【作者】 夏春燕；

【导师】 丛文铭； 刘会敏；

【作者基本信息】 第二军医大学 ， 病理学与病理生理学， 2007， 博士

【摘要】 面临OLT术后多种的严重并发症,正确的诊断和鉴别诊断是临床医师面临的棘手问题。肝穿刺活检现已成为OLT术后监测的重要手段之一,目前被公认为排异诊断的“金标准(gold standard)”。然而,肝穿刺活检也存在局限性,这是由于OLT后受各方面因素影响,多种病变重叠出现时病理表现复杂,给病理诊断和鉴别诊断造成困难。因此,对各种并发症的组织学特点要有较明确的区分,并密切结合发生时间、肝功能变化、临床表现、影像学检查,以及特异分子标志物等综合分析,按病变程度依次做出病理诊断,才能充分发挥移植病理的重要作用。本课题目的在于归纳OLT术后主要并发症的诊断谱,并对相关的分子标志物进行初步研究。研究内容共分以下三个部分:第一部分667例次OLT后肝穿刺标本的临床病理学研究目的:通过对667例次OLT术后肝穿刺标本的回顾性分析,筛选出与术后主要并发症密切相关的病理学及临床指标。方法:选择2003年6月～2006年6月之间我院病理科存档的OLT术后穿刺标本病理诊断病例为研究对象,均为B超引导下经皮肝穿刺组织。所有病例均需符合以下条件:(1)肝穿刺活检组织在显微镜下能见到连续4个以上汇管区结构;(2)临床及实验室检查资料完整。共415例,667例次。选择移植术后穿刺标本常见的病理指标,对所有病理片进行回顾性病理观察,使用SPSS11.0统计软件对各项病理指标及临床实验室指标与各类术后并发症的相关性进行统计分析。结果:OLT术后并发症发生率居前5位的依次是:急性排异、胆管并发症、保存性损伤、药物性肝损害及肝炎病毒感染/肝炎复发,占肝穿刺总例次的85.7%。其中急性排异以轻度及交界性为主,占急性排异的87.5%,保存性损伤以轻、中度为主,占保存性损伤的92.6%。诊断时间出现最早的5种并发症依次为:保存性损伤,急性排异,肝血管并发症,巨细胞病毒感染及胆道并发症,并且在保存性损伤,急性排异,肝血管并发症,巨细胞病毒感染及胆道并发症,并且在发生时间上,多数并发症存在重叠现象。大部分(93.2%)尤其是高级别的(92.5%)的急性排异发生在术后三个月内,高峰(49.5%)时间段在移植后8～30天。各病理观察指标在五种主要术后并发症中的相关性(P<0.05)结果显示:与急性排异密切相关的指标依次为:凋亡小体、核分裂、小胆管炎、终末肝静脉炎/细胞脱失以及嗜酸性细胞数量;与保存性损伤密切相关的指标依次为:核分裂、胆汁淤积及小胆管炎;与胆道狭窄密切相关的指标依次为:胆汁淤积、小胆管数量、小胆管炎、汇管区纤维增生和汇管区水肿;与药物损伤密切相关的指标依次为:肝细胞水变性和胆汁淤积;与病毒性肝炎密切相关的指标依次为:肝细胞水变性、单纯终末肝静脉周细胞脱失、凋亡小体、核分裂、胆汁淤积、小胆管炎和汇管区纤维增生。临床指标相关性检验结果显示:性别,年龄,原发病,手术方式,热、冷缺血时间,无肝期等在主要并发症各组间均未见显著差异(P>0.05)。在生化指标对肝穿次时间所作的波动曲线中显示:ALT在急性排异、保存性损伤组均在肝穿前有相似的上升幅度,之后很快下降,胆道狭窄组变化不明显; AST在急性排异、保存性损伤组有一小幅上升后下降,而胆道狭窄组有一个较小的持续下降的趋势;γ-GT在保存性损伤组升高幅度最大,之后呈缓慢下降,胆道狭窄组升高后保持在一个较高的水平,而急性排异组仅有一个小幅的升高; DBIL在急性排异、保存性损伤有较小升幅,而胆道狭窄组有一个持续向上的升幅,并且整体水平远在其他两组之上,总胆红素与DBIL表现相似。结论:从病理指标的相关性分析结果得到五种OLT术后主要并发症的病理诊断谱,可作为OLT术后并发症的病理诊断及鉴别诊断的依据。临床影像学、血清学资料以及生化指标的波动曲线,均对提高病理诊断的准确性具有重要参考价值。第二部分C4d在OLT术后肝穿刺标本中的沉积及意义目前研究表明,OLT后排异反应中体液排异对细胞排异起协同作用。肾移植病理研究中发现,C4d在移植肾间质毛细血管壁上弥漫沉积可以作为排异反应的一个特异性指标,心脏移植病理中也有同样现象。有文献报道C4d在汇管区毛细血管壁的沉积与OLT术后急性排异密切相关。目的:观察C4d在OLT术后穿刺活检组织中的沉积情况,确定其在急性排异的肝组织中是否有特异性及在病理鉴别诊断中的意义。方法:在第一部分肝穿刺标本中随机抽取OLT术后发生急性排异、胆管狭窄/阻塞,保存性损伤及肝炎病毒感染的石蜡组织各20例,药物性损伤10例,急性排异合并胆管狭窄及合并保存性损伤的各8例,急性阑尾炎标本作阴性对照。采用免疫组织化学法进行兔抗人C4d抗体染色,结果进行统计学分析。结果:经χ2检验比较显示:总体阳性率在急性排异、肝炎病毒感染、急性排异合并保存性损伤组较其它组有显著差异(P<0.05)。但在不同分布区域的阳性率显示:急性排异、肝炎病毒感染、急性排异合并胆管狭窄及急性排异合并保存性损伤这四组在汇管区的阳性率较其它三组并发症高(P<0.05),在肝窦区域显示,急性排异组比其它各组阳性率要高(P<0.05),在中央静脉区域几组间的阳性率差异无统计学意义。结论:结合病理诊断的特点,C4d在肝血窦壁的沉积可以作为肝移植急性排异鉴别诊断一个比较特异的指标。保存性损伤组存在一定比例的阳性病例,可能与同时伴有体液性免疫损伤有关。C4d在汇管区的沉积还与感染有关,如肝炎病毒感染病例的汇管区阳性率较高。第三部分ACE/ACE2在保存性损伤发生机制中的作用保存性损伤可引起的术后并发症有早期肝失功、胆道损伤、提高排异反应的发生率及影响移植肝长期存活等,是目前OLT领域面临的一大问题。至今,肝保存性损伤的确切发生机制并未完全揭示。过去认为肾素-血管紧张素的主要成员ACE、ACE2主要发挥的是系统性作用,但现在逐渐认识到它们对某些脏器(心、脑,胰及肝等)局部的血流调节亦起着十分重要的作用,并且发现ACE2对局部组织缺氧敏感及有促进细胞增殖等新功能。尽管对于ACE、ACE2在OLT中的表达情况国内外均未见报道,但它们可能在OLT中发挥重要作用。目的:观察大鼠OLT术后ACE、ACE2在保存性损伤及急性排异肝组织中的表达情况,了解其与肝保存性损伤的相关性及在肝移植中的意义。方法:建立大鼠OLT模型,设立保存性损伤组,急性排异组及对照组,保存性损伤组又根据冷保存时间分为轻、中、重3个小组,采用real-time PCR法测定OLT术后不同时间点ACE和ACE2的mRNA,并结合组织学动态变化进行分析。结果:ACE2mRNA在保存性损伤组的轻(6h)、中(15 h)、重(24 h) 3个小组间存在显著差异(P<0.01,P<0.05),并且与急性排异组之间存在显著差异(P<0.01,P<0.05)。而ACEmRNA在保存性损伤组组内,及急性排异组间均未见显著差异。组织学损伤动态观察显示:肝组织损伤程度与冷保存时间密切相关,并在OLT术后24～48 h最为严重。ACEmRNA的变化过程与组织学的相平行。ACE2mRNA的升高早于ACEmRNA。结论:ACE2mRNA的表达与保存性损伤程度密切相关,可作为保存性损伤病理诊断的一个候选指标;ACE可能与组织的炎症机制有关。更多还原

【Abstract】 Definite diagnosis is a core in the treatments to complications following OLT. Liver puncture biopsy has been an important role in diagnosis and management of most of the complications after OLT, especially in rejection. However, sometimes it was difficult to make a differential diagnosis by liver puncture biopsy, because the histopathological changes were so complicated with the overlap of two or more etiological factors. Accordingly, to identify specific pathological features of the complications is important to differential diagnosis, in addition ,the clinical features, biochemical abnormalities ,imageologic examination, and specific markers are considerable .Our aim is to summarize the spectra of pathological features of the major complications post OLT ,and to search for the possible markers.The current research project is comprised of the following three parts.Part 1 Clinicopathological Analysis of 667 Liver Puncture Biopsies Following OLTObjectives: To explore the pathological and clinical features of the major complications after OLT by retrospective analysis of 667 liver puncture biopsies.Methods : Collection of liver allograft biopsies in our department of pathology from June 2003 to June 2006. 667 liver biopsies were obtained from 415 patients with liver dysfunction .All tissues were consisted with the following: (1) successive 4 portal structures per sample microscopically; (2) integrity data of clinical and laboratory examination.Useing SPSS 11.0 for statistical analysis on the comparison between pathological, clinical features and major complications after OLT.Results: The most commonly five complications seen in 667 episodes were: acute cellular rejection(ACR) ( 210 episodes,31.5 %) , biliary complication (BC)(161 episodes, 24.1%) , preservation injury(PI)(125 episodes,18.7 %) , drug toxicity(52 episodes,7.8%), infection or recurrence of hepatitis virus B (24 episodes,3.6%) . In ACR, the indeterminate and mild episodes were seen 183 in 210(87.5%). In PI, the mild and moderate episodes were seen 116 in 125 (92.6%).The most early five complications occuring after OLT were:PI, ACR, vascular complication, CMV infection, BC. Most of ACR episodes, especially those grade as moderate and severe occured within the first three months after translplantation .The most risk period for ACR is 8～30day after OLT, and there are 104 episodes in 210( 49.5%) .The correlatetions of pathological features with five major complications after OLT showed that: ACR is close correlative with apoptotic body, karyodieresis, angiocholitis, hepatic vein inflammation, cell drop out, and eosinophils (P<0.05). PI is close correlative with cholestasis, karyodieresis, and angiocholitis (P<0.05). BC is close correlative with cholestasis, bile duct quantity, angiocholitis, portal fibrosis and hydropsia (P<0.05). Drug toxicity is close correlative with hydropic degeneration and cholestasis(P<0.05). Infection or recurrence of hepatitis virus B is close correlative with fibrosis, liver cell drop out, apoptotic body, karyodieresis, cholestasis, angiocholitis, and hydropic (P<0.05).The clinical features such as gender, age, primary diseases, operation modus, the ranging of cold and warm ischemic time, anhepatic phase, and so on , did not correlate with the five major complications (P>0.05).The fluctuation curve of biochemical indicator according to the time after OLT showed that ALT went up in ACR and PI groups before Liver puncture, but did not in BC group. AST went up at first and then descended in ACR and PI groups, but had a continuous descend in BC group.γ-GT step up greatly and then descended slowly in PI group. In BC groupγ-GT maintained a high level after step up. DBIL and TB kept on rising in BC group, but step up a little then descended in ACR and PI groups.Conclusion: The spectra of specific pathological features of the major complications after OLT is the basis of differential diagnosis. In addition, clinical imageological examination, serological evidence, and the fluctuation curve of biochemical indicator are valuable to a definite diagnose.Part 2 Significance of C4d Deposition in Liver Puncture Biopsies After OLTA recent study has shown synergistic actions between humoral and cellular rejections in liver transplantation. In a pathological study of kidney transplantation, C4d diffuse deposition in renal interstitial capillary walls seems to serve as a specific marker of rejection. The same phenomenon was also observed in cardiac transplantation. It is not clear whether such a phenomenon occurs in hepatic transplantation.Objectives: To determine the specificity of C4d in ACR and the significance in the differential diagnosis of complications after OLT by observation of C4d deposition in liver puncture biopsies .Methods : Selected 106 liver puncture biopsies from Part 1 including the complications as ACR (20 biopsies ),BC (20 biopsies ),PI (20 biopsies ), infection or recurrence of hepatitis virus B (20 biopsies ) , drug toxicity (10biopsies), ACR co-existing with PI (8 biopsies),and ACR co-existing with BC (8 biopsies).The sample of acute appendicitis was taken as negative control. C4d deposition was detected by immunohistochemical staining (using rabbit anti-human C4d antibody). The differences were analyzed usingχ2 test, P<0.05 was accepted as significant.Results: The positive ratio of C4d deposition in ACR , infection or recurrence of hepatitis virus B, and ACR co-existing with PI ware higher than that in other complications (P<0 .05). C4d deposition in different Sites showed that: in portal areas the positive ratio in ACR , Infection or recurrence of hepatitis virus B, ACR co-existing with BC ,and ACR co-existing with PI were higher than that in other complications (P<0 .05).In hepatic sinusoidal walls, the positive ratio of C4d deposition in ACR was higher than that in other complications(P<0 .05). In central veins walls, there was no significant difference in all complications.Conclusion: C4d deposition in hepatic sinusoidal walls may be served as a specific marker in the diagnosis of liver graft ACR.Some biopsies in PI would be associated with humoral immunity injury. C4d deposition in portal areas may be associated with infection.Part 3 The Role of ACE/ACE2 in the Mechanism of preservation injuryIt was found that in addition to the classic circulating renin-angiotensin system(RAS), increasing evidence supports the existence of local tissue RAS (heart, brain and kidney etc) that would be provided some new function in regulating blood flow of organs, responsing sensitively to tissue hypoxia, and promoting cellular proliferation, etc.Objectives:To explore the significance of RAS in rat liver transplantation , and the correlatetion of ACE /ACE2 with preservation injury after OLT by detection ACE and ACE2 mRNA in rat allograft.Methods: The male Wistar and Sprague-Dawley rats were allocated to five groups: mild PI group (n=15,SD→SD, cold ischemia time is 6 hours); moderate PI group (n=15,SD→SD, cold ischemia time is 15 hours);severe PI group(n=15,SD→SD, cold ischemia time is 24 hours);ACR group (n=15, Wistar→SD, no cold ischemia time);control group (n=15,SD→SD, no cold ischemia time). The observation of dynamic histologic change was performed. The mRNA of ACE and ACE2 were detected by real-time PCR.Results: ACE2 mRNA was detectable at higher level in PI groups than that in ACR group(P<0.01,P<0.05). The expression of ACE2 mRNA in PI groups showed that: the level of ACE2 mRNA in the moderate and the severe were significantly higher than that in the mild(P<0.01,P<0.05). However, the level of ACEmRNA was no significant deviation in all groups during first 48h after OLT . But the dynamic tendency of ACEmRNA was parallel to that of histologic changes.Conclusion: The expression of ACE2mRNA was close correlative with the PI grades, possibly response to hepatocellular hypoxia ,and ACE2 would be a potential marker of PI. It implied that ACE is correlative with the inflammationary injury of liver.更多还原