【作者】 谢小军；

【导师】 郑树森；

【作者基本信息】 浙江大学 ， 外科学， 2011， 博士

【摘要】 研究目的：急性排斥反应是肝脏移植术后常见的严重并发症,也是导致慢性排斥和移植物失功的重要原因之一,研究肝脏移植后免疫调节的机制有助于为急性排斥反应提供新的治疗手段。辅助性T细胞17(T helper 17 cells, Th17)以表达IL-17而得名,是不同于Thl、Th2的CD4+T细胞新亚群,在自身免疫性疾病、机体抗感染、肿瘤发生和器官移植排斥中发挥重要作用。业已证明,转化生长因子-β(transforming growth factor beta, TGF-β)与IL-6联合作用能诱导原始CD4+T细胞分化为Th17细胞。而Kupffer细胞作为体内最大的定居型巨噬细胞群,在活化状态下既可吞噬肠道来源的病原菌以及内毒素,又可释放大量生物活性物质,具有强大的抗原递呈功能,为淋巴细胞的分化、增殖、成熟以及凋亡等提供各种调节信号。鉴于肝脏中Kupffer细胞在病理生理条件下分泌大量TGF-p与IL-6等细胞因子,我们认为此时形成的肝脏局部免疫微环境有利于Thl7细胞的分化和增殖,而Th17细胞可能在肝脏免疫性疾病及排斥反应中发挥重要作用。我们通过建立大鼠肝脏移植急性排斥模型,探讨Th17细胞在肝移植急性排斥反应中的作用及机制,同时利用氯化钆(gadolinium trichloride,GdCl3)抑制Kupffer细胞功能,观察其对移植排斥反应的作用及Th17细胞的影响,并通过体外实验进一步证实Kupffer细胞对Th17细胞分化的调控作用。研究方-法：实验动物采用近交系雄性Dark Agouti(DA)和Brown Norway(BN)大鼠,体重200-300g,改良Kamada法建立大鼠原位肝脏移植模型。实验分组:(A)异基因组：DA大鼠作为供体,BN大鼠作为受体；(B)GdCl3预处理组：DA大鼠作为供体,BN大鼠作为受体,供体术前24小时阴茎静脉注射GdCl3(10 mg/kg)；(C)同基因组：供受体均采用BN大鼠。每组各18对,6只观察生存期,其余于术后5天和10天处死,留取外周血和肝组织标本,分别检测肝功能、肝脏病理组织学分析,流式细胞术检测外周血和肝脏淋巴细胞Thl7细胞比例,使用荧光定量聚合酶链锁反应(polymerase chain reaction,PCR)、蛋白质印迹(Western blot)、免疫组化以及酶联免疫吸附(enzyme-linked immunosorbent assay,ELISA)法检测肝组织IL-17、IL-6、TGF-βmRNA和蛋白表达水平。在体外将通过原位胶原酶灌注结合离心淘洗法分离的大鼠Kupffer细胞与使用免疫磁珠法分选出的脾脏初始CD4+T淋巴细胞进行混合培养,然后经流式细胞术分析其淋巴细胞表型。研究结果：与同基因移植大鼠比较,异基因移植大鼠出现典型急性排斥反应改变,肝脏Kupffer细胞与淋巴细胞浸润明显,其中Th17细胞比例增高,同时肝内IL-8、髓过氧化物酶(myeloperoxidase,MPO)、TGF-β和IL-6表达水平明显上升。注射氯化钆的大鼠肝脏Kupffer细胞数量明显下降,急性排斥反应明显改善,生存时间延长,并且肝内与外周血Th17水平显著降低,同时肝内TGF-β和IL-6表达下降。可见氯化钆能够有效地抑制Kupffer细胞的免疫活性和Thl7细胞增殖,从而抑制大鼠同种异基因肝移植后急性排斥反应。分别分离急性排斥大鼠和同基因移植大鼠肝脏Kupffer细胞,与正常DA大鼠脾脏原始CD4+T细胞混合培养,流式细胞术检测发现前者Th17细胞比例也明显高于后者(30.8% vs 8.1%),ELISA检测发现急性排斥大鼠Kupffer细胞培养上清TGF-β和IL-6水平明显较同基因组高。研究结论：肝脏Kupffer细胞通过分泌TGF-β和IL-6等细胞因子诱导Th17细胞的分化,促进肝脏移植急性排斥反应的发生。更多还原

【Abstract】 Objective:Orthotopic liver transplantation (OLT) is applied as a valuable method for treating end-stage liver diseases. However, acute rejection is one of its most serious complication reported in～about 30% of liver transplants performed. T lymphocytes and Kupffer cells are the dominant cell types that are found to infiltrate the liver following transplantation. Consistent with these observations, CD4+ T cells have been proposed to play a crucial role in determining transplant rejection or tolerance. T helper (Th) 17, recently identified as a new subset of CD4+ T cells, has been discovered to implicate in transplant rejection. Data from animal experiments and clinical trials confirmed that Thl 7 cells have a role in allograft rejection of solid organs, which previously thought to be driven by Th1-mediated inflammation. Kupffer cells are macrophage that are present in liver and localize to portal area and within sinusoidal lumen. Kupffer cells have previously been implicated in the pathogenesis of hepatic allograft rejection based on their antigen presentation and cytokine production. As antigen presenting cells (APC) are associated with liver, Kupffer cells interact with lymphocytes to induce T-cell proliferation and cytokine synthesis. Furthermore, under pathophysiological conditions, Kupffer cells have been shown to secrete high levels of IL-6 and TGF-β, which are key factors for Th17 cell differentiation. Therefore, we wonder if it is possible that Kupffer cells induce Th17 cell differentiation via production of IL-6 and TGF-βto promote acute liver allograft rejection. To test this hypothesis, the role of Kupffer cells and Th17 cells in acute liver transplant rejection was investigated using a rat OLT model.Methods:A rat model of allogeneic liver transplantation from Dark Agouti (DA) to Brown Norway (BN) was established with or without pretreatment with gadoliniumchloride (GdCl3), and isogeneic liver transplantation (BN to BN) was used as a control. The expression of kupffer cells cell-derived Th17-related cytokines in liver and peripheral blood were determined by immunohistochemistry, flow cytometry or ELISA, and survival differences were compared between groups. Furthermore, Kupffer cells in liver grafts were isolated and cocultured with naive CD4+ T cells for 5 days, then T cells were collected and the frequency of Th17 differentiation was determined using flow cytometry.Results:Both Kupffer cells and Th17 cells were found to infiltrate into liver allografts accompanied by increase of IL-6 and TGF-βlevels. Pretreatment with GdCl3 attenuated intragraft-infiltration of Th17 cells as well as Kupffer cells, decreased IL-6 and TGF-βlevels in grafts, improved liver function and prolonged the survival time(16.33±0.96 days vs 11.50±0.99 days, p<0.01). In vitro, the Kupffer cells from allograft liver secreted significantly more IL-6, TGF-βand induced Th17 differentiation more effectively comparing with those from isograft (30.8% vs 8.1%).Conclution:Kupffer cells potentially function as inducers of Th17 cells by secreting IL-6 and TGF-β, and promote acute liver allograft rejection.更多还原