冷保存大鼠部分肝移植模型的建立

目的　建立稳定的大鼠部分肝移植模型 ,研究冷保存对部分肝移植后肝再生的影响。方法　采用雄性SD大鼠 ,双袖套法行部分肝移植 ,观察不同冷保存时间受体生存状况以及各组手术情况。结果　各组移植肝平均体积为 62 % -64 % ,无肝期为 14 -15min。长时间冷保存后大鼠 7d存活率明显下降 ,生存分析显示各组间差异有显著性。 8h冷保存后大鼠 7d存活率为 40 % ,与临床辟裂式肝移植 1年生存率相似。结论　 8h保存大鼠部分肝移植模型是研究冷保存对部分肝移植肝再生影响的较好模型     

Amphiregulin Stimulates Liver Regeneration After Small‐for‐Size Mouse Liver Transplantation

This study investigated whether amphiregulin (AR), a ligand of the epidermal growth factor receptor (EGFR), improves liver regeneration after small‐for‐size liver transplantation. Livers of male C57BL/6 mice were reduced to ～50% and ～30% of original sizes and transplanted. After transplantation, AR and AR mRNA increased in 50% but not in 30% grafts. 5‐Bromodeoxyuridine (BrdU) labeling, proliferating cell nuclear antigen (PCNA) expression and mitotic index increased substantially in 50% but not 30% grafts. Hyperbilirubinemia and hypoalbuminemia occurred and survival decreased after transplantation of 30% but not 50% grafts. AR neutralizing antibody blunted regeneration in 50% grafts whereas AR injection (5 μg/mouse, iv) stimulated liver regeneration, improved liver function and increased survival after transplantation of 30% grafts. Phosphorylation of EGFR and its downstream signaling molecules Akt, mTOR, p70S6K, ERK and JNK increased markedly in 50% but not 30% grafts. AR stimulated EGFR phosphorylation and its downstream signaling pathways. EGFR inhibitor PD153035 suppressed regeneration of 50% grafts and largely abrogated stimulation of regeneration of 30% grafts by AR. AR also increased cyclin D1 and cyclin E expression in 30% grafts. Together, liver regeneration is suppressed in small‐for‐size grafts, as least in part, due to decreased AR formation. AR supplementation could be a promising therapy to stimulate regeneration of partial liver grafts.     

大鼠小体积肝移植后肝再生障碍的实验研究

目的探讨部分肝移植后肝再生状况和小体积移植物发生肝再生障碍的可能机制。方法实验分为大鼠全肝移植组（OLT）、50%部分肝移植组（50%PLT）和30%部分肝移植组（30%PLT）。分析各组术后肝功能的变化,通过免疫组化观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察。结果各组ALT和AST于术后24hr达到峰值,且30%PLT组上升显著。Cyclin D1和PCNA的免疫组化表达中,50%PLT组表达明显上调,而30%PLT组表达则明显抑制。电镜观察发现30%PLT组术后微观结构改变显著。结论 30%PLT组术后肝细胞增殖再生受到明显抑制,推测与肝细胞微环境的恶化和叠加的缺血再灌注损伤有关。     

A Novel Organ Preservation for Small Partial Liver Transplantations in Rats: Venous Systemic Oxygen Persufflation With Nitric Oxide Gas

The prognosis for recipients of small liver grafts is poor. The aim of this study was to determine the impact of venous systemic oxygen persufflation (VSOP) with nitric oxide (NO) gas for 30% partial liver preservation and transplantation in rats. After we determined optimal NO concentration as 40 ppm in vitro with the isolated perfused rat liver model, we assessed liver injury and regeneration in vivo at 1, 3, 24 and 168 h after transplantation in the following three groups after 3 h‐cold storage (n = 20 per group): control group = static storage; VSOP group = oxygen persufflation and VSOP+NO group = oxygen with NO persufflation. The liver graft persufflation was achieved with medical gas via the suprahepatic vena cava; In comparison with control group after transplantation, VSOP+NO preservation (1) increased portal circulation, (2) reduced AST and ALT release, (3) upregulated hepatic endothelial NO synthase, (4) reduced hepatocyte and bileductule damage and (5) improved liver regeneration. These results suggest that gaseous oxygen with NO persufflation is a novel and safe preservation method for small partial liver grafts, not only alleviating graft injury but also improve liver regeneration after transplantation.     

大鼠小体积肝脏移植后早期肝内细胞因子的变化

目的 研究不同冷缺血条件下大鼠小体积肝移植(30%标准体积)后早期肿瘤坏死因子α(TNF-α)及白细胞介素6(IL-6)的变化,及其与肝脏再生的关系.方法 建立Lewis大鼠30%标准体积的原位肝移植模型.根据供肝在UW液中冷保存时间的不同,将受者分为3组:冷缺血1 h组、冷缺血8 h组和冷缺血16 h组,每组均为20只.观察受者存活情况至术后第7天,并分别在移植肝恢复血流后90 min、1 h、2 h、4 h和7 d收集样本,检测移植肝组织中TNF-α和IL-6表达情况,肝细胞DNA的合成情况,进行移植肝的形态学观察.结果 大鼠肝移植手术成功率均为100%.移植后第7天,冷缺血1 h和8 h组受鼠的存活率均为100%.冷缺血16 h组受鼠的存活率较低,移植后第7天无受鼠存活.冷缺血1 h组TNF-α和IL-6的表达水平较低,冷缺血8 h组和冷缺血16 h组TNF-α和IL-6的表达则高于冷缺血1 h组(F=58.81和F=184.12,P<0.05).冷缺血8 h组和冷缺血16 h组间TNF-α和IL-6的表达的差异无统计学意义.冷缺血1 h组增殖细胞数目明显高于冷缺血8 h组,差异有统计学意义(t=5.59,P<0.05).移植术后24h,冷缺血1 h组移植肝有轻度的组织学损伤;冷缺血8 h组移植肝有轻度的窦状隙扩张和轻度的炎症;冷缺血16 h组移植肝有局部淤血,存在肝细胞崩解和坏死等改变.结论 在小体积肝移植后早期,TNF-α和IL-6的上调表达对肝脏再生有重要意义.不同冷缺血时间的小体积肝脏移植物内存在早期启动肝脏再生的信号.     

Rapamycin Attenuates Liver Graft Injury in Cirrhotic Recipient—The Significance of Down-Regulation of Rho-ROCK-VEGF Pathway

To investigate whether rapamycin could attenuate hepatic I/R injury in a cirrhotic rat liver transplantation model, we applied a rat orthotopic liver transplantation model using 100% or 50% of liver grafts and cirrhotic recipients. Rapamycin was given (0.2 mg/kg, i.v.) at 30 min before graft harvesting in the donor and 24 h before operation, 30 min before total hepatectomy and immediately after reperfusion in the recipient. Rapamycin significantly improved small-for-size graft survival from 8.3% (1/12) to 66.7% (8/12) (p = 0.027). It also increased 7-day survival rates of whole grafts (58.3%[7/12] vs. 83.3%[10/12], p = 0.371). Activation of hepatic stellate cells was mainly found in small-for-size grafts during the first 7 days after liver transplantation. Rapamycin suppressed expression of smooth muscle actin, which is a marker of hepatic stellate cell activation, especially in small-for-size grafts. Intragraft protein expression and mRNA levels of vascular endothelial growth factor (VEGF) were down-regulated by rapamycin at 48 h both in whole and small-for-size grafts. Consistently, mRNA levels and protein expression of Rho and ROCK I were decreased by rapamycin during the 48 h after liver transplantation. In conclusion, rapamycin attenuated graft injury in a cirrhotic rat liver transplantation model by suppression of hepatic stellate cell activation, related to down-regulation of Rho-ROCK-VEGF pathway.     

骨髓间充质干细胞促进大鼠小体积肝移植后肝再生的实验研究

目的 探讨骨髓间充质干细胞能否促进大鼠小体积肝移植后移植肝的再生.方法 体外分离、培养、鉴定大鼠骨髓间充质干细胞.在30％大鼠部分肝移植的模型基础上,实验分为对照组(30％ PLT)和骨髓间充质干细胞干预组(30％PLT+ MSC).比较两组肝移植术后的存活率,分析肝功能的变化,通过免疫组化来观察移植肝标本Cyclin D1和PCNA的表达,并对移植肝组织结构进行电镜观察.结果 骨髓间充质干细胞的干预,能够改善小体积肝移植术后肝功能状况,减轻组织学损伤,并能够提高存活率,30％ PLT组与30％ PLT+ MSC组一周存活率分别为16.7％和58.3％.而在Cyclin D1和PCNA的免疫组化表达中,30％ PLT组表达明显抑制,30％ PLT+ MSC组表达上调.结论 骨髓间充质干细胞可以存进大鼠小体积肝移植后移植肝的再生,改善肝功能,并提高存活率.     

Successful transplantation of a 20% partial liver graft in rats: a technical innovation

BACKGROUND: Graft-size disparity negatively impacts the postoperative outcome in the setting of adult-to-adult living donor liver transplantation. MATERIALS AND METHODS: As a model for partial liver transplantation in rats using a minimized graft volume, we orthotopically transplanted a 20% reduced-size liver with the two-cuff method. Blood chemistry, graft weight restoration, and labeling indices of 5-bromo-2'-deoxyuridine were estimated. RESULTS: We achieved 100% 2-week survival. Hepatic mass was restored rapidly with early normalization of biochemical parameters, although albumin synthesis capacity required more time to recover. CONCLUSION: This technique has great potential for research as well as a therapeutic approach to regeneration and functional recovery of small liver grafts under various posttransplant circumstances.     

减体积肝移植术后甘氨酸对肝脏再生的作用

目的 观察甘氨酸在大鼠减体积肝移植中对移植后大鼠生存率、脏器损伤及肝脏再生的影响.方法 雌性Sprague-Dawley大鼠(200～230 g)分为甘氨酸组和对照组.甘氨酸组:术前经静脉给予供体甘氨酸1.5 ml(300 mmol/L),对照组给予1.5 ml的林格氏液.50%大鼠肝脏在HTK液经过2 h的冷保存后行原位肝移植术.肝移植术后观察大鼠生存率、血清转氨酶、肝脏组织学及肝脏再生情况.结果大鼠减体积肝移植(50%)术后,甘氨酸显著延长大鼠生存率.减体积肝移植术后8 h对照组血清中的谷草转氨酶(AST)、谷丙转氨酶(ALT)及乳酸脱氢酶(LDH)分别升至(4045.4 ±614.5)、(1972.3±338.2)、(13 079.8±2608.4)U/L,而甘氨酸可降低血清转氨酶40%～50%(P<0.05).肝脏组织学也证明了甘氨酸的保护作用(P<0.05).移植术后8 h和24 h,肝脏的再生率和肝组织中的Ki-67表达在两组中差异有统计学意义.结论 甘氨酸在大鼠减体积肝移植术后可以延长生存率,减轻肝脏损伤,并且不阻碍肝脏的再生.Kuffer细胞的失活是其主要机制.     

Graft function and survival depend primarily on host factors in compromised recipient models of orthotopic liver transplantation in the rat

BACKGROUND: Experimental models of liver transplantation use normal recipients, although most patients undergoing liver transplantation suffer from acute or chronic liver failure. This study was designed to analyze the outcome of orthotopic liver transplantation in compromised rat hosts. METHODS: Recipient animals were either rats with D-galactosamine-induced acute or rats with chronic liver failure secondary to common bile duct ligation. Liver damage was evaluated by monitoring enzymes, bilirubin, ammonia levels, prothrombin, thrombin time, and cytokines. In vivo function of hepatocytes and sinusoidal endothelial cells were evaluated by indocyanine green and hyaluronic acid uptake. Transplantation was performed in normal, acute, and chronic liver failure rats at different time points using either freshly harvested or cold-preserved syngeneic livers. RESULTS: Survival with fresh grafts decreased significantly when transplants were performed 48 hr after the induction of acute liver failure. No rats with acute liver failure survived transplantation with grafts stored for 12 or 24 hr although in chronic failure survival was more 80%. Survival of acute liver failure rats receiving 6 hr preserved grafts was 16.6% compared with 83.3% observed with fresh grafts transplanted at the same time point after D-galactosamine injection. Elevated tumor necrosis factor-alpha and interleukin-1beta levels as well as impaired sinusoidal endothelial cell function were detected in acute liver failure rats with 6 h preserved grafts. CONCLUSION: These results suggest that preoperative status and different host factors have a significant effect on outcome and graft function after liver transplantation in rats.     

冷缺血对大鼠小体积肝移植肝再生的影响

目的探讨冷缺血对大鼠小体积肝移植术后肝再生的影响.方法本组在2002年9月～2004年8月利用大鼠肝总量30%原位肝移植模型,实验分为肝总量70%肝切除组(C组)和冷缺血1、3、5 h组(E1、E2、E3组),观察1w生存率及肝重量/受体原肝重量比值(EGW/RLW),并检测术后1、2、3、7d肝细胞增殖活性及肝组织学变化.结果E1组1w生存率及EGW/RLW分别达100%和95%,均明显高于E2、E3组(P均＜0.05);E1、E 、E3组均于术后2d达到增殖高峰,其中E1组峰值显著高于E2、E3组(P＜0.001),且与C组峰值无差异(P＞0.05);组织学检查见E1组肝细胞核分裂明显活跃.结论冷缺血1 h的小体积供肝和70%肝切除后肝脏具有同样的增殖活性,仅增殖高峰稍晚;冷缺血超过3h严重影响小体积供肝的再生能力和受者的存活率.     

NIM811, a Mitochondrial Permeability Transition Inhibitor, Prevents Mitochondrial Depolarization in Small-for-Size Rat Liver Grafts

ATP decreases markedly in small-for-size liver grafts. This study tested if the mitochondrial permeability transition (MPT) underlies dysfunction of small-for-size livers. Half-size livers were implanted into recipients of about twice the donor weight, resulting in quarter-size liver grafts. NIM811 (5 microM), a nonimmunosuppressive MPT inhibitor was added to the storage solutions. Mitochondrial polarization and cell death were assessed by confocal microscopy of rhodamine 123 (Rh123) and propidium iodide (PI), respectively. After quarter-size transplantation, alanine aminotransferase (ALT), serum bilirubin and necrosis all increased. NIM811 blocked these increases by >70%. After 38 h, BrdU labeling, a marker of cell proliferation and graft weight increase were 3% and 5%, respectively, which NIM811 increased to 30% and 42%. NIM811 also increased survival of quarter-size grafts. In sham-operated livers, hepatocytes exhibited punctate Rh123 fluorescence. By contrast, in quarter-size grafts at 18 h after implantation, mitochondria of most hepatocytes did not take up Rh123, indicating mitochondrial depolarization. Nearly all hepatocytes not taking up Rh123 continued to exclude PI at 18 h, indicating that depolarization preceded cell death. NIM811 and free radical-scavenging polyphenols strongly attenuated mitochondrial depolarization. In conclusion, mitochondria depolarized after quarter-size liver transplantation. NIM811 decreased injury and stimulated regeneration, probably by inhibiting free radical-dependent MPT onset.     

Selective neutralization of the chemokine TCA3 reduces the increased injury of partial versus whole liver transplants induced by cold preservation

BACKGROUND: Given the shortage of liver donors and the development of techniques for partial liver transplantation, we compared chemokine expression and inflammatory cell infiltration of partial versus whole grafts in a mouse syngeneic liver transplant model. METHODS: Orthotopic liver transplantation, using whole or partial murine liver grafts, was performed following cold preservation in ViaSpan solution for periods of one to eight hours. RESULTS: Partial grafts showed more severe cold ischemia/reperfusion injury and greater inflammatory cell infiltration than whole grafts, and was accompanied by the marked intrahepatic upregulation of multiple chemokines. Quantitative analysis showed that compared with expression in whole grafts harvested after the same period of cold ischemia, partial grafts had eightfold more T-cell activation gene (TCA)-3 (CCL1) chemokine messenger RNA (mRNA) expression (P<0.01) and sixfold more inducible protein (IP)-10 chemokine (CCL10) mRNA expression (P<0.01), as well as increased expression of the chemokine receptors CCR8 (receptor for TCA3) and CXCR3 (receptor for IP-10; P<0.01). Blockade of TCA3 by neutralizing monoclonal antibody significantly decreased intragraft IP-10 expression (P<0.05) but not tumor necrosis factor-alpha or interleukin-6 expression in partial grafts, and significantly decreased cold ischemia/reperfusion injury (P<0.05) and associated neutrophil and T-cell infiltration (P<0.01). CONCLUSIONS: These data demonstrate that the chemokine TCA3/CCL1 is important to the pathogenesis of ischemic injury of experimental partial liver grafts, and that its therapeutic targeting within such grafts can overcome the deleterious effects of prolonged cold preservation and restore liver function to the level achieved using whole liver grafts.     

Induction of Rejection After Small-for-Size Liver Transplantation: Size Matters

Background: Reduced-size liver transplantation is associated with liver regeneration. This study was designed to analyze the influence of graft size on liver rejection and liver regeneration. Methods: Reduced-size liver transplantations were performed in the rejecting ACI to Lewis and the graft acceptance BN to Lewis strain combination. The BN to Lewis control group was treated with the immunosuppressive drug FK506. Results: An accelerated liver rejection in the ACI to Lewis strain combination was found in small-for-size partial liver grafts. Graft weight to recipient liver weight ratio (GW/RLW) showed a positive correlation with survival time. In the BN to Lewis strain combination, lethal rejection was seen in small-for-size partial liver grafts. A critical immunologic GW/RLW of 33% was calculated. In rats dying from lethal rejection, GW/RLW and survival time showed a positive correlation. However, GW/RLW showed a negative correlation with hepatocellular proliferation. In regenerating livers, MHC II upregulation was also observed in the control group. All control animals survived small-for-size liver transplantation. Conclusions: The relative graft size seems to be a decisive factor influencing the kinetic of liver rejection and the induction of liver rejection. Relative critical immunologic liver mass was determined to be 33%.     

中度脂肪变性供肝肝移植的早期疗效与安全性研究

目的研究总结浙江大学医学院附属第一医院肝移植中心在脂肪变性供肝肝移植方面的经验并对其临床价值进行评估。方法分析2003年5月至2005年6月采用中度脂肪变性供肝（大泡性脂肪变性肝细胞占30％-60％）的24例成人肝移植受者（观察组）相关临床和随访数据,根据年龄、性别、终末期肝病模型评分和冷缺血时间进行匹配后,将24例采用无或轻度脂肪变性（大泡性脂肪变性肝细胞〈30％）供肝的肝移植受者作为对照组,将两组受者资料和移植肝相关数据进行分析比较。结果移植后1周内两组间肝肾功能变化差异无统计学意义（P〉0．05）,两组间早期移植物功能不全和早期急性肾损伤的发生率以及术后3个月、6个月及1年受者存活率差异均无统计学意义（均P〉0．05）。结论中度脂肪变性供肝在移植后早期可以发挥足够的功能,在排除其他危险因素的前提下可以安全地作为供肝系统的一部分。     

Improved Hepatic Regeneration With Reduced Injury by Redox Factor-1 in a Rat Small-Sized Liver Transplant Model

Redox factor-1 (Ref-1) has been shown to function in a redox-dependent manner in the cell. This study was designed to examine the effects of Ref-1 on liver regeneration as well as protection against postischemic injury in a rat model of 20% partial liver transplantation. Adenovirus carrying the full length of Ref-1 gene was introduced into liver grafts by ex vivo perfusion via the portal vein during preservation. Liver graft weights were assessed, as well as graft histology, serum levels of alanine aminotransferase (ALT)/bilirubin, DNA binding activities of AP-1 and Stat3. Redox factor-1 successfully expressed in the liver graft, improved regeneration by promoting cell proliferation. Overexpression of Ref-1 protein also reduced post-transplant injury and inflammatory reactions in the grafts. The increased serum levels of ALT and bilirubin observed after transplantation were significantly reduced by Ref-1 overexpression. Furthermore, adenovirally overexpressed Ref-1 in mouse liver successfully promoted liver regeneration after simple partial hepatectomy. Interestingly, Ref-1 significantly increased DNA binding of Stat3, but not AP-1. Overexpressed Ref-1 effectively promoted graft regeneration and reduced postischemic injury in a small-sized liver transplantation model. The results of the present study may open a new avenue to clinical transplantation of disproportionately sized grafts in living-related liver transplantation.     

大鼠小体积肝移植后肝细胞再生的研究

目的 探讨小体积肝移植术后肝再生的情况。方法 建立大鼠30％原位肝移植模型,实验分为肝切除（PH）组、全肝移植（0LT）组和30％小体积肝移植（30％POLT）组,观察1w生存率,并于术后1、2、3、7d检测肝细胞增殖活性、肝功能和肝组织学变化。结果 各组1w生存率均为100％;30％POLT组术后2d达到增殖高峰,峰值与PH组无差异（P〉0．05）;其肝功能酶学指标在术后1、3d明显升高,组织学见肝细胞核分裂极其活跃。结论 冷缺血1h的30％供肝和肝切除后肝脏具有同样的增殖活性,仅增殖高峰稍晚;较短的冷缺血时间以及熟练的手术技术可能对小体积供肝的再生起重要作用。