【作者】 高峰；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2007， 博士

【摘要】 第一部分拓展边缘性供肝应用的临床研究序言随着移植外科技术的日臻完善、器官保存方法的不断改进、新型免疫抑制剂的成功研制及移植免疫理论的不断发展，器官移植已取得了巨大成就，成为挽救各类终末期脏器疾病的有效手段，创造了巨大的社会和经济效益，其成就已为医学界和社会所肯定。迄今全球已有近80万病人接受了器官移植术。临床肝脏移植(LT)经过40年的发展，已成为终末期肝病的唯一有效治疗方法。目前许多中心肝移植受体的一年生存率可以达到90％以上。肝移植越来越被人们所接受，如此高的生存率使近年来肝移植的受体选择标准不断得到扩大，一些原来是肝移植相对禁忌症甚至禁忌症的疾病也成了手术适应症。这就导致了要求接受肝移植的患者人数的增加、等待时间的延长，进而引起了患者病情变化甚至丧失手术时机等一系列问题。尽管2000年一年中美国开展了4800例肝移植术，而登记的等待肝移植的患者有将近17000人。近10年来，全球所进行的肝移植术的数量远跟不上需要肝移植患者人数的增长速度。目前，这一矛盾日渐突出，供肝短缺已经成为比器官移植后的排斥反应、外科并发症及感染更为严峻的全球性问题。为此，各国的学者正在致力于如何扩大供体来源，包括边缘性供肝的应用以及外科手术技巧的创新如活体肝移植(LDLT)、劈裂式肝移植(SLT)等。到目前为止，对边缘性供肝仍没有一个普遍认同的明确定义。一般说来，供体的一些因素与早期移植物功能不良(IPF)和原发性移植物无功能(PNF)的发生密切相关，进而可以导致移植物生存率下降，凡是具有这些危险因素的肝脏均可以被认为是边缘性供肝。目前公认的几个因素有：脂肪变性、老年性肝脏、缺血时间延长、无心跳供肝等。如何选择合适的边缘性肝脏作为供体，使其规范化并采取适当措施减轻其损伤仍是器官移植医师面临的重要课题。本研究主要着眼于供肝脂肪变性和冷缺血时间延长两方面来探讨边缘性供肝在我国是否有其应用价值。对象和方法1、研究对象2003年1月到2005年9月，我单位连续实施了204例成人(年龄大于16岁)尸肝肝移植。2、研究方法供肝的脂肪变性程度通过有经验的移植外科医师在获取和保存过程中的视诊和触诊进行判断。如初步判断脂肪变性程度较为严重则行冰冻切片检查，快速评估供肝脂肪变性程度。如为重度脂肪变性，则摒弃不用。脂肪空泡占据了细胞质大部分空间，细胞核被挤到边缘认为是大泡性肝细胞脂肪变性。大泡性脂肪变性的分级：Ⅰ级，没有脂肪变性；Ⅱ级，轻度脂肪变性(＜30％)；Ⅲ级，中度脂肪变性(30％-60％)；Ⅳ级，重度脂肪变性(＞60％)。研究变量为：供体年龄、性别、供肝重量、供肝重量与供肝受体重量比(GRBW)、冷缺血时间，受体年龄、性别、体重、原发疾病、移植前肝功能(总胆红素[TB]，谷丙转氨酶[ALT]，谷草转氨酶[AST]，凝血酶原时间[PT])及肾功能(肌酐[Scr]，尿素氮[BUN])，移植前终末期肝病的评分(MELD评分)，移植后肝功能及肾功能。移植前的数据在移植术前24h内收集，移植后的肝功能和肾功能在移植后一周内每日观测。所有病人均在门诊常规随访。早期移植物功能不全(EAD)定义为术后2-7天内至少出现以下一种情况：TB＞10mg／dL，PT≥17s，肝性脑病。早期肾功能不全(ERD)定义为术后一周内出现SCr≥2md／DL和／或需要透析治疗维持。3、统计分析所有实验数据经SPSS 13.0软件进行统计学处理，P＜0.05判定有统计学差异。结果供肝脂肪变性分布比例如下：非中重度脂肪变性组，(n=160；78.4％)；中度脂肪变性组，(n=44；21.6％)。肝移植术后1周内肝肾功能动态变化两组间变化趋势一致，1-7d两组间变量比较无明显差异。ALT和AST术后1d最高，5d内呈明显下降趋势，5d后可基本恢复至正常水平；TB在术后1-2d达峰值，以后逐渐下降至较低水平；PT于3d后可基本恢复正常水平；SCr波动于0.8-1.2mg／dL，呈小幅度变化；而BUN相对维持在较高水平。PNF发生率为0％。EAD、ERD，病人累计生存率及移植物累计生存率在两组间均无显著差异(P＞0.05)。59例病人(28.9％)接受CIT延长供肝。肝移植术后1周内肝肾功能动态变化在CIT延长组及CIT正常组间变化趋势相似，1-7d两组间变量比较均无明显差异。CIT延长组的EAD发生率明显高于CIT正常组(30.4％vs．18.3％，P＜0.05)。病人累积生存率及移植物累积存活率在CIT延长组均明显差于CIT正常组(P均＜0.05)。中度脂肪变性合并CIT延长组的EAD发生率明显高于非中重度脂肪变性合并CIT正常组，病人生存率及移植物存活率均低于非中重度脂肪变性合并CIT正常组(P＜0.05)。结论1．中度脂肪变性供肝并不影响肝移植病人术后早期肝肾功能的恢复，也不影响病人和移植物的存活率，可以安全的作为供肝系统的一部分。2．CIT延长供肝早期移植物功能不良的发生率高，且病人的生存率及移植物的存活率均显著下降。3．中度脂肪变性供肝合并冷缺血时间延长时，对受体和移植物预后较差，应当常规丢弃。第二部分边缘性供肝移植的实验研究序言国外一些移植中心提出所谓边缘性肝脏的概念，指一些处于边缘应用状态的供体肝脏，如脂肪变性供肝、酒精性肝病供肝、热缺血或冷缺血时间延长供肝以及年龄大于50岁的老年供肝等。先前的临床研究已经得出了仅仅中度脂肪变性供肝并不影响移植物功能及病人生存率，而合并冷缺血时间延长供肝将显著影响移植物及病人的结果。为了进一步在前瞻性研究中证实这一结果，并深入研究其发生机制，从而探索边缘性移植物的保护性干预，减轻移植物损伤、提高联合移植疗效，我们开展了边缘性供肝的实验研究。目前引起瞩目的保护性基因A20编码7锌指结构蛋白，具有调节炎性反应和免疫应答，发挥抗细胞凋亡和抗组织损伤的强大作用，其体外实验观察到，多种炎症刺激因子或细胞因子如TNFα、IL-1、脂多糖等都能诱导A20表达，其诱导依赖于核转录因子NF-κB的激活，同时又对NF-κB的激活起负反馈调节作用，阻断NF-κB的活化，抑制NF-κB依赖性的iNOS等一系列前炎性基因的表达。我们利用基因组学研究通过比较观察脂肪变性供肝、脂肪变性合并冷缺血时间延长供肝和正常供肝大鼠在移植后不同时点A20的表达，探讨边缘性供肝对移植物功能及生存率的影响机制。材料与方法1．动物模型1．1大鼠脂肪变性模型的建立健康成年雄性Lewis大鼠喂养胆碱-蛋氨酸缺乏饲料后6W后随机挑选5只处死检测，观察脂肪变性程度，并检测肝脏组织中GSH、MDA、MPO指标。1．2大鼠原位肝移植模型的建立采用本单位改良的Kamada-Calne方法行大鼠原位肝移植。2．动物模型分组采用雄性Lewis大鼠分别作为供体和受体，共分成3组：脂肪变性供肝移植组(Steatotic donor liver group，S组)，脂肪变性+冷缺血时间延长(6h)供肝移植组(Steatotic+prolonged cold ischemia time donor liver group，SPC组)，正常供肝移植组(normal donor group，N组)。3．生存率观察，根据以上分组分别设立生存率观察亚组：(Ss组、SPCs组、Ns组)。4．标本采集：各组大鼠分别于再灌注后24、48、72h处死，取材检测。5．生化学检测：大鼠血清生化检测。6．肝组织谷胱甘肽(GSH)、丙二醛(MDA)、髓过氧化物酶(MPO)检测。7．肝脏病理组织检查：光学显微镜及电子显微镜下观察组织学变化。8．末端脱氧核苷酸转移酶介导的缺口末端生物素标记法(TUNEL)检测凋亡。9．肝脏内保护性基因A20 mRNA的逆转录聚合酶链式反应(RT-PCR)检测10．统计学处理，所有实验数据经SPSS 13.0软件进行统计学处理，P＜0.05判定有统计学差异。结果Ss组、SPCs组和Ns组大鼠的7天存活率分别为70％、40％和90％，Kaplan-Meier生存率曲线显示Ns组大鼠的存活率明显高于SPCs组(P＜0.05)，而Ss组与SPCs组比较或于Ns组比较均无显著差异(P＞0.05)。受体大鼠ALT、AST和TB等肝功能指标均在移植后72h内不断升高。与其他两组相比，SPC组肝功能指标处于较高水平。与N组相比，移植后24h，SPC组ALT显著高(P＜0.05)；移植后48h，SPC组AST、TB值显著高(P＜0.05)；移植后48h，SPC组ALT、AST、TB值显著高(P＜0.05)。S组与N组移植后3个时点的肝功能指标未见统计学差异(P＞0.05)。24h时点SPC组GSH明显高于N组(P＜0.05)，S组MDA明显高于N组(P＜0.05)，48h时点的SPC组MDA明显高于N组(P＜0.05)。24h时点SPC组及N组GSH均明显高于S组(P＜0.05)。S组和SPC组各时点出现严重的小梁结构破坏，肝窦扩张，肝细胞肿胀变性，尤以SPC组为重，N组大鼠术后各时点小梁结构无殊，细胞形态基本正常。N组大鼠肝脏细胞凋亡少，S组大鼠肝脏可见少量凋亡小体，多于N组。而SPC组大鼠肝脏内可见大量凋亡细胞。移植后24h，N组大鼠肝脏内即有A20的表达，而S组和SPC组大鼠肝脏内无表达；移植后48h，N组大鼠肝脏内A20表达强，S组大鼠肝脏内A20有表达，而SPC组表达弱；移植后72h，3组大鼠肝脏内A20均有表达。结论1．单纯中度脂肪变性供肝安全有效，不影响移植后受体肝功能及生存率，可用作肝移植的供肝。2．中度脂肪变性基础上合并冷缺血时间延长供肝严重影响移植后受体肝功能及生存率，可能与肝细胞抗氧化能力下降、细胞大量凋亡、保护性基因A20表达延迟等相关，此类供肝不能用于肝移植。更多还原

【Abstract】 Part I Clinical study of liver transplantaion using marginal donorsOrthotropic liver transplantation has been accepted as an established treatment modality for patients with all forms of end-stage liver diseases. The number of patients on waiting lists has increased dramatically, meanwhile, donations of organs have not increased proportionally, and the discrepancy between available livers and request for transplantation has widened. With innovative surgical techniques and improved immunosuppression, liver transplantation (LT) continues to be the only definitive mode of therapy for patients with end-stage liver diseases. However, the major factor limiting the application of liver transplantation remains the scarcity of donor organs, which presently accounts for a 10 per cent waiting list mortality rate. This has forced many centers to use livers that are considered marginal in terms of performance and survival after implantation. Steatosis is a common feature that identifies marginal liver function, and investigations to evaluate steatosis have reported controversial results. Prolonged cold ischemia time is another feature thatidentifies marginal liver function.The aim of the study here is to present our experience with the use of marginal donor liver, which have resulted in a large increase in the number of liver transplantations.Materials and methodsPatient characteristicsFrom January 2003 to September 2005, 204 adult patients (age > 16 years) receiving cadaveric related liver transplantation at the First Affiliated Hospital, Zhejiang University School of Medicine, China, were enrolled in this retrospective study.During the period of the study, frozen section was not routinely used at the time of procurement for the grading of steatosis. The assessment of donor livers by the donor surgeon was based on visual inspection, palpation, and appearances after flushing with preservation solution. Donor livers that were felt to have moderate steatosis by the donor surgeon were used if there was felt to be a suitable recipient. Postreperfusion liver biopsies were obtained after liver implantation for histologic evaluation of donor liver steatosis. All biopsy specimens were semi-quantitatively graded by the same pathologists after haematoxylin-eosin staining. Fatty infiltration in liver grafts was differentiated in macrovesicular and microvesicular steatosis. Fat droplets displacing the hepatocyte nucleus and occupying the majority of the cytosol were considered macrovesicular steatosis. Data analysisThe following variables were recorded for study population: donor age, donor gender, donor weight, recipient age, cold ischemia time (CIT), recipient gender, recipient weight, etiology of the hepatopathy; pre-transplant liver function [total bilirubin (TB),aminoleucine transferase (ALT), aspartate aminotransferase (AST) and prothrombin time (PT)], pre-transplant kidney function [serum creatinine (SCr) and blood urea nitrogen (BUN)], pre-transplant model for end-stage liver diseases (MELD) score, post-transplant liver function, post-transplant kidney function, immunosuppressive regime (cyclosporine or tacrolimus). The pre-transplant data were collected 24-hour before transplantation. The post-transplant liver and kidney function were observed during the first week of post-transplant. All patients were routinely followed up closely at the outpatient clinic. Patient and graft survival was analyzed at 3 months, 6 months, 1-year or 2-year, using inadequate follow-up as the exclusion criterion.Early allograft dysfunction (EAD) was defined by the presence of at least one of the following characteristics: TB > 10 mg/dl, PT> 17 s, and hepatic encephalopathy from day 2 to day 7 post-transplantation. Early renal dysfunction (ERD) was defined by SCr > 2 mg/dl and/or the need of renal replacement therapy in the first post-transplant week. Statistical analysisThe Kolmogorov—Smirnov test was used to check for normality. Quantitative variables were expressed as mean ± SD with normal distribution and were expressed as median without normal distribution. Categorical variables were presented as values and percentages. The Student’s t test or Wilcoxon’s rank sum test was used to compare quantitative variables and the Chi-square test was used to compare categorical variables. Kaplan-Meier method and log-rank test were used for survival analysis. SPSS for Windows version 13.0 (SPSS Inc., Chicago, IL) was used to complete all the analyses, and a P value of < 0.05 was considered statistically significant.ResultsThe steatosis scores of the total number of allografts transplanted were as follows: no steatosis and mild steatosis, (n =160; 78.4%); moderate steatosis, (n =44; 21.6%). No donor liver with severe steatosis was used for transplantation. Liver and kidney functionAfter the transplantation, recipient’s biochemistry parameters were recorded and the dynamic changes of liver and kidney functions in the first post-transplant week were described. The serum levels of TB, PT, ALT and AST decreased well in the whole week and almost decreased to normal range on the post-transplant day 7. The serum levels of SCr and BUN showed no decline. From the post-transplant day 2 to day 7, the median of serum markers were compared without any significant difference. Patient and graft outcomePNF was really a rare complication in our center (0%). Patients with CIT ≥ 12h presented a significant higher incidence of EAD and lower patient and graft survival than patients with CIT < 12h (P < 0.05). Patients with moderate steatosis presented no significant differences on incidence of EAD, patient and graft survival compared with Patients with no or mild steatosis (P > 0.05). The incidence of EAD was higher in patients with moderate steatosis combined with CIT ≥ 12h than that others (P < 0.05). The 3-month and 6-month patient and graft survival rates were significantly higher in patients with moderate steatosis combined with CIT ≥ 12h than patients with no or mild steatosis combined with CIT < 12h (all P < 0.05).Conclusion1. Moderate steatotic liver can be used to expand the donor pool.2. Allograft with prolonged CIT is associated with poor patient and graft outcome3. Moderate steatotic liver with prolonged CIT is associated with poor patient and graft outcome and should be routinely discarded.Part II Experimental study of liver transplantation using marginal donorsThe discrepancy between available livers and request for transplantation has forced many centers to use livers that are considered marginal donors such as fatty liver. The shortage of organs has led clinicians to continually modify criteria to expand donor pool, particularly those in the so-called expanded or marginal donor pool. As we have mentioned before moderate steatotic liver with prolonged CIT are considered as poor patient and graft outcome should be routinely discarded.Apoptosis or programmed cell death is a genetically controlled response of the cell to commit suicide. Apoptosis is the physiological process for cell deletion in normal, reorganizing, or involuting tissue. A20 was described as a protective gene product induced by tumour necrosis factor a (TNF-α) in human endothelial cells. Both in vivo and in vitro studies have proved its functions in inhibiting apoptosis and inflammatory responses, and its role as a negative regulator of nuclear factor-kappa B (NF-κB) activation has been illustrated recently. NF-κB constitutes a family of more than 150 transcription factors that play a key role in the control of apoptosis and regulation of immune and inflammatory responses. A20 may thus offer itself as a potential therapeutic target for the treatment of various diseases where apoptosis and/or inflammatory responses constitute components of the pathophysiology.The aim of study is to investigate the potential mechanism of the negative effect of steatosis and prolonged cold ischemia time donor on outcome.Materials and methods1. Animal model1.1 Fatty liver rat model :Healthy adult male Lewis rats were fed with a choline-methionine deficient diet for 6 weeks. Five rats were randomly selected and killed for liver biochemical assessment. The pathological change of fatty livers and the activity of liver GSH, MDA, MPO were studied.1.2 In vivo model of rat liver transplantationThe rat liver transplantation was performed according to .Kamada-Calne method with some modification2. Experimental groupsThe present study included male Lewis rats as donor and recipient. Rats were randomly divided into three groups: steatotic donor liver group, steatotic+prolonged cold ischemia time donor liver group, normal donor group. The rates of survive in each group were recorded and rats from each group were killed 24, 48 and 72 hrs after reperfusion. The serum and liver specimens were collected from the killed animals.3. Relevant studies :3.1 Serum biochemistry examination3.2 Liver GSH, MDA, MPO activity assays.3.3 Histology: liver specimens were examined under light microscopy and electron microscopy.3.4 Apoptosis was also quantified by the TUNEL assay.3.5 RT-PCR was used to assess the protective gene A20 mRNA in the liver..4. Statistical analysisData were analyzed using SPSS 13.0 software. Statistical significance was regarded as P value of 0.05 or less.ResultsSurvival study showed the 7-day survival rate of 90 % in N group and 70 % in S group and 40% in SPC group, respectively. The significant difference was investigated between N group and SPC group (P <0.05).Biochemical data showed that the levels of hepatic enzymes and TB reached the peak at 72 hours after reperfusion in each group. At 24 and 48 hours after transplantation, the levels of ALT, AST and TB in SPC group were significantly higher than those in N group (P <0.05). Swelling of hepatocytes, structural derangement, necrosis, regional sinusoidal dilation, congestion and detached epithelial lining were seen in S group and SPC group, especially in the latter. Apoptosis cells in SPC group are much more than those in N group. RT-PCR results showed that the expression of A20 mRNA in SPC group was obviously decreased at each time point after transplantation compared with those in N group.Conclusion1. Moderate steatotic liver can be used to expand the donor pool.2. Moderate steatotic liver with prolonged CIT is associated wth poor patient and graft outcome and should be routinely discarded.3. The negative effect of steatosis combined with prolonged CIT donor might be due to the down-regulated expression of A20, decreased antioxidation ability and apoptosis.更多还原