microsurgery.1996;17(1):21-24.
59. Spiegel HU, Palmes D. Surgical techniques of orthotopic rat liver transplantation. J Invest Surg.1998;11 (2):83-96.
60. Spiegel HU, Schleimer K, Kranz D, Diller R. Orthotopic rat liver transplantation and bile duct reconstruction by a splint technique. Eur Surg Res.1997;29(6):421-428.
61. Kamada N, Calne RY, Wight DG, Lines JG. Orthotopic rat liver transplantation after long-term preservation by continuous perfusion with fluorocarbon emulsion. Transplantation.1980;30(1):43-48.
62. Kitakado Y, Tanaka K, Asonuma K, et al. A new bioabsorbable material for rat orthotopic liver transplantation. Eur Surg Res.1991;23(5-6):366-370.
63. Ma Y, Wang GD, Guo ZY, Guo ZG, He XS, Chen GH. Surgical techniques of arterialized orthotopic liver transplantation in rats. Chin Med J (Engl). 2007;120(21):1914-1917.
64. Li N, Cai CJ, Wu YR, Lu MQ. A technique of recipient portal venoplasty and cuff insertion for portal revascularization in orthotopic rat liver transplantation. J Surg Res.2012;176(1):317-320.
65.杜晓宏,王广义,孟伟,赵军,张舵.改进的二袖套法大鼠原位肝移植术.中华器官移植杂志.2003；24(5)：316-317.
66.马毅,何晓顺,陈规划.大鼠原位肝移植模型的手术技巧及并发症的预防.中华显微外科杂志.2003；26(1)：45-47.
67.张莹,别平,石承先,张玉君,吴乔,李昆.“二袖套法”制备大鼠原位肝移植模型的方法改进.消化外科.2005；4(6)：444-446.
68. Lee SH, Fisher B. Portacaval shunt in the rat. Surgery.1961;50:668-672.
69. Tomita Y, Zhang QW, Yoshikawa M, Uchida T, Nomoto K, Yasui H. Improved technique of heterotopic cervical heart transplantation in mice. Transplantation.1997;64(11):1598-1601.
70. Lee S. An improved technique of renal transplantation in the rat. Surgery. 1967;61(5):771-773.
71.陈忠华,夏穗生.改进的大鼠原位肝移植术.中华器官移植杂志.1984；05(2)：50-53.
72.曾琪华,孙君泓,吴孟超.三袖套法大鼠原位肝移植.中华器官移植杂志.1989；10(4)：159-161.
73.马毅,何晓顺,陈规划,陈细桃,黄洁夫.重建肝动脉血供大鼠原位肝移植模型的术式探讨.中华实验外科杂志.2004；21(1)：110-111.
74.陈志宇,张玉君,王槐志,董家鸿.改良法重建肝动脉血供的大鼠原位肝移植模型.中华实验外科杂志.2005；22(8)：1016-1017.
75. Wang H, Li C, Hu J, et al. Effect of different suprahepatic vena cava reconstruction methods on the hemodynamics of rats after liver transplantation. PLoS One.2013;8(9):e72695.
76. de Vera ME, Lopez-Solis R, Dvorchik I, et al. Liver transplantation using donation after cardiac death donors:long-term follow-up from a single center. Am J Transplant.2009;9(4):773-781.
77. Mathur AK, Heimbach J, Steffick DE, Sonnenday CJ, Goodrich NP, Merion RM. Donation after Cardiac Death Liver Transplantation:Predictors of Outcome. Am J Transplant.2010; 10(11):2512-2519.
78. Monbaliu D, Pirenne J, Talbot D. Liver transplantation using Donation after Cardiac Death donors. JHepatol.2012;56(2):474-485.
79.中华医学会器官移植学分会.中国心脏死亡器官捐献工作指南(第2版).中华器官移植杂志.2011；32(12)：756-758.
80.霍枫,汪邵平,李鹏,et al心脏死亡器官捐献获取流程探讨.中国普外基础与临床杂志.2012；19(5)：468-472.
81.郑树森,屠振华.心脏死亡器官捐献肝移植.中国普外基础与临床杂志.2012；19(5)：461-463.
82. Ikeda T, Yanaga K, Kishikawa K, Kakizoe S, Shimada M, Sugimachi K. Ischemic injury in liver transplantation:difference in injury sites between warm and cold ischemia in rats. Hepatology.1992;16(2):454-461.
83. Imamura H, Rocheleau B, Cote J, Huet PM. Long-term consequence of rat orthotopic liver transplantation with and without hepatic arterial reconstruction: a clinical, pathological, and hemodynamic study. Hepatology. 1997;26(1):198-205.
84. Ishak K, Baptista A, Bianchi L, et al. Histological Grading and Staging of Chronic Hepatitis. J Hepatol.1995;22(6):696-699.
85. Steering Committee of the Istanbul S. Organ trafficking and transplant tourism and commercialism:the Declaration of Istanbul. Lancet.2008;372(9632):5-6.
86. Cameron AM, Ghobrial RM, Yersiz H, et al. Optimal utilization of donor grafts with extended criteria:a single-center experience in over 1000 liver transplants. Ann Surg.2006;243(6):748-753; discussion 753-745.
87. Durand F, Renz JF, Alkofer B, et al. Report of the Paris consensus meeting on expanded criteria donors in liver transplantation. Liver Transpl. 2008;14(12):1694-1707.
88. McCormack L, Petrowsky H, Jochum W, Mullhaupt B, Weber M, Clavien PA. Use of severely steatotic grafts in liver transplantation:a matched case-control study. Ann Surg.2007;246(6):940-946; discussion 946-948.
89. Matsuno N, Kobayashi E. Challenges in machine perfusion preservation for liver grafts from donation after circulatory death. Transplantation research. 2013;2(1):19.
90. Olthoff KM, Kulik L, Samstein B, et al. Validation of a current definition of early allograft dysfunction in liver transplant recipients and analysis of risk factors. Liver Transpl.2010;16(8):943-949.
91. Miyagawa Y, Imamura H, Soeda J, et al. Fate of hepatocyte and sinusoidal lining cell function and kinetics after extended cold preservation and transplantation of the rat liver. Liver Transpl.2002;8(4):370-381.
92. Yu WM, Coddington D, Bitter-Suermann H. Rat liver preservation. I. The components of UW solution that are essential to its success. Transplantation. 1990;49(6):1060-1066.
93. Gao W, Lemasters JJ, Thurman RG. Development of a new method for hepatic rearterialization in rat orthotopic liver transplantation. Reduction of liver injury and improvement of surgical outcome by arterialization. Transplantation.1993;56(1):19-24.
94. de Groot H, Rauen U. Ischemia-reperfusion injury:processes in pathogenetic networks:a review. Transplant Proc.2007;39(2):481-484.
95. Massip-Salcedo M, Rosello-Catafau J, Prieto J, Avila MA, Peralta C. The response of the hepatocyte to ischemia. Liver Int.2007;27(1):6-16.
96. Bilzer M, Gerbes AL. Preservation injury of the liver:mechanisms and novel therapeutic strategies.J Hepatol. 2000;32(3):508-515.
97.黄志强.胆道的解剖生理学与肝移植后胆道并发症.中华办科杂志.2006；44(5).
98.董家鸿.肝移植的胆道并发症.腹部外科.2008；21(3)：175-177.
99. Sanchez-Urdazpal L, Gores GJ, Ward EM, et al. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology. 1992;16(1):49-53.
100. Noack K, Bronk SF, Kato A, Gores GJ. The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation. Transplantation. 1993;56(3):495-500.
101. Huet PM, Nagaoka MR, Desbiens G, et al. Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver. Hepatology.2004;39(4):1110-1119.
102. McGuire PG, Howdieshell TR. The importance of engraftment in flap revascularization:confirmation by laser speckle perfusion imaging. J Surg Res. 2010;164(1):e201-212.
103. Friedrich-Rust M, Ong MF, Herrmann E, et al. Real-time elastography for noninvasive assessment of liver fibrosis in chronic viral hepatitis. AJR Am J Roentgenol.2007;188(3):758-764.
104. Friedrich-Rust M, Schwarz A, Ong M, et al. Real-time tissue elastography versus FibroScan for noninvasive assessment of liver fibrosis in chronic liver disease. Ultraschall Med.2009;30(5):478-484.
105. Ochi H, Hirooka M, Koizumi Y, et al. Real-time tissue elastography for evaluation of hepatic fibrosis and portal hypertension in nonalcoholic fatty liver diseases. Hepatology.2012;56(4):1271-1278.
106. Millonig G, Friedrich S, Adolf S, et al. Liver stiffness is directly influenced by central venous pressure. J Hepatol.2010;52(2):206-210.
107. Babcock T, Helton WS, Espat NJ. Eicosapentaenoic acid (EPA):an antiinflammatory omega-3 fat with potential clinical applications. Nutrition. 2000；16(11-12):1116-1118.
108. Gaiva MH, Couto RC, Oyama LM, et al. Diets rich in polyunsaturated fatty acids:effect on hepatic metabolism in rats. Nutrition.2003; 19(2):144-149.
109. Qi K, Fan C, Jiang J, et al. Omega-3 fatty acid containing diets decrease plasma triglyceride concentrations in mice by reducing endogenous triglyceride synthesis and enhancing the blood clearance of triglyceride-rich particles. Clin Nutr.2008;27(3):424-430.
110. Levy JR, Clore JN, Stevens W. Dietary n-3 polyunsaturated fatty acids decrease hepatic triglycerides in Fischer 344 rats. Hepatology. 2004;39(3):608-616.
111. Valenzuela R, Espinosa A, Gonzalez-Manan D, et al. N-3 long-chain polyunsaturated fatty acid supplementation significantly reduces liver oxidative stress in high fat induced steatosis. PLoS One.2012;7(10):e46400.
112. Alwayn IP, Andersson C, Zauscher B, Gura K, Nose V, Puder M. Omega-3 fatty acids improve hepatic steatosis in a murine model:potential implications for the marginal steatotic liver donor. Transplantation.2005;79(5):606-608.
113. Rollins MD, Scaife ER, Jackson WD, Meyers RL, Mulroy CW, Book LS. Elimination of soybean lipid emulsion in parenteral nutrition and supplementation with enteral fish oil improve cholestasis in infants with short bowel syndrome. Nutr Clin Pract.2010;25(2):199-204.
114. Jurewitsch B, Gardiner G, Naccarato M, Jeejeebhoy KN. Omega-3-enriched lipid emulsion for liver salvage in parenteral nutrition-induced cholestasis in the adult patient. JPEN J Parenter Enteral Nutr.2011;35(3):386-390.
115. Burns DL, Gill BM. Reversal of parenteral nutrition-associated liver disease with a fish oil-based lipid emulsion (Omegaven) in an adult dependent on home parenteral nutrition. JPEN J Par enter Enteral Nutr.2013;37(2):274-280.
116. Diamond IR, Sterescu A, Pencharz PB, Kim JH, Wales PW. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. JPediatr Gastroenterol Nutr.2009;48(2):209-215.
117. Beyazit Y, Purnak T, Kekilli M. Omega-3 fatty acids:a promising alternative treatment approach for patients with nonalcoholic fatty liver disease. Dig Dis Sci.2010;55(8):2422-2423; author reply 2423.
118. Shapiro H, Tehilla M, Attal-Singer J, Bruck R, Luzzatti R, Singer P. The therapeutic potential of long-chain omega-3 fatty acids in nonalcoholic fatty liver disease. Clin Nutr.2011;30(1):6-19.
119. Kim K, Jung N, Lee K, et al. Dietary omega-3 polyunsaturated fatty acids attenuate hepatic ischemia/reperfusion injury in rats by modulating toll-like receptor recruitment into lipid rafts. Clin Nutr.2013;32(5):855-862.
120. Zuniga J, Venegas F, Villarreal M, et al. Protection against in vivo liver ischemia-reperfusion injury by n-3 long-chain polyunsaturated fatty acids in the rat. Free radical research.2010;44(8):854-863.
121. Zuniga J, Cancino M, Medina F, et al. N-3 PUFA supplementation triggers PPAR-alpha activation and PPAR-alpha/NF-kappaB interaction: anti-inflammatory implications in liver ischemia-reperfusion injury. PLoS One. 2011;6(12):e28502.
122. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Modern pathology. an official journal of the United States and Canadian Academy of Pathology, Inc.1998;11(2):155-168.
123. Geuken E, Visser D, Kuipers F, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol. 2004;41(6):1017-1025.
124. Hoekstra H, Porte RJ, Tian Y, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology. 2006;43(5):1022-1031.
125. Chen G, Wang S, Bie P, Li X, Dong J. Endogenous bile salts are associated with bile duct injury in the rat liver transplantation model. Transplantation. 2009;87(3):330-339.
126. Lee SI, Valim C, Johnston P, et al. Impact of fish oil-based lipid emulsion on serum triglyceride, bilirubin, and albumin levels in children with parenteral nutrition-associated liver disease. Pediatr Res.2009;66(6):698-703.
127. Babcock TA, Helton WS, Hong D, Espat NJ. Omega-3 fatty acid lipid emulsion reduces LPS-stimulated macrophage TNF-alpha production. Surgical infections.2002;3(2):145-149.
2. Adam R, Hoti E. Liver transplantation:the current situation. Semin Liver Dis. 2009;29(1):3-18.
3. Merion RM. Current Status and Future of Liver Transplantation. Seminars in Liver Disease.2010;30(4):411-421.
4. Lentsch AB, Kato A, Yoshidome H, McMasters KM, Edwards MJ. Inflammatory mechanisms and therapeutic strategies for warm hepatic ischemia/reperfusion injury. Hepatology.2000;32(2):169-173.
5. Zhai Y, Busuttil RW, Kupiec-Weglinski JW. Liver ischemia and reperfusion injury:new insights into mechanisms of innate-adaptive immune-mediated tissue inflammation. Am J Transplant.2011;11(8):1563-1569.
6. Zhai Y, Petrowsky H, Hong JC, Busuttil RW, Kupiec-Weglinski JW. Ischemia-reperfusion injury, in liver transplantation-from bench to bedside. Nat Rev Gastro Hepat.2013;10(2):79-89.
7. Dyerberg J, Bang HO, Stoffersen E, Moncada S, Vane JR. Eicosapentaenoic acid and prevention of thrombosis and atherosclerosis? Lancet. 1978;2(8081):117-119.
8. Daviglus ML, Stamler J, Orencia AJ, et al. Fish consumption and the 30-year risk of fatal myocardial infarction. The New England journal of medicine. 1997;336(15):1046-1053.
9. Hu FB, Bronner L, Willett WC, et al. Fish and omega-3 fatty acid intake and risk of coronary heart disease in women. JAMA.2002;287(14):1815-1821.
10. Krauss RM, Eckel RH, Howard B, et al. AHA Dietary Guidelines:revision 2000:A statement for healthcare professionals from the Nutrition Committee of the American Heart Association. Circulation.2000;102(18):2284-2299.
11. Yokoyama M, Origasa H, Matsuzaki M, et al. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS):a randomised open-label, blinded endpoint analysis. Lancet. 2007;369(9567):1090-1098.
12. Spadaro L, Magliocco O, Spampinato D, et al. Effects of n-3 polyunsaturated fatty acids in subjects with nonalcoholic fatty liver disease. Dig Liver Dis. 2008;40(3):194-199.
13. de Meijer VE, Gura KM, Le HD, Meisel JA, Puder M. Fish oil-based lipid emulsions prevent and reverse parenteral nutrition-associated liver disease:the Boston experience. JP EN J Par enter Enteral Nutr.2009;33(5):541-547.
14. Puder M, Valim C, Meisel JA, et al. Parenteral fish oil improves outcomes in patients with parenteral nutrition-associated liver injury. Ann Surg. 2009;250(3):395-402.
15. Gura KM, Duggan CP, Collier SB, et al. Reversal of parenteral nutrition-associated liver disease in two infants with short bowel syndrome using parenteral fish oil:implications for future management. Pediatrics. 2006;118(1):e197-201.
16. Fallon EM, Le HD, Puder M. Prevention of parenteral nutrition-associated liver disease:role of omega-3 fish oil. Curr Opin Organ Transplant. 2010;15(3):334-340.
17. Calder PC. N-3 polyunsaturated fatty acids and inflammation:from molecular biology to the clinic. Lipids.2003;38(4):343-352.
18. Schmocker C, Weylandt KH, Kahlke L, et al. Omega-3 fatty acids alleviate chemically induced acute hepatitis by suppression of cytokines. Hepatology. 2007;45(4):864-869.
19. Hao W, Wong OY, Liu X, Lee P, Chen Y, Wong KK. omega-3 fatty acids suppress inflammatory cytokine production by macrophages and hepatocytes. JPediatr Surg.2010;45(12):2412-2418.
20. Clavien PA, El-Badry AM, Moritz W, Contaldo C, Tian YH, Graf R. Prevention of reperfusion injury and microcirculatory failure in macrosteatotic mouse liver by omega-3 fatty acids. Hepatology.2007;45(4):855-863.
21. Lee S, Kim S, Le HD, et al. Reduction of hepatocellular injury after common bile duct ligation using omega-3 fatty acids. J Pediatr Surg. 2008;43(11):2010-2015.
22. Gonzalez-Periz A, Planaguma A, Gronert K, et al. Docosahexaenoic acid (DHA) blunts liver injury by conversion to protective lipid mediators: protectin D1 and 17S-hydroxy-DHA. Faseb J.2006;20(14):2537-2539.
23. Song BJ, Moon KH, Olsson NU, Salem N, Jr. Prevention of alcoholic fatty liver and mitochondrial dysfunction in the rat by long-chain polyunsaturated fatty acids. J Hepatol. 2008;49(2):262-273.
24. Coelho AM, Machado MC, Takahashi HK, et al. Ischemic preconditioning-like effect of polyunsaturated fatty acid-rich diet on hepatic ischemia/reperfusion injury. J Gastrointest Surg.2011;15(10):1679-1688.
25. Marsman HA, Heger M, Kloek JJ, Nienhuis SL, ten Kate FJ, van Gulik TM. Omega-3 fatty acids reduce hepatic steatosis and consequently attenuate ischemia-reperfusion injury following partial hepatectomy in rats. Dig Liver Dis.2011;43(12):984-990.
26. Qiu YD, Wang S, Yang Y, Yan XP. Omega-3 polyunsaturated fatty acids promote liver regeneration after 90% hepatectomy in rats. World J Gastroenterol.2012;18(25):3288-3295.
27. Marsman HA, de Graaf W, Heger M, et al. Hepatic regeneration and functional recovery following partial liver resection in an experimental model of hepatic steatosis treated with omega-3 fatty acids. Br J Surg. 2013;100(5):674-683.
28. Chen CC, Ho CY, Chaung HC, et al. Fish omega-3 fatty acids induce liver fibrosis in the treatment of bile duct-ligated rats. Dig Dis Sci. 2013;58(2):440-447.
29. Zhu XH, Wu YF, Qiu YD, Jiang CP, Ding YT. Liver-protecting effects of omega-3 fish oil lipid emulsion in liver transplantation. World J Gastroenterol. 2012;18(42):6141-6147.
30. Zhu X, Wu Y, Qiu Y, Jiang C, Ding Y. Effects of omega-3 fish oil lipid emulsion combined with parenteral nutrition on patients undergoing liver transplantation. JPEN J Par enter Enteral Nutr.2013;37(1):68-74.
31. Lee S, Charters AC, Chandler JG, Orloff MJ. A technique for orthotopic liver transplantation in the rat. Transplantation.1973;16(6):664-669.
32. Lee S, Charters AC,3rd, Orloff MJ. Simplified technic for orthotopic liver transplantation in the rat. Am J Surg.1975; 130(1):38-40.
33. Kamada N, Calne RY. Orthotopic liver transplantation in the rat. Technique using cuff for portal vein anastomosis and biliary drainage. Transplantation. 1979;28(1):47-50.
34. Zimmermann FA, Butcher GW, Davies HS, Brons G, Kamada N, Turel O. Techniques for orthotopic liver transplantation in the rat and some studies of the immunologic responses to fully allogeneic liver grafts. Transplant Proc. 1979;11(1):571-577.
35. Miyata M, Fischer JH, Fuhs M, Isselhard W, Kasai Y. A simple method for orthotopic liver transplantation in the rat. Cuff technique for three vascular anastomoses. Transplantation.1980;30(5):335-338.
36. Kamada N, Calne RY. A surgical experience with five hundred thirty liver transplants in the rat. Surgery.1983;93(1 Pt 1):64-69.
37. Settaf A, Gugenheim J, Houssin D, Bismuth H. Cuff technique for orthotopic liver transplantation in the rat. A simplified method for the suprahepatic vena cava anastomosis. Transplantation.1986;42(3):330-331.
38. Tsuchimoto S, Kusumoto K, Nakajima Y, et al. Orthotopic liver transplantation in the rat. A simplified technique using the cuff method for suprahepatic vena cava anastomosis. Transplantation.1988;45(6):1153-1155.
39. Knoop M, Hutchinson IV. Suprahepatic vena cava cuffs in rat liver transplantation--a simplified method? Transplantation.1989;47(3):576.
40. Xu HS, Rosenlof LK, Selby JB, Jones RS. A simple method for bile duct anastomosis and interval bile collection in the liver-transplanted rat. J Surg Res.1992;53(5):520-523.
41. Kashfi A, Mehrabi A, Pahlavan P, et al. A review of various techniques of orthotopic liver transplantation in the rat. Transpl P.2005;37(1):185-188.
42. Harihara Y, Sanjo K, Idezuki Y. A modified cuff technique for suprahepatic vena cava anastomosis in rat liver transplantation. Transplantation. 1992;53(3):707-709.
43. Tan F, Chen Z, Zhao Y, Liang T, Li J, Wei J. Novel technique for suprahepatic vena cava reconstruction in rat orthotopic liver transplantation. Microsurgery.2005;25(7):556-560.
44. Oldani G, Maestri M, Gaspari A, et al. A Novel Technique for Rat Liver Transplantation Using Quick Linker System:A Preliminary Result. J Surg Res. 2008;149(2):303-309.
45. Ariyakhagorn V, Schmitz V, Olschewski P, et al. Improvement of Microsurgical Techniques in Orthotopic Rat Liver Transplantation. J Surg Res. 2009;153(2):332-339.
46. Delriviere L, Gibbs P, Kobayashi E, Goto S, Kamada N, Gianello P. Detailed modified technique for safer harvesting and preparation of liver graft in the rat. Microsurgery.1996;17(12):690-696.
47. Delriviere L, Gibbs P, Kobayashi E, Goto S, Kamada N, Gianello P. Technical details for safer venous and biliary anastomoses for liver transplantation in the rat. Microsurgery.1998; 18(1):12-18.
48. Hori T, Nguyen JH, Zhao XD, et al. Comprehensive and innovative techniques for liver transplantation in rats:A surgical guide. World J Gastroentero. 2010;16(25):3120-3132.
49.董家鸿,黄志强.精准肝切除——21世纪肝脏外科新理念.中华外群杂志.2009；47(21)：1601-1605.
50. Dong J, Yang S, Zeng J, et al. Precision in liver surgery. Semin Liver Dis. 2013;33(3):189-203.
51. Kilkenny C, Browne WJ, Cuthill IC, Emerson M, Altman DG. Improving Bioscience Research Reporting:The ARRIVE Guidelines for Reporting Animal Research. Plos Biology.2010;8(6).
52.李文滨,李国林,褚忠华,et al单人裸眼建立大鼠原位肝移植模型.中华实验外科杂志.2011；28(11)：1994-1996.
53. Holzen JP, Palmes D, Langer M, Spiegel HU. Microsurgical training curriculum for learning kidney and liver transplantation in the rat. Microsurgery.2005;25(8):614-623.
54. Ozkan O, Ozgentas HE. Open guide suture technique for safe micro vascular anastomosis. Ann Plast Surg.2005;55(3):289-291.
55.宇汝胜.大鼠原位肝移植模型的手术操作技巧探讨.肝胆胰外科杂志.2008；20(1)：7-9.
56.吕和平,薛涛,张启瑜,廖毅,施红旗.单人直视下大鼠减体积肝移植模型的技术改进.中华实验外科杂志.2011；28(2)：208.
57. Sanchez-Patan F, Blanco R, Aller MA, Anchuelo R, Roman FS, Arias J. End-to-side portacaval shunt:a simplified technique. J Invest Surg. 2007;20(2):135-138.
58. Marni A, Ferrero ME, Forti D. End-to-side anastomosis in heterotopic rat organ transplantation. Microsurgery.1996;17(1):21-24.
59. Spiegel HU, Palmes D. Surgical techniques of orthotopic rat liver transplantation. J Invest Surg.1998;11 (2):83-96.
60. Spiegel HU, Schleimer K, Kranz D, Diller R. Orthotopic rat liver transplantation and bile duct reconstruction by a splint technique. Eur Surg Res.1997;29(6):421-428.
61. Kamada N, Calne RY, Wight DG, Lines JG. Orthotopic rat liver transplantation after long-term preservation by continuous perfusion with fluorocarbon emulsion. Transplantation.1980;30(1):43-48.
62. Kitakado Y, Tanaka K, Asonuma K, et al. A new bioabsorbable material for rat orthotopic liver transplantation. Eur Surg Res.1991;23(5-6):366-370.
63. Ma Y, Wang GD, Guo ZY, Guo ZG, He XS, Chen GH. Surgical techniques of arterialized orthotopic liver transplantation in rats. Chin Med J (Engl). 2007;120(21):1914-1917.
64. Li N, Cai CJ, Wu YR, Lu MQ. A technique of recipient portal venoplasty and cuff insertion for portal revascularization in orthotopic rat liver transplantation. J Surg Res.2012;176(1):317-320.
65.杜晓宏,王广义,孟伟,赵军,张舵.改进的二袖套法大鼠原位肝移植术.中华器官移植杂志.2003；24(5)：316-317.
66.马毅,何晓顺,陈规划.大鼠原位肝移植模型的手术技巧及并发症的预防.中华显微外科杂志.2003；26(1)：45-47.
67.张莹,别平,石承先,张玉君,吴乔,李昆.“二袖套法”制备大鼠原位肝移植模型的方法改进.消化外科.2005；4(6)：444-446.
68. Lee SH, Fisher B. Portacaval shunt in the rat. Surgery.1961;50:668-672.
69. Tomita Y, Zhang QW, Yoshikawa M, Uchida T, Nomoto K, Yasui H. Improved technique of heterotopic cervical heart transplantation in mice. Transplantation.1997;64(11):1598-1601.
70. Lee S. An improved technique of renal transplantation in the rat. Surgery. 1967;61(5):771-773.
71.陈忠华,夏穗生.改进的大鼠原位肝移植术.中华器官移植杂志.1984；05(2)：50-53.
72.曾琪华,孙君泓,吴孟超.三袖套法大鼠原位肝移植.中华器官移植杂志.1989；10(4)：159-161.
73.马毅,何晓顺,陈规划,陈细桃,黄洁夫.重建肝动脉血供大鼠原位肝移植模型的术式探讨.中华实验外科杂志.2004；21(1)：110-111.
74.陈志宇,张玉君,王槐志,董家鸿.改良法重建肝动脉血供的大鼠原位肝移植模型.中华实验外科杂志.2005；22(8)：1016-1017.
75. Wang H, Li C, Hu J, et al. Effect of different suprahepatic vena cava reconstruction methods on the hemodynamics of rats after liver transplantation. PLoS One.2013;8(9):e72695.
76. de Vera ME, Lopez-Solis R, Dvorchik I, et al. Liver transplantation using donation after cardiac death donors:long-term follow-up from a single center. Am J Transplant.2009;9(4):773-781.
77. Mathur AK, Heimbach J, Steffick DE, Sonnenday CJ, Goodrich NP, Merion RM. Donation after Cardiac Death Liver Transplantation:Predictors of Outcome. Am J Transplant.2010; 10(11):2512-2519.
78. Monbaliu D, Pirenne J, Talbot D. Liver transplantation using Donation after Cardiac Death donors. JHepatol.2012;56(2):474-485.
79.中华医学会器官移植学分会.中国心脏死亡器官捐献工作指南(第2版).中华器官移植杂志.2011；32(12)：756-758.
80.霍枫,汪邵平,李鹏,et al心脏死亡器官捐献获取流程探讨.中国普外基础与临床杂志.2012；19(5)：468-472.
81.郑树森,屠振华.心脏死亡器官捐献肝移植.中国普外基础与临床杂志.2012；19(5)：461-463.
82. Ikeda T, Yanaga K, Kishikawa K, Kakizoe S, Shimada M, Sugimachi K. Ischemic injury in liver transplantation:difference in injury sites between warm and cold ischemia in rats. Hepatology.1992;16(2):454-461.
83. Imamura H, Rocheleau B, Cote J, Huet PM. Long-term consequence of rat orthotopic liver transplantation with and without hepatic arterial reconstruction: a clinical, pathological, and hemodynamic study. Hepatology. 1997;26(1):198-205.
84. Ishak K, Baptista A, Bianchi L, et al. Histological Grading and Staging of Chronic Hepatitis. J Hepatol.1995;22(6):696-699.
85. Steering Committee of the Istanbul S. Organ trafficking and transplant tourism and commercialism:the Declaration of Istanbul. Lancet.2008;372(9632):5-6.
86. Cameron AM, Ghobrial RM, Yersiz H, et al. Optimal utilization of donor grafts with extended criteria:a single-center experience in over 1000 liver transplants. Ann Surg.2006;243(6):748-753; discussion 753-745.
87. Durand F, Renz JF, Alkofer B, et al. Report of the Paris consensus meeting on expanded criteria donors in liver transplantation. Liver Transpl. 2008;14(12):1694-1707.
88. McCormack L, Petrowsky H, Jochum W, Mullhaupt B, Weber M, Clavien PA. Use of severely steatotic grafts in liver transplantation:a matched case-control study. Ann Surg.2007;246(6):940-946; discussion 946-948.
89. Matsuno N, Kobayashi E. Challenges in machine perfusion preservation for liver grafts from donation after circulatory death. Transplantation research. 2013;2(1):19.
90. Olthoff KM, Kulik L, Samstein B, et al. Validation of a current definition of early allograft dysfunction in liver transplant recipients and analysis of risk factors. Liver Transpl.2010;16(8):943-949.
91. Miyagawa Y, Imamura H, Soeda J, et al. Fate of hepatocyte and sinusoidal lining cell function and kinetics after extended cold preservation and transplantation of the rat liver. Liver Transpl.2002;8(4):370-381.
92. Yu WM, Coddington D, Bitter-Suermann H. Rat liver preservation. I. The components of UW solution that are essential to its success. Transplantation. 1990;49(6):1060-1066.
93. Gao W, Lemasters JJ, Thurman RG. Development of a new method for hepatic rearterialization in rat orthotopic liver transplantation. Reduction of liver injury and improvement of surgical outcome by arterialization. Transplantation.1993;56(1):19-24.
94. de Groot H, Rauen U. Ischemia-reperfusion injury:processes in pathogenetic networks:a review. Transplant Proc.2007;39(2):481-484.
95. Massip-Salcedo M, Rosello-Catafau J, Prieto J, Avila MA, Peralta C. The response of the hepatocyte to ischemia. Liver Int.2007;27(1):6-16.
96. Bilzer M, Gerbes AL. Preservation injury of the liver:mechanisms and novel therapeutic strategies.J Hepatol. 2000;32(3):508-515.
97.黄志强.胆道的解剖生理学与肝移植后胆道并发症.中华办科杂志.2006；44(5).
98.董家鸿.肝移植的胆道并发症.腹部外科.2008；21(3)：175-177.
99. Sanchez-Urdazpal L, Gores GJ, Ward EM, et al. Ischemic-type biliary complications after orthotopic liver transplantation. Hepatology. 1992;16(1):49-53.
100. Noack K, Bronk SF, Kato A, Gores GJ. The greater vulnerability of bile duct cells to reoxygenation injury than to anoxia. Implications for the pathogenesis of biliary strictures after liver transplantation. Transplantation. 1993;56(3):495-500.
101. Huet PM, Nagaoka MR, Desbiens G, et al. Sinusoidal endothelial cell and hepatocyte death following cold ischemia-warm reperfusion of the rat liver. Hepatology.2004;39(4):1110-1119.
102. McGuire PG, Howdieshell TR. The importance of engraftment in flap revascularization:confirmation by laser speckle perfusion imaging. J Surg Res. 2010;164(1):e201-212.
103. Friedrich-Rust M, Ong MF, Herrmann E, et al. Real-time elastography for noninvasive assessment of liver fibrosis in chronic viral hepatitis. AJR Am J Roentgenol.2007;188(3):758-764.
104. Friedrich-Rust M, Schwarz A, Ong M, et al. Real-time tissue elastography versus FibroScan for noninvasive assessment of liver fibrosis in chronic liver disease. Ultraschall Med.2009;30(5):478-484.
105. Ochi H, Hirooka M, Koizumi Y, et al. Real-time tissue elastography for evaluation of hepatic fibrosis and portal hypertension in nonalcoholic fatty liver diseases. Hepatology.2012;56(4):1271-1278.
106. Millonig G, Friedrich S, Adolf S, et al. Liver stiffness is directly influenced by central venous pressure. J Hepatol.2010;52(2):206-210.
107. Babcock T, Helton WS, Espat NJ. Eicosapentaenoic acid (EPA):an antiinflammatory omega-3 fat with potential clinical applications. Nutrition. 2000；16(11-12):1116-1118.
108. Gaiva MH, Couto RC, Oyama LM, et al. Diets rich in polyunsaturated fatty acids:effect on hepatic metabolism in rats. Nutrition.2003; 19(2):144-149.
109. Qi K, Fan C, Jiang J, et al. Omega-3 fatty acid containing diets decrease plasma triglyceride concentrations in mice by reducing endogenous triglyceride synthesis and enhancing the blood clearance of triglyceride-rich particles. Clin Nutr.2008;27(3):424-430.
110. Levy JR, Clore JN, Stevens W. Dietary n-3 polyunsaturated fatty acids decrease hepatic triglycerides in Fischer 344 rats. Hepatology. 2004;39(3):608-616.
111. Valenzuela R, Espinosa A, Gonzalez-Manan D, et al. N-3 long-chain polyunsaturated fatty acid supplementation significantly reduces liver oxidative stress in high fat induced steatosis. PLoS One.2012;7(10):e46400.
112. Alwayn IP, Andersson C, Zauscher B, Gura K, Nose V, Puder M. Omega-3 fatty acids improve hepatic steatosis in a murine model:potential implications for the marginal steatotic liver donor. Transplantation.2005;79(5):606-608.
113. Rollins MD, Scaife ER, Jackson WD, Meyers RL, Mulroy CW, Book LS. Elimination of soybean lipid emulsion in parenteral nutrition and supplementation with enteral fish oil improve cholestasis in infants with short bowel syndrome. Nutr Clin Pract.2010;25(2):199-204.
114. Jurewitsch B, Gardiner G, Naccarato M, Jeejeebhoy KN. Omega-3-enriched lipid emulsion for liver salvage in parenteral nutrition-induced cholestasis in the adult patient. JPEN J Parenter Enteral Nutr.2011;35(3):386-390.
115. Burns DL, Gill BM. Reversal of parenteral nutrition-associated liver disease with a fish oil-based lipid emulsion (Omegaven) in an adult dependent on home parenteral nutrition. JPEN J Par enter Enteral Nutr.2013;37(2):274-280.
116. Diamond IR, Sterescu A, Pencharz PB, Kim JH, Wales PW. Changing the paradigm:omegaven for the treatment of liver failure in pediatric short bowel syndrome. JPediatr Gastroenterol Nutr.2009;48(2):209-215.
117. Beyazit Y, Purnak T, Kekilli M. Omega-3 fatty acids:a promising alternative treatment approach for patients with nonalcoholic fatty liver disease. Dig Dis Sci.2010;55(8):2422-2423; author reply 2423.
118. Shapiro H, Tehilla M, Attal-Singer J, Bruck R, Luzzatti R, Singer P. The therapeutic potential of long-chain omega-3 fatty acids in nonalcoholic fatty liver disease. Clin Nutr.2011;30(1):6-19.
119. Kim K, Jung N, Lee K, et al. Dietary omega-3 polyunsaturated fatty acids attenuate hepatic ischemia/reperfusion injury in rats by modulating toll-like receptor recruitment into lipid rafts. Clin Nutr.2013;32(5):855-862.
120. Zuniga J, Venegas F, Villarreal M, et al. Protection against in vivo liver ischemia-reperfusion injury by n-3 long-chain polyunsaturated fatty acids in the rat. Free radical research.2010;44(8):854-863.
121. Zuniga J, Cancino M, Medina F, et al. N-3 PUFA supplementation triggers PPAR-alpha activation and PPAR-alpha/NF-kappaB interaction: anti-inflammatory implications in liver ischemia-reperfusion injury. PLoS One. 2011;6(12):e28502.
122. Allred DC, Harvey JM, Berardo M, Clark GM. Prognostic and predictive factors in breast cancer by immunohistochemical analysis. Modern pathology. an official journal of the United States and Canadian Academy of Pathology, Inc.1998;11(2):155-168.
123. Geuken E, Visser D, Kuipers F, et al. Rapid increase of bile salt secretion is associated with bile duct injury after human liver transplantation. J Hepatol. 2004;41(6):1017-1025.
124. Hoekstra H, Porte RJ, Tian Y, et al. Bile salt toxicity aggravates cold ischemic injury of bile ducts after liver transplantation in Mdr2+/- mice. Hepatology. 2006;43(5):1022-1031.
125. Chen G, Wang S, Bie P, Li X, Dong J. Endogenous bile salts are associated with bile duct injury in the rat liver transplantation model. Transplantation. 2009;87(3):330-339.
126. Lee SI, Valim C, Johnston P, et al. Impact of fish oil-based lipid emulsion on serum triglyceride, bilirubin, and albumin levels in children with parenteral nutrition-associated liver disease. Pediatr Res.2009;66(6):698-703.
127. Babcock TA, Helton WS, Hong D, Espat NJ. Omega-3 fatty acid lipid emulsion reduces LPS-stimulated macrophage TNF-alpha production. Surgical infections.2002;3(2):145-149.