【作者】 郑峻松；

【导师】 吴军；

【作者基本信息】 第三军医大学 ， 外科学， 2003， 博士

【摘要】 器官移植是现今挽救终末期器官功能衰竭患者生命的最终有效途径。如何控制器官移植后排斥发应，诱发宿主对移植物的免疫耐受，一直是移植免疫领域研究的热点及难题。迄今，防治移植排斥的主要对策仍然是通过各种途径抑制宿主的免疫功能，但现在临床使用的各种免疫抑制剂都存在非特异性抑制的突出弱点。近年来提出的特异性免疫耐受理论为移植免疫的研究指明了方向：即通过诱导机体仅对移植物抗原产生耐受，而保留和不干预机体的其他免疫功能，达到既延长移植物存活时间，又使对宿主自身免疫功能的影响降到最低限度的效果。国内外在这方面做过的研究包括：( 胸腺中注射供体细胞或MHC抗原；( 诱导嵌合体和微嵌合体形成；" 基因改造修饰供体MHC抗原；1/4 阻断T细胞辅助刺激信号途径等。而这些方法或者由于手术操作复杂、诱导时间长或者过程本身对患者进一步造成损伤，以及应用效果不稳定等原因而限制了其直接过渡到临床应用。T淋巴细胞是机体细胞免疫的重要执行和调节细胞，是参与同种/异种器官移植急性排斥反应的主要效应细胞，诱导受体T淋巴细胞对供者的特异性细胞免疫耐受是解决器官移植急性排斥反应的最主要最有效手段。由于免疫干预措施的局限性，诱导异基因组织器官移植（含皮肤移植）终生存活仍然仿佛是童话世界中的海市蜃楼。在大面积深度烧伤患者的临床治疗中，自体皮肤移植仍然是封闭治愈深度烧伤创面的唯一最终有效途径。而大面积烧伤后，封闭烧伤创面的自体皮源往往严重不足，采用皮肤代用品暂时覆盖受损创面便成为烧伤治疗中被迫而必须采取的补救措施。异体（异种）皮由于其生理结构、性能等方面与人体皮肤存在的极大相似性，被认为是皮肤代用品中的首选。但是在其临床应用中，已经发现其存活时间相对较短。在大面积深度烧伤患者治疗过程中，患者微存的少量健康皮肤作为供皮区切取以后，未完全愈合待第二切取时，移植的异体（种）皮多数已被排斥。所以，其自体皮源的供应必然存在或长或短的时相缺隙。大面积深度烧伤后，机体免疫功能尤其是细胞免疫功能本已严重受抑，采用非选择性的免疫抑制药物延长创面异基因移植皮肤存活时间，将势必进一步加重患者本身的免疫功能抑制，促成感染等其他并发症的发生。因此，采用免疫抑制药物延长烧伤<WP=11>创面移植皮肤存活时间几乎被视为临床禁忌，探索一种选择性干预和抑制排斥皮肤的移植免疫功能而主要是抑制针对移植皮肤特异性的T细胞功能的治疗措施势在必行。免疫自稳机制是机体免疫系统恒久存在的的三大原始功能之一。调节性T细胞（Regulatory T cell, Treg）对正常机体中未被胸腺“阴性选择”克隆清除的“自身反应性” T细胞的“休眠状态”的维持和对部分效应性T细胞的功能抑制是个体维持免疫自稳的重要机制之一。通过特异性抗原预先致敏的Treg细胞对效应性T细胞的抑制具有抗原特异性。调节性T细胞虽然在自身免疫性疾病和肿瘤免疫研究领域被发现，但由于其对效应性T细胞的选择性抑制，使其在移植领域的研究和应用越来越受到重视。本课题拟在建立稳定的Treg细胞富集分离方法的基础上，充分探讨Treg细胞对效应性T 细胞的作用机制，同时采用皮肤移植模型，从Treg细胞参与维持正常机体免疫自稳的机制出发，以供体抗原遭遇型(antigen experienced)受体Treg细胞体内应用的方法，选择性抑制移植抗原特异性效应性T 细胞，分析其对受体正常免疫功能的影响及其介导移植免疫耐受的免疫学机制，为推动Treg细胞作为应用性致耐工具细胞奠定实验基础。本项目研究中，我们所获得的主要结果和结论归纳如下：1． 在Treg细胞分选方法上，通过T细胞纯化柱分离T淋巴细胞，并采用MACS磁板分选装置，以CD8-D&CD25-P组合方式分选CD4+CD25+ Treg细胞是经优化后的分离富集Treg细胞的理想方法。2． 胸腺微环境是影响Treg细胞发育形成的重要因素， IL-10和TGF-β1是Treg细胞体内外诱导增殖及促生长的细胞因子，以IL-10对Treg细胞的促生长作用更显著。成年期胸腺仍然是培育Treg细胞形成发育的重要载体器官。胸腺切除将导致Treg细胞外周比例的明显下降。3． Treg细胞能分泌细胞因子IL-10和TGF-β1。其膜表面共刺激分子表达中，CTLA4表达水平明显高于CD28，这种表达特征与效应性T细胞正好相反。CTLA4的高表达可能是Treg细胞施行细胞间接触抑制和其抑制高效性的分子基础。Treg细胞对效应性T细胞的功能抑制有细胞间接触机制和细胞因子依赖机制，但以细胞间接触机制占主导；而细胞因子机制中，IL-10的作用强于TGF-β1。Treg细胞对效应性T细胞的功能抑制具有抗原特异性。4． Treg细胞对同种及异种混合淋巴细胞反应的抑制呈现明显的细胞剂量效应关系，Treg细胞对同种混合淋巴细胞反应的抑制有明显的剂量饱和特性。在体皮肤移植实验同样证明，Treg细胞应用对介导同种及异种皮肤移植存活时间存在细胞剂量与耐受强度（以平均存活时间表示）的剂量时间依赖关系。一次性Treg细胞应用对介导二次植皮平均存活时间较不采用任何干预措施所获得的正常对照二次植皮平均存活时间（3-4天）长9-10天。我们推测，Treg应用后，<WP=12>5． 可能会带来机体naive T细胞向更多还原

【Abstract】 Transplantation has become a mainstay of therapy for most patients with end-stage organ dysfunction. During the past 30 years, organ transplantation has developed from a highly experimental procedure into an important part of routine clinical practice. This is reflected by the fact that graft survival time has been significantly prolonged, and such survival depends on a number of factors but the most significant of these is the administration of powerful immunosuppressive drugs. Transplantation between genetically disparate individuals (transplantation of an allograft or xenograft) evokes a rapid and potentially destructive immune response that, if left unchecked, can lead to almost complete destruction of the transplanted organ within a matter of days. Administration of immunosuppressive drugs (such as the purine analogue azathioprine, the steroid methylprednisolone, the cyclic peptide cyclosporin A and a variety of anti-lymphocyte antibody preparations) attenuates this response and thus prevents acute graft rejection. However, continued graft survival depends on life-long immunosuppression because, except for a very small number of cases (the majority of which involve liver transplantation and should probably be regarded as a special case), withdrawal of immunosuppression results in re-activation of the rejection response, leading to rapid graft destruction. Although the immunosuppressive drugs that are currently available and very effective in the short term, substantial problems in four specific areas indicate a pressing need to develop alternative and more sophisticated ways of preventing graft rejection. These areas can be summarised as follows: ①chronic graft rejection, ②transplant-associated vasculopathy, ③infection and ④ cancer.Clinical success continues to be limited by the ongoing need in the majority of patients for nonspecific immunosuppressive therapy that reduces the risk of graft rejection but also brings with it unwanted effects, such as increased susceptibility to infection and malignancy. In addition, the majority of immunosuppressive drugs in current clinical use act by inhibiting T cell activation and thus prevent graft rejection; however, this may be counter-productive, as under appropriate circumstances, T cell activation may lead to the induction of processes facilitating the development of graft-specific tolerance. The final<WP=6>destruction of an allograft might involve most, or perhaps all, of the cellular and non-cellular components of the immune system, but many studies have shown that T lymphocytes and particularly CD4+ T cells play an essential pivotal role in graft rejection. Therefore, the majority of protocols aimed at providing true transplantation tolerance are designed to tolerise T cells. In order to assess the possibilities for the induction of T-cell tolerance to transplanted organs, it is necessary to consider some fundamental aspects of T-cell function and examine the basis of tolerance to self. That is to say, all strategies for transplantation tolerance must target T cells, and the major aim of transplantation immunology is to develop protocols that prevent immune responses towards the graft but leave the rest of the immune system intact.Most important of all is that, the immunosuppressive agents currently used in clinical transplantation are non-specific in nature and cannot distinguish between beneficial immune responses against infectious pathogens and destructive immune responses against the graft. Thus, the administration of immunosuppressive drugs necessary to prevent graft rejection can lead to an increased risk of opportunistic infection. While current procedures for burn injury management have improved patient prognosis, increased morbidity and mortality remain major concerns. Thus, identification of the mechanisms responsible for post-burn immune dysfunction and increased susceptibility to subsequent sepsis and multiple organ failure under such conditions are essential for the development of improved treatment modalities. Thermal injury induces a更多还原