【作者】 陈松；

【导师】 陈实；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 因输血、妊娠、多次移植等原因成为预致敏的高危受体病人，在接受同种异体器官移植后易发生较严重的体液性排斥反应，如超急性、加速性或急性血管性排斥反应。且随着器官移植开展历史的延长，等待接受二次、甚至三次移植的预致敏患者将越来越多。目前临床针对这些预致敏患者的“脱敏”治疗，价格昂贵、副作用大、效果不稳定且仅适用于部分病人。我们拟建立非人灵长类动物皮肤预致敏后肾脏移植的动物研究模型，尝试应用“适应”策略诱导移植肾的长期存活，并阐述其相关机制。准确的鉴定非人灵长类的血型是非人灵长类动物器官移植研究的基础。非人灵长类的ABO血型抗原都表达在组织器官内，而不是在红细胞上，这给非人灵长类血型的鉴定带来很大的困难。为找到更加简捷、准确鉴定非人灵长类动物类人ABO血型的方法，采用近年来临床上广泛应用的卡式微柱凝胶正、反定型法对38只猕猴和26只食蟹猴的血型进行了鉴定，并与肾组织免疫组化法的检测结果进行比较。结果显示：卡式微柱凝胶正定型法的检测结果中无一例为阳性结果；血浆中的纤维蛋白原和人-猴种属间非特异性抗体都会对卡式微柱凝胶反定型法的部分检测结果产生干扰；采用经正常人O型红细胞吸附处理后的清亮血清，卡式微柱凝胶反定型法的检测结果明确，与免疫组化法判定结果一致。由此得出：采用经人正常O型红细胞吸附处理后的清亮血清作为样本，卡式微柱凝胶反定型法可以准确的判定猕猴或食蟹猴的血型，是个更为简单、快捷的方法，推荐为非人灵长类血型检测的常规方法。建立非人灵长类预致敏后肾脏移植加速性排斥反应模型。取血型相容的正常猕猴配对，预先将供体腹部全层皮肤移植到受体背部，使受体预致敏。2周后再将同一供体的左侧肾脏移植到受体腹腔内，同时切除受体猴自体双肾。以未致敏的猴肾移植作为对照，观察术后受体猴血肌酐水平变化，移植物存活时间及病理特点；观察临床常用的CsA为基础的三联免疫抑制治疗(CsA+MMF+强的松)是否可以延长该模型移植物存活时间。结果显示：4例未致敏肾移植猴(对照组，第1组)的存活时间分别为9、1 8、8和7天；3例致敏后肾移植猴(致敏后未用药组，第2组)的存活时间分别为3、3和4天；3例致敏后肾移植猴行CsA三联免疫抑制治疗(致敏后用药组，第3组)，存活时间分别为2、3和4天。我们得出结论：①受体猴被供体皮肤预致敏后再行肾脏移植，可以加速移植物的排斥反应，是首个更加接近于人类预致敏后排斥反应特点的大动物研究模型。②该模型模拟的预致敏后加速性排斥反应不能被CsA三联免疫抑制治疗方案逆转。观察非人灵长类动物预致敏后肾脏移植加速性排斥反应的免疫学及病理学变化。采用非人灵长类动物皮肤预致敏后肾脏移植加速性排斥反应模型，检测皮肤移植预致敏前后及肾移植后受体猴血清内供体特异性抗体的变化，并在发生排斥反应时对移植肾进行免疫组化(测定补体、抗体的沉积和各类型淋巴细胞浸润情况)及病理学分析。结果显示3例致敏未用药受体猴均发生了加速性排斥反应。2例受体猴在预致敏后血清中供体特异性抗体明显增加，对供体的淋巴毒反应明显升高；肾移植后受者血清中供体特异性抗体及针对供体的淋巴毒进一步升高。HE染色显示被排斥的移植肾内明显的动脉坏死、血栓形成、间质出血、中性粒细胞浸润；免疫组化及荧光染色显示移植肾内有大量的补体、抗体沉积(主要为IgG)，而少见各类型的淋巴细胞浸润。1例受体猴体内的供体特异性抗体及对供体淋巴毒反应的升高程度不如前2例明显，病理学变化以肾小管损伤为主。与致敏不用药组相比，用药组的3例受体猴移植肾被排斥的病理学变化较轻，没有典型的动脉坏死、血栓形成或中性粒细胞大量浸润的情况，但仍有一定量的抗体和补体沉积，同时伴有一定量的淋巴细胞浸润。我们得出结论：综合本部分的研究，我们得出以下结论：①非人灵长类动物预致敏后肾脏移植加速性排斥反应的免疫学机制为：皮肤移植预致敏可以诱导受体猴产生程度不等的预存抗体，导致大部分移植肾在术后早期发生主要由抗体和补体介导的严重的急性体液性排斥反应。②其病理学特点为：以大量出血、坏死、血栓形成、中性粒细胞浸润为主；有大量抗体和补体片段沉积；较少的淋巴细胞浸润为特点。③CsA三联免疫抑制治疗方案虽然不能延长预致敏非人灵长类动物的移植肾存活时间，但可以明显减轻移植肾的病理学变化，提示继续改进和加强免疫抑制治疗方案仍有可行性。针对预存抗体导致的体液性排斥反应，在非人灵长类动物预致敏后同种异体肾移植模型中研究短期补体抑制诱导“适应”的可行性及相关机制，以期为临床预致敏高危移植探索出一种简便易行的新方法。以猕猴为实验动物，先行供体皮肤移植致敏14天后再行肾移植术，建立预致敏同种异体肾移植模型。在CsA三联治疗的基础上加用国产高纯度眼镜蛇毒因子(CVF)短期抑制补体活性(第4组)(n=5)。观察移植肾存活时间，检测抗供体特异性抗体及淋巴毒的变化，定期穿刺活检标本或按计划切取的移植肾标本行HE染色以及一系列免疫组化检测。结果显示，在移植后2周内加用CVF治疗几乎可以完全清除受者循环中的补体C3，移植肾存活时间显著延长(41，140，＞250，＞500，＞700天)，与前3组比较有显著性差异(P＜0．05)。免疫学及病理学研究显示，加用CVF治疗的第4组在移植肾活检组织中仅有一定的抗体沉积，补体成分的沉积极少，虽然停用CVF后受者血清补体C3水平和CH50均恢复到正常水平，但移植肾仍能长期存活，另外可见保护性基因Bcl-2和补体调节蛋白CD46、CD59的表达明显上调。我们得出结论：在CsA三联免疫抑制维持治疗基础上，短期抑制受者补体活性显著延长预致敏后肾移植存活，大部分动物的移植肾在补体水平恢复正常后仍能存活良好，成功诱导出“适应”，其机制可能与移植物内保护性基因Bcl-2和补体调节蛋白的明显上调表达有关。为解决临床预致敏高危移植的难题提供了极具参考价值的实验依据。在以往的研究基础上，我们发现眼镜蛇毒因子(CVF)在器官移植领域有良好的应用前景。为此，我们对其理化性质和免疫原性进行深入研究。采用的云南孟加拉种眼镜蛇蛇毒因子(Y-CVF)较文献报道的其它各种CVF具有更高的活性和较少的用药量。为探讨Y-CVF静脉使用是否诱导非人灵长类动物体内产生特异性中和抗体和异种天然抗体，本研究给2只正常食蟹猴每2周静脉注射一次治疗剂量的Y-CVF(0．05mg／kg)，共4次，检测不同时间点血清内补体C3水平、总补体活性(CH50)、抗Y-CVF抗体和抗猪内皮细胞异种抗体的变化。结果显示，前2次注射Y-CVF均有良好的清除补体效果，第3次注射Y-CVF时补体仅被部分灭活，第4次注射Y-CVF则基本无效。Western Blot和ELISA均证实特异性抗Y-CVF抗体产生，且其滴度随着Y-CVF注射次数增加而递增。多次注射Y-CVF后，并没有在血清内检测到明显的抗猪内皮细胞抗体的变化。同时，在本研究中，我们还观察了2例前期研究中长期存活的猕猴。在这2例猕猴的用药方案中，Y-CVF和CsA+MMF+Pred联合使用，且仅在术后早期2周内多次小剂量使用。我们在猕猴体内的补体水平恢复正常后，对血清内的抗Y-CVF抗体进行了检测，发现并没有很高滴度的中和抗体存在。在整个Y-CVF治疗过程中，补体C3水平一直维持在极低水平，也没有Y-CVF注射无效的现象。我们因此得出结论：①Y-CVF是一种具有更高抑制补体活性的蛇毒因子。②Y-CVF具有一定的免疫原性。单独使用能诱导非人灵长类动物产生中和抗体，从而导致Y-CVF失效。③Y-CVF未诱导抗猪抗体(含抗α-Gal抗体)的水平升高。④在联合使用免疫抑制剂的基础上，短期多次小剂量使用Y-CVF，可以明显降低中和抗体的产生，具有良好的临床应用前景。总之，我们首先创建了简单的、准确的鉴定非人灵长类动物血型的新方法，成功的建立了非人灵长类动物的预致敏肾脏移植加速性排斥反应模型。并在此基础上，首次在非人灵长类预致敏后成功获得同种肾脏移植的长期存活，并诱导出“适应”现象。我们也对该“适应”现象的机制进行了一定的探讨，发现其机制可能与保护基因BcL-2的高表达有关。另外，我们也首次在大动物“适应”现象中，观察到补体调节蛋白CD46和CD59的高表达现象，这可能是“适应”现象的另一个机制。更多还原

【Abstract】 Transplantation of grafts into presensitized recipients,which may result from multipleblood transfusions,previous pregnancy,or prior transplantation,results in severe humoralrejection,including hyperacute rejection,accelerated rejection and acute vascular rejection.Along with the increase of cases in transplantation,more and more presensitized recipientsare waiting for a second,even third transplantation.The present costly“desensitization”therapy to these presensitized patients has some unmanageable adverse side effects and isonly effective in some patients.In this study,we tried to establish a renalallotransplantation model in skin-presensitized non-human primates and induce long-timerenal allograft survival by an“accommodation”strategy.To know ABO blood types of monkeys exactly is the first step of the reaserch innon-human primate animal transplantation area.Monkeys do not express ABO antigens onred blood cells(RBCs),but on tissues,which is an obstacle of its ABO typing.In this studywe describe a simple and efficient method to blood type monkeys.Rhesus monkeys(n=38)and cynomolgus monkeys(n=26)were blood typed by the direct and reverse gel system,which is widely used in clinics in recent years.Based on the results ofimmunohistochemical staining,we evaluated the feasibility and the interference factors ofthe gel system to blood type monkeys.The results revealed that the direct gel system hadnone positive report,the fibrinogen and non-specific anti-human antibodies in monkeyblood interfered with the reverse gel system in some samples.Being in accord with theresults of immunohistochemical staining,using clear sera,which were pre-absorbed onnomal human type O RBCs,the reverse gel system gave an accurate blood typedetermination of monkeys.We concluded that the reverse gel system could be used forABO typing of monkeys,and the fibrinogen and non-specific anti-human antibodies werethe major interference factors,which could be eliminated by using clear and pre-absorbed sera.To establish an accelerated rejection model of renal allotransplantation in presensitizedmonkeys.Recipient rhesus monkeys were presensitized by grafting of 2 full-thicknessabdominal skin grafts from ABO-compatible donor monkeys and subsequently received akidney from the same donor two weeks later.In comparision with non-presensitised controlgroup,the changes of serum creatinine levels,survival time and the pathology of graftswere observed and studied.The efficiency of a regular clinical immunosuppressive regimenwith CsA+MMF+Prednisone on this presensitized transplant model was also evaluated.The results revealed that,four renal grafts in non-presensitized control group survived for 7,8,9,and 18 days respectively;In contrast,3 presensitized recipients without treatment(untreated presensitized group)rejected allografts in 3,3,4 days respectively;3presensitized recipients with triple therapy(treated presensitized group)rejected allograftsin 2,3,4 days respectively.We concluded that,presensitization by donor skin canacceletate allograft rejection,which can not be reversed by the administration of CsA incombination with MMF and prednisone.We described,for the first time,a non-humanprimate animal model of accelerated humoral rejection,which will enable us to moreextensively study in the case ofpresensitized or hyperimmunized patients.To study the immunologic and pathologic features of an accelerated rejection model ofrenal allotransplantation in presensitized monkeys.The accelerated rejection model of renalallotransplantation was established in presensitized monkeys,which received donor skintransplantation in advance.The changes of donor specific antibody(DSA)levels in therecipient monkeys before/after skin and kidney transplantation were measured.The kidneygrafts were examined for routine pathology,antibody and complement depositions,virouslymphocyte subsets infiltration by HE stainning,immunofluorescence,orimmunohistochemistry.The results revealed that,all renal allografts in presensitizeduntreated monkeys developed accelerated rejetion within 4 days.In 2 presentized monkeys,the levels of DSA and their mediated complement- dependent cytotoxicity(CDC)significantly increased after skin transplantation,and further markedly elevated at the timeof kidney graft rejection.In the rejected renal grafts,massive C3,C4,C5b-9 and IgGdeposits with few lymphocytes infiltration were found.Typical pathologic changes included severe arterionecrosis,thrombosis,interstitial hemorrhage,and infiltration of neutrophils.In the rest one presentized monkey,the levels of DSA and CDC only marginally increased,and the pathological changes of the rejected renal graft characterized mainly by the injuryof renal tubules.To compare with the results of presensitized untreated group,thepathological damage in renal allograft was much slighter in presensitized treated group.Weconcluded that,presensitization by donor skin transplantation could elevate the levels ofDSA and CDC in recipient monkeys,which resulted in severe antibody mediated acutehumoral rejection in most of the following renal transplants;The typical pathologic featureswere characterized by evidence of acute tissue injury,such as hemorrhagic lesions,necrosis,thrombosis and polynuclear cell infiltratioin;CsA+MMF+Pred administration did notprolonged graft survival,but could mitigate the pathological damage,which gave us a clue.The immunologically sensitized recipients with donor-specific antibody(DSA)areconsidered to be at greater risk for the development of acute humoral rejection followingtransplantation.Induction of self-protection against DSA-mediated humoral injury knownas accommodation may allow an allograft survives continuously in presensitizated recipient.The present study was undertaken to determine whether renal allograft accommodationcould be induced in donor skin-presensitized monkeys by short-term inhibition ofcirculating complement,and to investigate its possible mechanisms via analysis of certainintragraft protective genes expression.ABO-compatible and MHC-mismatched rhesusmonkeys were paired as donor and recipient.Donor skin allografts were transplanted topresensitize recipients 14 days prior to renal transplantation.Based on the CsA+MMF+Predtherapy,we added CVF administration(CVF+CsA+MMF+Pred group)(CVF,0.05 mg/kgiv.on day -2 and -1,then 0.02 mg/kg iv.every other day from POD 0-13,n=5).Serum DSAand its mediated CDC were detected by FACS using donor lymphocytes as targets.Intragraft expression of the antiapoptotic and complement regulatory proteins at differenttime-points was determined by immunohistochemical stainning and western blot.Theresults revealed that,triple therapy with CsA+MMF+Pred adding a short-term CVF therapyin the early period(2 weeks)significantly prolonged graft survival to a median survival of145 days(41,140,＞250,＞500,＞700 days;P＜0.01 vs.other groups).CVF treatment led tovery dramatic fall in serum C3 level and CH50 for at least 2 weeks,and 3 of 5 recipient monkeys received this therapy survived continuously with normal renal function andhistology even after the serum C3 levels returned to normal following the cessation of CVFtherapy.The expression of Bcl-2,CD46 and CD59 were significantly elevated in long-termsurvival grafts via analysis of immunohistochemistry at different time-points(especially onPOD 50 and 100).We concluded that,additionally using CVF in the early critical periodallowed long-term renal allograft survival in most presensitized recipients and most likelyinduced successful accommodation.The establishment of accommodation in this modelmay be associated with up-regulation of certain protective genes and complementregulatory proteins in grafts.These encouraging results indicate that the complementinhibition-based strategy may be valuable in future clinical cross-match positive orABO-incompatible transplantation.The preceding results presented an exciting possible application of Cobra venomfactor(CVF)in clinic.So a deeper research of its property and immunogenicity innon-human primates was necessary.In comparision with various CVF reported previously,Yunnan-cobra venom factor(Y-CVF)has higher anticomplement activity and lowertherapeutic dosage.To investigate whether Y-CVF could induce the specific neutralizedanti-Y-CVF antibody and xenoantibodies in non-human primates,two cynomolgusmonkeys were intravenous injected with Y-CVF(0.05mg/kg)every 2 weeks for 4 times.The changes of serum C3,CH50,anti-Y-CVF antibody and xenoantibody levels weremeasured.The results revealed that the first two injection of Y-CVF resulted in effectivedepletion of complement C3,the third injection had only incomplete effect and the fourthinjection was almost ineffective.The results of Western Blot and ELISA confirmed theproduction of anti-Y-CVF antibody,and its titre was increasing progressively along with theinjection times of Y-CVF.Additionally no significant changes of anti porcine endotheliacell xenoantibodies were found measured by flow cytometry.In addition,we also measuredthe level of anti Y-CVF antibody in the sera of two longtime survival rhesus monkeys,which accepted a short-term Y-CVF therapy in the early period(2 weeks)in combinationwith CsA+MMF+Pred.We found the level of the specific antibody was not high,and thelevel of C3 was steady low during the period of Y-CVF injection.We concluded that,Y-CVF was a more effective;Y-CVF had its immunogenicity,and multiple injection of Y-CVF stimulated anti-Y-CVF antibody production in monkeys,which resulted in theinvalid of Y-CVF;The induction of anti-alpha -Gal xenoantibody by Y-CVF in primateswas not observed;In combination with other immunosupressive agents,a short-termY-CVF therapy in the early period could markedly reduce the production of neutralizingantibody and had a good application prospect in clinic.In sum,we described,for the first time,a simple and reliable method for ABO typingof monkeys and a non-human primate animal model of accelerated humorat rejection as apreclinical model.And we successfully induced a long-time allograft survival andaccommodation in these presensitized recipient monkeys by early inhibition of complementin combination with CsA triple maintenane therapy.The accommodation may be associatedwith up-regulation of intragraft Bcl-2,CD46 and CD59.更多还原