【作者】 孙尔维；

【导师】 陈忠华；

【作者基本信息】 华中科技大学 ， 器官移植， 2008， 博士

【摘要】 背景器官移植是终末期器官功能衰竭的重要治疗手段,移植排斥反应是影响预后的关键因素。临床上主要通过免疫抑制疗法防治移植排斥反应,但存在感染、严重毒副反应和费用高昂等缺点。理论上,诱导针对移植抗原的特异性免疫耐受是防治移植物排斥反应的最佳途径。诱导移植免疫耐受的前提是:①使受者免疫系统接触供者的移植抗原;②对受者免疫系统进行调控,使其耐受供者抗原。迄今为止所建立的免疫耐受诱导方法在小动物模型均取得一定疗效,但在大动物模型尚未见成功报道,也难以临床应用。因而,亟待探索诱导移植免疫耐受的新策略。近10年来,凋亡细胞对免疫系统的调节作用受到高度关注。现已证明,凋亡细胞可诱导移植免疫耐受,其理论基础为:(1)抗原提呈细胞(APC)可快速摄取凋亡细胞;(2)APC可更有效地提呈凋亡细胞所携带的供者抗原;(3)局部微环境的作用。作者的课题组既往的研究结果发现:①凋亡细胞在体外可显著抑制T细胞活化,静脉输注供者凋亡细胞可抑制针对供者移植抗原的特异性淋巴细胞增殖(MLR);②静脉注射供者凋亡细胞可明显延长心脏、皮肤和肝脏移植物存活,而无需应用免疫抑制剂;③受者吞噬细胞是供者凋亡细胞诱导免疫耐受的重要介导细胞等。国内外其他实验室也相继证明输注供者凋亡细胞是诱导移植耐受的有效策略。应用供者凋亡细胞诱导免疫耐受是一个新的研究领域,有许多问题尚待探讨。如输注的供者凋亡细胞在受者体内如何分布,被体内何种细胞吞噬,抗原提呈如何进行,受者体内的T细胞功能亚群如何变化及其机制和意义,以及能否应用到临床等。最重要的是对于供者凋亡细胞诱导移植免疫耐受这一现象的深刻理论意义及其对自身免疫性疾病、肿瘤等疾病和移植物排斥的治疗意义。为此,本课题拟对上述问题进行研究。课题分为以下四个部分一、供者凋亡细胞在受者体内的分布及其局部免疫调节作用目的:研究供者凋亡细胞输注后在受者体内的分布、吞噬和诱导器官内局部免疫调节的情况。方法:近交系C57BL(H-2~b)和Balb/c(H-2~d)分别作为供者和受者。首先将C57BL脾细胞以2μM CFSE染色,然后以1μM地塞米松(Dex,Sigma)诱导凋亡。部分实验中凋亡细胞以Annexin-V免疫磁珠(Miltenyi)进行富集。将2×10~7 C57BL脾细胞以CFSE标记后诱导凋亡,静脉注射给Balb/C小鼠,在不同时间采集受者肝脏、脾脏和血清标本。应用组织学和流式细胞术分析供者凋亡细胞在受者体内分布,同时应用Bioplex或ELISA的方法测定肝脏和脾脏组织,以及血清内细胞因子浓度。与此同时,将CFSE~+的凋亡C57BL脾细胞与从Balb/C小鼠肝脏内分离的APC在体外共培养,并以荧光显微镜、免疫组织化学和流式细胞术分析不同APC吞噬供者凋亡细胞的情况。结果:供者凋亡细胞主要分布于受者的肝脏,而脾脏内分布很少。静脉注射后30—60分钟,供者凋亡细胞聚集在肝脏,并且在90分钟之内被清除。而输注受者凋亡细胞在其自身体内的分布情况与供者凋亡细胞类似,表明供者凋亡细胞在受体体内的分布主要取决于凋亡的特性,而非供者特性。体内和体外实验均表明,肝脏内的三种APC,包括肝窦状内皮细胞(LSEC),枯否氏细胞(KC)和树突状细胞(DC)均能有效地吞噬供者凋亡细胞。输注供者凋亡细胞后,不同时间检测肝脏、脾脏和血清内的IL-1β,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,TNF-α,IFN-γ,GMCS和TGF-β,发现尽管在肝脏和脾脏内均出现Th1反应,但是在受者体内诱导一个强烈和持久的Th2反应,而在其血清内未检测到类似反应。结论:输注供者凋亡细胞后诱导移植耐受的机制之一是被肝脏内的吞噬细胞所吞噬,并在局部诱导Th2反应。二、供者凋亡细胞对受者体内细胞毒性杀伤和调节性T细胞(Treg)的影响目的:明确供者凋亡细胞对受者体内细胞毒性杀伤和Treg有何影响。方法:近交系雌性C57BL/6j(H-2~b)和Balb/c(H-2~d)分别作为供者和受者。用不同浓度的CFSE对供者和受者细胞进行染色后,将其混合并作为靶细胞输注入受者体内,一定时间后,采外周血流式细胞术测定标记供者细胞的清除情况,应用标记供者细胞清除率与标记受者细胞的清除率来计算供者细胞的杀伤率。并在同种异体皮肤移植模型、免疫抑制模型和裸鼠模型中验证体内淋巴细胞毒性方法。成功建立体内细胞毒性测定方法后,观察了输注供者凋亡细胞对受者体内细胞毒性杀伤和Treg的影响。给Balb/c受鼠注射2×10~7 C57小鼠凋亡脾细胞(活细胞、坏死细胞作对照),一周后采集外周血标本,用流式细胞仪的方法检测Treg。同时,给受者注射CFSE标记的供者和受者脾细胞混合物,2小时后采外周血,流式细胞仪测定体内细胞毒性杀伤。结果:在体内细胞毒性杀伤模型的建立方面,发现输注1×10~7-1×10~8CFSE~+的细胞后,可以在受者体内方便地检测到标记细胞。在同种异体供者皮肤移植预先致敏的受者,供者细胞在1-2小时内被清除,而未经致敏的动物清除速度明显低于致敏动物,4小时后才开始被清除,24小时显著清除,72小时才全部被清除。受者体内清除供者细胞的能力与同种异体排斥反应的发生时相一致。在免疫抑制或裸鼠体内,对供者细胞的清除降低。预先注射活细胞和坏死细胞的受者动物,供者靶细胞很快被清除,而输注凋亡细胞的受者动物体内,供者靶细胞不被杀伤。表明注射供者活细胞和坏死细胞后,受者体内针对供者细胞的毒性明显增强,而注射凋亡细胞的动物,毒性没有增强。注射供者凋亡细胞后,受者动物体内Treg比例也明显增加。结论:体内细胞毒性检测方法是一个准确、方便、快捷的体内细胞毒性杀伤检测方法。与活细胞和坏死细胞相比,注射供者凋亡细胞后受者体内细胞毒性杀伤功能明显受到抑制,其机制可能与受者体内Treg比例增加有关。三、供者凋亡细胞对同种异体恒河猴肾移植存活的影响及对Treg的影响目的:了解输注供者凋亡细胞能否预防恒河猴同种异体肾移植排斥反应,延长移植物存活,解析Treg在凋亡细胞延长移植物存活中的意义。方法:首先分析了恒河猴的类人类血型情况,对比了常规单克隆抗体法、凝集抑制试验法和反向定型法。结果发现反向定型法,即不测定红细胞上的抗原,而测定血清中的抗体,可以鉴定恒河猴的血型情况。恒河猴同种异体肾移植模型的建立,采用保留受者自体肾的方法。供者手术:麻醉后摘取左肾,HCA液灌洗后备用。受者手术:麻醉后将供肾静脉和动脉分别与受者下腔静脉和腹主动脉吻合,输尿管与膀胱吻合,术后输尿管内置双J管。供者细胞凋亡的诱导方法,我们摸索了高能X线、Co60γ射线和紫外线照射法。确定大量制备供者凋亡细胞的最佳条件为:培养皿直径6cm、10%小牛血清培养基、UVC照射距离为20cm、照射时间为60分钟。为了探讨供者凋亡细胞对恒河猴同种异体肾移植存活的影响。我们给受者静脉输注7.2-7.4×10~7凋亡的供者外周血单个核细胞(PBMC),一周后行供者特异性肾移植手术,对照恒河猴给予等量PBS。术后采用甲基强的松龙(MP)+环磷酰胺(CHX)+雷帕霉素(RAPA)三联免疫抑制,其中RAPA术后每日一次,共14天,剂量约为临床剂量的五分之一。手术后以B超、CT等观察受者移植肾存活情况,同时连续观察受者外周血Treg的比例。结果:对比单克隆抗体法、凝血抑制实验法和反向定型法,发现反向定型法测定恒河猴类人类血型敏感、有效。43只恒河猴类中人类血型的分布依次为B型(25只,占58.14%),AB型(15只,占34.88%),A型(3只,占7%)。未发现类人O型。对于恒河猴同种异体肾移植模型,4只恒河猴手术过程顺利,术中及术后未用免疫抑制剂,术中见有泌尿,术后2天移植肾彩超血供正常。一只猴第3天出现移植肾区明显肿大,检查发现移植肾静脉血栓,予以切除。一例术后7天因排斥并发肠梗阻死亡。其余两例移植肾存活5天排斥。3只用于研究供者凋亡细胞输注对肾移植存活的影响的恒河猴均接受相同的三联免疫抑制治疗(MP+CHX+RAPA)。输注供者凋亡细胞两只猴中一只移植物存活14天,1只移植物存活33天,一只猴仅输注PBS而没有输注供者凋亡细胞,移植肾存活14天。检测受者猴PBMC中Treg的比例显示,输注供者凋亡细胞的两只动物,PBMC中Treg的比例明显高于未输注供者凋亡细胞的动物,其中Treg升高幅度最高者的移植物存活时间最长。结论:反向定型法是检测恒河猴类人类血型的可靠有效方法。输注供者凋亡细胞有可能延长恒河猴肾移植物存活,其机制可能与受者体内Treg的比例的提高有关。四、“细胞死亡”免疫识别模型的建立目的:建立新的“细胞死亡”免疫识别模型。方法:文献调研、结合本课题组10年的研究,进行理论论证。结果:建立“细胞死亡”免疫识别模型的四项基本原则,并与传统的“自我非我”选择模型、以及最近提出的“危险信号”模型和“感染性非我”模型进行对比。应用“细胞死亡”免疫识别模型可以更加方便地解释自身免疫、肿瘤免疫、移植免疫、胚胎免疫、肝脏免疫以及免疫系统中Treg的作用等。观点:机体对所接触抗原产生免疫应答或免疫耐受,不取决于抗原性质是否“自我非我”,也不取决与是否存在感染或有无危险信号,而是取决于抗原提呈过程中的细胞死亡方式:细胞凋亡诱导免疫耐受,而细胞坏死诱导免疫应答。更多还原

【Abstract】 BackgroundOrgan transplant is one of the most effective treatments for patients with end-stage diseases,while rejections constitute the major obstacle for long-term graft survival. Immunosuppression has been the only applicable clinical method for preventing and treating transplant rejections,although it usually leads to increased opportunistic infections, severe drug toxicity and high medical expense.Theoretically,induction of donor antigen specific tolerance is the ideal way to prevent transplant rejections.Two principles are important for transplant tolerance induction.First,donor antigens should be accessible to the appropriate immune cells residing in the recipient immune system,and second,the immune system should be regulated accordingly.However,the methods successfully established in murine transplant models are hardly transferable to non-human primates and patients,appealing novel tolerance strategies to deal with clinical transplant rejections.In past 10 years,the immune regulatory property of apoptotic cells became a major focus in immunological studies and apoptotic cells were demonstrated to induce tolerance. Rapid phagocytosis of apoptotic cells by antigen presenting cells(APC),more efficient presentation of apoptotic cell-derived antigens to na(i|¨)ve T cells and local immunosuppressive environment initiated by apoptotic cells and their phagocytes all contribute to tolerance induction. The authors also found that apoptotic cells inhibited T cell activation in vitro and intravenous infusion of donor apoptotic cells suppressed donor antigen specific mixed lymphocyte reaction(MLR).More importantly,administration of donor apoptotic cells prolonged heart,skin,and liver graft survival under no immunosuppression,in which phagocytes in the recipients played an important role.Reports in the literature also supported that administration of donor apoptotic cells is an effective strategy to induce transplant tolerance.Many questions remain obscure concerning the mechanisms on tolerance induction by donor apoptotic cells,such as the distribution of infused donor apoptotic cells,their phagocytosis,antigen presentation,and the function of recipient T cells subsets.More importantly,it is critical to understand the theoretical significance of apoptotic cell-induced tolerance in immune recognition and to develop more effective and clinically applicable measures for the treatment of autoimmune diseases,tumors or transplant rejections.The whole project includes four parts:1.The distribution of intravenously infused donor apoptotic cells in recipient organs and its significance in immune regulationPurposes:To investigate the distribution of intravenously infused donor apoptotic cells, and to explore the significance of phagocytotic cells and local cytokines in the induction of local immune regulatory environment.Methods:Inbred mice C57BL(H-2~b) and Balb/C(H-2~d) were chosen to be donors and recipients,respectively.C57BL donor splenocytes were first stained with 2μM CFSE and treated with Dexamethasone(Dex) to induce apoptosis.In some experiments,donor apoptotic cells were enriched with Annexin-V beads(Mitenyl).In vivo,2×10~7 C57BL apoptotic splenocytes were injected intravenously into Balb/C recipients,and liver,spleen and blood samples were collected at different time intervals after injection.The cells were analyzed by fluorescence histochemistry and flow cytometry to examine the in vivo distribution of apoptotic donor cells.Meanwhile,the cytokine concentrations in the tissues of liver and spleen,and in serum were determined with Bioplex system or ELISA. In vitro,CFSE~+ apoptotic C57BL splenocytes were co-cultured with APCs isolated from Balb/C liver and phagocytosis of apoptotic cells by liver APC was analyzed by fluorescence microscopy,immunohistochemistry,and flow cytometry.Results:Apoptotic donor cells were mainly deposited in the liver,but hardly found in the spleen.Thirty to sixty minutes after the injection,donor apoptotic cells mainly accumulated in the liver and were rapidly cleared,usually within 90 minutes.Both donor and recipient apoptotic cells displayed similar distribution,indicating that the deposit of donor apoptotic cells in the recipient liver depends on the nature of apoptosis,not on their allogenicity.In vivo and in vitro experiments revealed that all three kinds of liver APCs, including liver sinusoid endothelial cells(LSEC),Kupffer cells(KC) and dendritic cells (DC),effectively phagocytosed donor apoptotic cells.In the liver,spleen and serum,11 cytokines including IL-1β,IL-2,IL-4,IL-5,IL-6,IL-10,IL-12,TNF-α,IFN-γ,GMCS and TGF-β,were detected at different time intervals after the infusion of donor apoptotic cells. Although Th1 response was found both in the liver and spleen,a stronger,more rapid and sustained Th2 response revealed in the liver,but not in the spleen.In the serum,neither Th1 nor Th2 responses were found.Conclusion:The deposition of donor apoptotic cells in the recipient liver and the induction of a liver specific Th2 response might contribute to tolerance inductivity of donor apoptotic cells.2.The effect of donor apoptotic cells on in vivo cytotoxicity against donor cells and on regulatory T cells(Tregs)Purposes:To explore the influence and significance of donor apoptotic cells on the in vivo cytotoxicity against donor cells and on Tregs. Methods:Inbred mice C57BL(H-2~b) and Balb/C(H-2~d) were chosen to be donors and recipients.Donor and recipient splenocytes were stained with different concentrations of CFSE respectively and a mixture of CFSE stained donor and recipient cells were injected intravenously into recipient mice.Then at different time intervals,peripheral blood were harvested and analyzed by flow cytometry to determine the clearance rate of CFSE~+ donor cells in comparison to CFSE~+ recipient cells.In vivo cytotoxicity was calculated according to clearance rates of CFSE~+ donor cells and CFSE~+ recipient cells.The accuracy,specificity and sensitivity of the in vivo cytotoxicity test were verified in mouse skin allograft and immunosuppressive models,and in nude mice.For investigating the influence of donor apoptotic cells on in vivo cytotoxicity and Tregs,2×10~7 apoptotic, necrotic or living C57 splenocytes were intravenously injected into Balb/C mice,and at different time intervals,peripheral blood samples were collected and analyzed by flow cytometry for Tregs.And at the same time,a mixture of CFSE~+ donor and recipient cells was intravenously injected and peripheral blood collected for the evaluation of in vivo cytotoxicity against donor target cells.Results:An injection of CFSE~+ donor and recipient cells with cell number between 1×10~7 and 1×10~8 made those cells easily detected in recipient peripheral blood.In skin allograft sensitized recipients,CFSE~+ donor cells were rapidly eliminated within 1-2 hr, while in those un-sensitized animals the elimination tempo was significantly slower so that they only started to be cleared 4 hrs,significantly eliminated 24hrs and vanished 72 hrs after the injection.In immunosuppressed or nude mice,the elimination rate of test donor cells was significantly decreased.Although CFSE~+ donor cells were rapidly cleared in mice that had been treated with donor necrotic or living cells,they were not eliminated in mice treated with apoptotic cells,indicating that in vivo lymphocyte toxicity were increased in necrotic or living donor cell treated mice,but not increased in apoptotic donor cell treated animals.On the contrary,the proportion of Tregs in the peripheral blood was also increased in mice treated with apoptotic donor splenocytes. Conclusions:In vivo cytotoxicity is an accurate,simple and rapid method for the detection of lymphotoxicity.Donor apoptotic splenocytes,in contrast to necrotic or living donor cells,significantly inhibited lymphocytotoxicity in recipients,during which the increase of proportion of Tregs might play an important role.3.The effect of allogeneic donor apoptotic cells on renal allograft survival and on the proportion of Tregs in monkey recipientsPurpose:To examine the effect of intravenously infused allogeneic donor apoptotic cells on renal allograft survival in a rhesus monkey renal transplant model and to investigate the significance of Treg in donor apoptotic cell-induced immune regulation.Methods:First,we compared three methods to test the human-like blood types in rhesus monkey,including monoclonal antibody test,agglutination inhibition test and reverse blood group test.The results showed that reverse blood group test,i.e,testing A,B-antigen specific antibodies in the serum rather than the antigens on erythrocytes,could be used to identify blood types in rhesus monkeys.Rhesus monkey allogeneic renal transplantation was performed by planting donor left kidney in the lower abdomen without nephrectomy of their own kidneys.Usually,donor left kidney was procured and perfused ex vivo with cold HCA solution.For transplantation operation in the recipients,donor renal vein and artery were anastomosed to recipient infra vena cava and abdominal aorta,respectively.Graft ureter was anastomosed directly to the bladder and a double pig-tailed tube placed in its lumen with two ends entending to the renal graft pelvis and to the bladder.High energy X-ray,Co-60-γray,and ultraviolet C(UVC) were tested for the induction of peripheral blood mononuclear cells(PBMC) apoptosis in the rhesus monkeys. Consequently,large number of apoptotic PBMC can be induced by UVC irradiation(dish diameter 6cm,RMPI-1640 supplemented with 10%bovine serum,distance 20 cm and irradiation time 60 minutes). For evaluating the effect of donor apoptotic cells on renal allograft survival,7.2-7.4×10~7 allogeneic donor apoptotic PBMC,or PBS for the control animal,were intravenously injected into recipients one week before allogeneic renal transplantation.The animals were treated with combined immunotherapy,including methylprednisolon(MP), cycloheximide(CHX) and rapamycin(RAPA),in which RAPA was administrated daily for 14 days with a dose approximately about one fifth clinical dose.Ultrasound and CT scan were used to monitor allograft rejection and survival.At the same time,the proportion of Tregs in recipient PBMC was also analyzed weekly after the infusion of donor apoptotic cells.Results:In 43 rhesus monkey tested by reverse blood type method,B type was the most popular,accounting for 58.14%(25 monkeys),followed by AB(34.88%,15 monkeys) and A(7%,3 monkeys),while no O type found.Four monkeys underwent renal transplantation without immunosuppression.In 4 monkeys,the transplantation operation was finished smoothly with immediate urination and blood flow found normal 2 days after operation.One of the monkeys developed swelling of the abdomen 3 days after transplant surgery.Venous embolism was found and the graft removed immediately.Another monkey died 7 days later because of intestinal obstruction and the other two monkeys recovered smoothly although renal graft rejected 5 days after operation.Three monkeys selected for studying the effect of donor apoptotic cells on renal allograft survival were administrated triple immunotherapy, MP+CHX+RAPA.Among them,one week before transplant surgery,two monkeys were injected with 7.2×10~7 or 7.4×10~7 donor apoptotic cells,while the other monkey were infused PBS.In the two monkeys received donor apoptotie cells,renal allograft survival were 14 days and 33 days,while in the monkey infused with PBS,renal allograft survival was 14 days.The proportion of Tregs in the monkeys treated with donor apoptotic cells was higher than that in PBS treated animal.Renal allograft survival was the longest in the animal with highest proportion of Tregs. Conclusion:Reverse blood type test is a reliable and effective method for the detection of rhesus monkey blood types.Donor apoptotic cells may prolong monkey renal allograft survival,possibly by enhancing the proportion of Tregs in the recipients.4.Establishment of a "cell death" immune recognition modelPurpose:To establish an immune recognition model that can better explain various immune phenomena.Method:Theoretical analysis based on the data obtained in our laboratory in the past 10 years and on literature reports.Different immune recognition models,including classic "self nonself" model(clonal selection theory) and contemporary "danger" model and "infectious non-self" model,were compared.Results:"Cell death" immune recognition model mainly comprises four principles in immune recognition and can better explain autoimmunity,tumor immunity,transplant immunity,fetal immunity,liver immunity and the role of Tregs in immune responses.Conclusion:Active immune response or immune tolerance of the immune system to an antigen is neither dependent on whether the antigen is self or non-self,nor on the presence of "danger" or infection,but on the way of cell death during antigen presentation. Apoptotic cells induce tolerance while necrotic cells initiated immune response.更多还原