【作者】 卢彩宝；

【导师】 吴雄飞；

【作者基本信息】 第三军医大学 ， 外科学， 2008， 博士

【摘要】 肾脏移植是临床根治慢性终末期肾病的有效方法,随着配型技术和免疫抑制剂的进步,移植后急性排斥反应发生率降低,短期存活率大幅提高。但是移植后发生一定比例的急性排斥反应(acute rejection, AR)仍然是肾移植术后的主要并发症之一,也是导致慢性排斥反应(chronic rejection, CR)和移植肾失功的重要危险因素。同种异体肾移植术后发生排斥反应主要有两种机制,一是抗体介导的体液免疫,二是T细胞介导的细胞免疫,两种理论的根本区别在于移植物的损害是由抗体的活动所造成,还是直接的细胞毒作用,即由T细胞、NK细胞等细胞毒或迟发性超敏反应所造成的。如果受者体内存在或出现针对供者特异性的抗体,通常称为急性体液性排斥反应(acute humoral rejection,AHR)或血管性排斥反应,体液性排斥反应导致移植物失功仍是临床治疗中的难题。2001年,第六届Banff肾移植病理会议正式将同种异型抗体介导的肾移植排斥反应的病理诊断补充入Banff国际分型标准,急性抗体介导的移植肾排斥反应诊断标准包括3个基本特点:急性组织损伤的形态学证据;抗体作用的免疫病理学证据(如肾小管周围毛细血管C4d沉积);循环供体特异性HLA抗体或其他供体内皮细胞抗原特异性抗体的血清学证据。Colvin将抗体引起的移植物损伤进一步分为四种类型:超急性排斥反应、急性排斥反应、慢性排斥反应和无临床症状的适应状态。介导体液性排斥反应的抗体多为HLA抗体,我们临床检测多可以避免这种排斥反应,但那些未知抗体目前还没有引起足够的重视,虽然发生几率不高,但一旦发生,尚无特异治疗手段。因此,鉴别这些非抗HLA抗体并阐明其作用机制,对体液排斥反应的有效治疗显得非常重要。非HLA抗体包括抗MICA抗体、抗MICB抗体、抗内皮细胞抗体和新近发现的抗血管紧张素Ⅱ1型受体自身抗体(Autoantibodies against the angiotensinⅡtype 1 receptor, AT1-AA)等。AT1-AA于1999年在先兆子痫患者中发现的,直到2005年Dragun等才发现在发生顽固性血管性排斥反应的患者中存在AT1-AA,并认为AT1-AA在介导血管性排斥反应中起重要作用。为证明AT1-AA可引起血管性排斥反应,Dragun等将含有人AT1-AA的血清注射到肾移植大鼠。实验使用Fischer344-Lewis肾移植模型,移植后一周,移植肾显示动脉内膜炎和血管内浸润。但该实验采用人血清,并且不是纯化的抗体,不能完全排除存在异种抗原抗体反应的可能性,因此,我们认为需要采用大鼠源性AT1-AA针对大鼠进行实验,才能更直接阐明AT1-AA对移植肾脏的损伤作用。Dragun等报道的16例AT1-AA引起的血管性排斥反应患者中只有5例C4d阳性,说明其排斥进程与HLA抗体介导的血管性排斥反应有所区别。我们推测AT1-AA介导的血管性排斥反应机制并非如HLA抗体那样结合细胞表面抗原后激活补体导致细胞的损伤,其可能的机制是与靶细胞表面的AT1R结合后启动细胞内信号转导机制,促进细胞的增殖或凋亡。为验证以上假设,我们将观察内皮细胞(endothelial cell, EC)受到AT1-AA刺激后的功能改变,细胞间粘附分子-1(Intercellular adhesion molecule-1,ICAM-1)作为急性排斥反应和内皮激活的重要指标将作为检测对象。体液性排斥反应学说认为EC是抗体作用的最初靶点。血管内皮细胞在血液和组织液间有广泛的联系和物质交换,不仅具有选择性通透性、抗血栓性、血管紧张调节、毛细血管形成、产生细胞因子、粘附因子及抗肿瘤活性等功能。细胞间粘附分子-1是免疫球蛋白超家族中的一员,文献报道急性排斥反应时,肾小管上皮细胞和肾血管内皮细胞上ICAM-1表达明显增加,且肾小管上皮细胞上ICAM-1的表达与排斥的严重性呈正相关。ICAM-1不仅是急性排斥反应的始动因素,而且在免疫应答和移植物的排斥反应中发挥重要作用。AT1-AA是否会刺激内皮细胞增殖,导致ICAM-1的合成与分泌增加从而加重或启动血管性排斥反应尚无相关研究报道。本实验将用AT1-AA刺激培养的大鼠动脉内皮细胞,检测ICAM-1的合成和释放是否增加。有研究显示,AngII与AT1R结合后是通过MAPK、JAK/STAT等信号转导通路实现生物学功能,特别是在内皮细胞损伤、血管平滑肌细胞增殖和动脉粥样硬化的形成方面发挥作用。我们推测AT1-AA可能是通过与这些细胞表面的配体(AT1R)结合,转导细胞内信号。本实验将研究MAPKs信号转导通路在AT1-AA介导内皮细胞功能变化中的作用。对AT1-AA进行研究必须获得一定数量的AT1-AA,并对其进行检测和鉴定。我们拟用大鼠AT1R多肽免疫大鼠,观察是否可诱导产生AT1-AA;将AT1-AA注射到肾移植大鼠,观察是否引发排斥反应,探讨AT1-AA对肾血管内皮细胞的信号转导途径,然后选用可能的阻断方法。通过上述研究,阐明同种AT1-AA确可介导早期体液性排斥反应、缺血再灌注损伤的移植肾内皮细胞为抗体作用的主要靶点、AT1-AA与受体结合后的主要信号转导通路。本课题对于揭示AT1-AA介导体液性排斥反应的发生、作用机理具有重要意义,为临床监测和治疗这一特殊类型排斥反应提供理论依据。主要研究内容:1.本研究拟用大鼠源性AT1R多肽片段偶联血蓝蛋白制备抗原并免疫大鼠,监测免疫的大鼠体内是否产生AT1-AA,如有AT1-AA生成则对其进行分离、纯化和鉴定,并观察其对自身组织有无病理性损伤。2.采用制备的大鼠源性AT1-AA注射到肾移植受体大鼠,观察受体大鼠是否发生急性血管性排斥反应,并重点观察血管内皮细胞有无损伤;给予口服氯沙坦有无保护作用。3.在体外实验中观察AT1-AA对培养的内皮细胞的作用,监测内皮细胞受到AT1-AA刺激后ICAM-1的表达变化。研究AT1-AA诱导内皮细胞ICAM-1表达的信号通路,探讨AT1-AA介导内皮细胞ICAM-1变化的分子机制。使用氯沙坦阻断对信号转导通路及ICAM-1合成有无影响,探讨氯沙坦是否可作为临床预防和治疗AT1-AA所致排斥反应的措施。主要实验方法和结果:1.大鼠源性AT1R多肽偶联血蓝蛋白制备免疫原对大鼠进行免疫,第二次加强免疫后大鼠血清AT1-AA可疑阳性,第三次加强免疫后AT1-AA血清阳性率90 %,第四次加强免疫后,免疫组AT1-AA阳性率100 %,其中AT1-AA滴度在第13周达到高峰。使用protein G-sephrose 4B亲合层析纯化AT1-AA,所得到的抗体进行生物学鉴定,可以使培养的心肌细胞搏动速度加快。光镜及电镜未观察到明显的心脏、肾脏和肝脏病理改变。2.成功建立了大鼠肾移植模型,SD大鼠的MHC个体差异很小,组织相容性很好,移植肾病理改变轻微,术后第7天活检按Banff’97在ⅠA以下,肾小管、肾小球、微血管的损害很轻,可作为急性排斥反应的对照。3.肾移植大鼠注射AT1-AA,发生急性血管性排斥反应,光镜下表现为单核细胞、淋巴细胞浸润累及微血管,形成动脉内膜炎,肾小球毛细血管炎症细胞浸润,但未见透壁性纤维素样动脉内膜炎;电镜下显示内皮细胞肿胀,破裂形成碎片向管腔内突出、脱落,内皮细胞间间隙增宽,连接不完整;血管周围水肿,管腔内炎症细胞较多并向管腔外游出。肾移植大鼠注射AT1-AA的同时给予氯沙坦口服具有明显的保护作用,移植肾功能得到保护,移植肾损伤明显减轻。4.采用组织块移植法进行大鼠主动脉内皮细胞原代培养,并对培养的内皮细胞进行纯化和传代培养,经免疫荧光测定,其标志物vWF表达阳性,纯度高,可满足实验需求。5.用不同浓度的AT1-AA(1:200,1:100,1:50)刺激内皮细胞,在刺激后不同时间分别检测ICAM-1蛋白表达。结果显示,内皮细胞经AT1-AA作用后,ICAM-1水平在0-24h内随作用时间的延长逐渐升高,至24h时,ICAM-1水平达到最高,36h开始下降。不同浓度的AT1-AA所导致的ICAM-1水平升高程度有所不同,浓度越高,对内皮细胞内合成ICAM-1的影响越大,其中以稀释度为1:50的AT1-AA对其影响最大。6.使用制备的AT1-AA刺激培养的内皮细胞,在不同的时间点检测ERK1/2、p38MAPK和JNK/SAPK的水平。结果发现,AT1-AA能够激活ERK1/2和p38MAPK,在20分钟左右活化作用最强,随着时间的延长,ERK1/2、p38MAPK水平逐渐下降;AT1-AA对JNK/SAPK无明显活化作用。7.使用不同浓度的PD98059(30μM、60μM)和氯沙坦(10-6mol/L、10-5mol/L)可明显抑制ERK1/2活性,其中60μM PD98059和10-5mol/L氯沙坦对ERK的抑制最为显著。在p38MAPK通路的实验中显示不同浓度的SB203580(20μM、40μM)和氯沙坦(10-6mol/L、10-5mol/L)也在不同程度上抑制p38MAPK的激活,其中40μM SB203580和10-5mol/L氯沙坦效果较为明显。8.分别使用SB203580、PD98059和氯沙坦对内皮细胞进行预处理,在加入AT1-AA后24h收集内皮细胞检测ICAM-1的合成情况,结果发现氯沙坦和SB203580联合对内皮细胞预处理对ICAM-1的升高抑制最为显著,可以抑制约97%,单独加入PD98059或单独加入氯沙坦对AT1-AA引起的ICAM-1升高的抑制相对较低,分别为40%和55%。结论:1.大鼠源性AT1R多肽能够诱导大鼠产生AT1R自身抗体,此抗体对自身组织的损伤作用不明显。本实验证实了大鼠源性AT1-AA的存在,并成功的对抗体进行分离、纯化,为进一步研究其在器官移植中的作用提供了实验基础。2.采用制备的大鼠源性AT1-AA注射到肾移植受体大鼠,观察发现受体大鼠发生急性血管性排斥反应,光镜和电镜显示移植肾血管内皮细胞有明显损伤。3. AT1-AA可引起部分难治性血管性排斥反应,推测其可能机制是与内皮细胞表面AT1R结合后启动细胞内信号转导通路,造成内皮功能紊乱,表现之一是合成并分泌ICAM-1,进而导致白细胞浸润并进一步破坏内皮细胞结构和功能,形成恶性循环,最终导致移植物失功。使用AT1R阻断剂(如氯沙坦)可以减轻这种排斥反应,对移植肾有益,提示临床移植前后需要检测受体AT1-AA水平,特别是既往妊高症或恶性高血压患者,治疗AT1-AA引起的排斥反应可以早期使用ARBs。4.用AT1-AA刺激培养的大鼠动脉内皮细胞,可以诱导ICAM-1的合成和释放增加,AT1-AA可以活化ERK1/2、p38MAPK,使用ERK1/2通路阻断剂PD98059、p38MAPK通路阻断剂SB203580和氯沙坦能抑制ICAM-1的合成。提示我们预防和治疗AT1-AA介导排斥反应的手段可以考虑使用ARBs、ERK/p38MAPK通路阻断剂以及ICAM-1与LFA-1结合位点阻断,其作用点各不相同,我们把它们分别称为受体结合阶段、信号转导阶段和效应分子阶段,为将来AT1-AA的治疗提供重要理论参考。更多还原

【Abstract】 Renal transplantation has been an effective and radical treatment for end-stage renal diseases. With the development of technology in tissue matching and the wide application of immunodepressants, the incidence of acute rejection after renal transplantation has decreased and the short-term survival after transplantation has dramatically increased. However, acute rejection is still one of the major complications after renal transplantation, and is also the main risk factor leading chronic rejection and graft dysfunction. There are two main mechanisms of rejection after allograft renal transplantation, one is antibodies-mediated humoral immunity, the other is T cell-mediated cytoimmunity. The basic difference of these two mechanisms is that, for the former the graft injury is caused by antibodies, while for the latter the graft injury is caused by direct cytotoxic actions induced by T cell or NK cell or tardive hypersensitivity reaction. If there are preexisting or newly developed antibodies in the blood of recipient specifically against the donor, the acute rejection after renal transplantation is called acute humoral rejection(AHR) or vascular rejection. Humoral rejection induced graft dysfunction is still a problem in clinical practice. The standardized criteria for the pathological diagnosis of AHR was discussed on the Sixth Banff Conference on Allograft Pathology in 2001. Diagnostic criteria for acute antibody-mediated rejection includes: a) morphologic evidence of acute tissue injury; b)Immunopathologic evidence of antibody action; c)Serologic evidence of circulating antibodies against donor HLA or other anti-donor endothelial antigen. In the past decade, four forms of antibody-mediated graft injury have been defined as hyperacute rejection, acute humoral rejection, chronic humoral rejection and accommodation. Most of the antibodies that mediate humoral rejection are against HLA and can be detected, and the rejection mediated by them can be avoided. While, although the low incidence, there is still no specific treatment for the rejection mediated by other unknown antibodies. For this reason, it is important to identify and elucidate the mechanisms by which non anti-HLA antibodies contribute to humoral rejection to provide effective treatment for humoral rejection. Non anti-HLA antibodies include anti-MICA antibody, anti-MICB antibody, anti-endothelial cell antibody and autoantibodies against the angiotensinⅡtype 1 receptor(AT1-AA), which was discovered in 1999 in patients of preeclampsia. Until 2005 Dragun et al reported the presence of AT1-AA in recipients of renal allografts who had severe vascular rejection and concluded that AT1-AA played an important role in mediating humoral rejection. In order to find out whether AT1-AA can induce vascular rejection, the serum containing human AT1-AA was injected into rat recipients(Fischer344-Lewis renal transplantation model). One week after the surgery, endarteritis and intravascular infiltrates were observed in the graft. For the fact that human serum is heteroantigen for rat, it is still not clear whether it is AT1-AA which specifically causes this injury. A better strategy is to use rat-derived AT-AA to see whether this injury will happen again. We presume that the possible role of AT1-AA in mediating humoral rejection is to bind to AT1R on the target cell surface and then initiate intracellular signal transduction, to promote cell proliferation or apoptosis, which is different from the role of anti-HLA antibodies which activate complement after binding to cell surface antigen. In order to verify our presumption, functional exchange of endothelial cell(EC) stimulated by AT1-AA will be observed and intercellular adhesion molecule-1(ICAM-1), a marker of acute rejection and endothelial cell activation, will be detected.In the theory of humoral rejection, EC is the primary target of antibodies. EC is a type of cell equipped with unique biological functions, serving as a barrier between blood and tissue, a place where cells from the donor and the receptor first meet, where the battle between them first occurs. The rejection mediated by antibody post renal transplantation is characterized by inflammation in EC, and subsequent cell infiltration and broken EC, followed by the activation of EC and apoptosis. There are wide exchanges of molecules between EC and blood, or tissue fluid. EC has many biological functions, such as selective penetration, anti-thrombosis, regulation of tension of blood vessels, involved in formation of capillary, producing cytokines, adhesions and anti-tumor, etc. ICAM-1 is a member of immunoglobulin superfamily, and has been reported to be over-expressed by tubular epithelium and vascular EC during acute rejection. The increase expression of ICAM-1 is also positively correlated with the severeness of the rejection. This suggests that ICAM-1 is an important factor for acute rejection initiating and immunologic response. It is still unknown whether AT1-AA can promote EC proliferation and lead to increased ICAM-1 synthesis and secretion, accordingly trigger or aggravate humoral rejection. In this study, cultrued rat artery EC is stimulated by AT1-AA and whether ICAM-1 synthesis and secretion is increased will be detected. It has been reported that AngII exerts its biological functions through binding to AT1R and activating signal transduction pathways such as MAPK and JAK/STAT, playing a significant role in EC injury, vascular smooth muscle cell proliferation and atherosclerosis forming. We suspect that AT1-AA probably though binding to sell surface ligand(AT1R) to transducts intracellular signal. In this study the role of MAPKs signal transduction pathway in AT1-AA mediated EC functional changes is also evaluated.It is necessary to obtain adequate AT1-AA and to identify and detect it. In this study, rats are immumized with rat-derived AT1R polypeptide and whether AT1-AA can be induced is observed. Then AT1-AA is injected into rat recipients of renal allograft and whether there is a rejection is observed. The signal transduction pathways AT1-AA involved in kidney vascular EC are determined to help to find possible blocking methods. This study aims to demonstrate that homologous AT1-AA can definitely mediate early humoral rejection with graft EC undergoing ischemia-reperfusion injury as the main target cell and to find out the main signal pathways AT1-AA activated following binding to its receptor. The study will provide important evidence for elucidating the mechanism underpinning AT1-AA mediated humoral rejection and also offer theoretical support for the treatment of this type of rejection in clinical practice.Main contents:1. Rat-derived AT1R peptides were coupled with keyhole limpet hemocyanin(KLH) as the antigen to immunize rats. AT1-AA was examined, isolated, purified and identified from immunized rats serum. The toxicity of AT1-AA obtained was also evaluated by examining the tissue damage in immunized rats.2. Rat-derived AT1-AA was injected into rat recipients of renal allograft and whether acute vascular rejection occured were observed. Vascular EC injury was espcially monitored. Whether oarl administration of Losartan was protective was also observed.3. The effect of AT1-AA on cultured EC was observed in vitro studies and ICAM-1 expression change in EC stimulated by AT1-AA was detected. The signal pathways and molecular mechanism of ICAM-1 expression in EC stimulated by AT1-AA were studied. Whether Losartan had a blocking effect on signal pathways and ICAM-1 synthesis and whether it could contribute to prevention and treatment of AT1-AA mediated rejection were evaluated.Main methods and results:1.Rat-derived AT1R peptide coupled with KLH was used as immunogen to immunize rats. After the second enhanced immunization, suspicious positive result for AT1-AA was obtained from rat serum. After the third enhanced immunization, the positive rate for AT1-AA from serum was 90%; after the fourth, 100%. The concentration of AT1-AA peaked at the 13th week. AT1-AA was purified by protein G-sephrose 4B affinity chromatograph and subsequently identified by biological function. No significant changes were observed in heart, kidney and liver in rats as assessed by light microscopy and electon microscopy.2. Rat renal transplantation model was successfully established. Since little difference in MHC existed between individual SD rats, no obvious pathological changes were observed after the surgery. Kidney biopsy on the 7th day post-transplantation indicated a level lower than I A according to Banff’97. The injury in renal tubules, glomeruli, and microvessel was negligible, which was served as the negative control for acute rejection.3. Injection of AT1-AA into rat recipients of renal allograft induced acute vascular rejecion. Under light microscope, it was featured by infiltration of mononuclear cells and lymphocytes into microvessels, endarteritis occured, neutrophils and mononuclear cells in glomeruli, but not capillary thrombosis and not fibrinoid necrosis. Under electron microscope, it was featured by EC swelling, breaking into pieces and desquamating from vessel wall, enlargement of interspace among Ecs, uncompleted conjunction, edema around the vessels and external migration from vessel of inflammatory cells. Losantan exerted an obvious protective effect on AT1-AA induced pathology with improved renal function.4. Rat aorta EC was primary cultured using tissue transplant method, purified and then secondary cultured. The high quality of EC was ascertained by immunofluorescent staining of vWF.5. EC was stimulated by AT1-AA of different concentration(1:200,1:100,1:50) and expression of ICAM-1 was detected at different time after stimulation. The result revealed that expression of ICAM-1 was gradually upregulated as time passed within the first 24 hours, peaking at hour 24 and began to wane from hour 36. It was also found that this effect was AT1-AA dose-dependent: the higher the concentration of AT1-AA, the higher increase of expression of ICAM-1, with the concentration of 1:50 being the most effective one.6. AT1-AA was used to stimulate cultured EC. ERK1/2, p38MAPK and JNK/SAPK were examined respectively at different time point after stimulation. Results revealed that AT1-AA could activate ERK1/2 and p38MAPK with the most powerful activation at 20 minutes after stimulation. Thereafter the level of ERK1/2 and p38MAPK was attenuated. However AT1-AA had no obvious stimulating effect on JNK/SAPK.7. Different concentrations of PD98059(30μM and 60μM)and losantan (10-6mol/L and 10-5mol/L)could dramatically suppress the activity of ERK1/2, with 60μM PD98059 and 10-5mol/L losantan being the most potent. Different concentrations of SB203580(20μM and 40μM)and losantan (10-6mol/L and 10-5mol/L)could also suppress the activity of p38MAPK, with 40μM SB203580 and 10-5mol/L losantan being the most effective.8. EC was pretreated with SB203580, PD98059 and losantan. Then AT1-AA was added to stimulate the increasing expression of ICAM-1. EC was collected to examine the level of ICAM-1 24 hours after the addition of AT1-AA. Results revealed that the combination of losantan and SB203580 had the most powerful suppression on the increase of ICAM-1, which was 97%. While either PD98059 or losantan alone could only achieve 55% and 40%.Conclusion:1. Rat-derived AT1R peptide has the ability of inducing AT1R auto-antibodies, which has no apparent damaging effect on host tissues. The study confirms the exist of rat-derived AT1R and successful isolation and purification of AT1R auto-antibodies, which provides the experimental basis for further study on effect of AT1R auto-antibodies on transplantation.2. Injection of rat-derived AT1-AA into rat recipients of renal allograft can induce acute vascular rejection and obvious damages in vascular EC of the graft under light microscope and electron microscope. 3. AT1-AA can induce partly refractory vascular rejection. The possible mechanism of rejection might be AT1-AA binding to AT1R on the surface of EC and subsequent activation of intra-cellular signal transduction, which leads to EC dysfunction with increasing synthesis and secretion of ICAM-1 being one of the features. Increased ICAM-1 induces leukocytes infiltration and disorder of EC structure and function. The vicious circle finally leads to graft function failure. Losartan partly prevents vascular rejection and is effective on protection of the graft. It suggests that AT1-AA should be detected before and after transplantation, especially for patients who used to have pregnancy hypertension and malignant hypertension. ARBs is a good choice for early treatment of rejection induced by AT1-AA.4. Synthesis and secretion of ICAM-1 is increased in cultured rat aorta EC stimulated by AT1-AA. AT1-AA can activate ERK1/2 and p38MAPK, while ERK1/2 antagonist PD98059, p38MAPK antagonist SB203580 and Losartan can inhibit the increasing of ICAM-1. This study indicates the possible use in prevention and treatment of AT1-AA induced rejection of ARBs, ERK/p38MAPK antagonists and blocking the binding site of ICAM-1 and LFA-1, which lie in different stages of receptor binding stage, signal transduction stage and effectors stage. This will provide important theoretical references for further treatment by AT1-AA.更多还原