【作者】 何凡；

【导师】 陈知水；

【作者基本信息】 华中科技大学 ， 外科学， 2009， 博士

【摘要】 第一部分门静脉耐受中CD₄⁺T和CD₈⁺T细胞不同作用目的：探讨门静脉耐受中不同T细胞成分的作用。方法：门静脉输注经丝裂霉素处理过的C57小鼠脾细胞给Balb/C，建立门静脉输注模型，使用去增殖的C57小鼠淋巴细胞作为刺激细胞，PⅥ小鼠或正常Balb/c小鼠作为反应细胞，单向混培后，Alamar Blue检测细胞增殖。于门静脉输注后7d行C57和C3H小鼠心脏移植，观察心脏移植存活。流式分选分选门静脉输注小鼠中CD₃⁺、CD₄⁺、CD₈⁺T细胞，分选后测定分选细胞纯度；过继输注给另一BalB/C，并同时行C57或C3H来源的心脏移植，观察并比较心脏移植物存活时间。于移植后7d取心脏移植物，HE染色检测移植物中淋巴细胞浸润情况。结果：C57小鼠淋巴细胞刺激后，PⅥ鼠和正常鼠淋巴细胞增殖的比例分别为（14．5±3．5）％和（31．3±5．6）％，P＜0．01。C57小鼠淋巴细胞门静脉输注后，后续移植的C57小鼠心脏存活时间明显延长，而对第三系C3H供心存活时间无明显延长，门静脉输注同种抗原可以诱导抗原特异性的免疫耐受。过继输注PⅥ小鼠T细胞（CD₃⁺）可以转移门静脉输注所诱导的耐受。CD₄⁺、CD₈⁺T细胞过继输注证实，CD₄⁺T细胞转移抗原特异性的耐受，明显延长C57小鼠移植物存活时间，而CD₈⁺T细胞无此效应。移植物病理检测证实，过继输注PⅥ小鼠CD₄⁺T后，术后第7d，移植物中淋巴细胞浸润明显减轻，心肌结构基本正常。结论：门静脉输注同种抗原可以诱导抗原特异性的免疫耐受，过继输注耐受小鼠的CD₄⁺T细胞可以转移耐受，而过继输注耐受小鼠CD₈⁺T细胞无此效应。第二部分CD₄⁺CD₂₅⁺Foxp₃⁺调节性T细胞在门静脉耐受中的作用目的：探讨门静脉耐受模型中CD₄⁺CD₂₅⁺Foxp₃⁺调节性T细胞的比例，及其在诱导耐受中的作用及机制。方法：于门静脉输注同种抗原后第7，14及21天，取小鼠血、肝及脾脏，流式细胞仪检测CD₄⁺CD₂₅⁺Foxp₃⁺调节性T细胞的比例。流式细胞仪分选PⅣ小鼠CD₄⁺CD₂₅⁺T细胞，以不同的浓度加入C57所刺激的Balb/c小鼠淋巴细胞混培体系中，观察对细胞增殖的影响。分别于门静脉输注后第1，3，5天给予CD₂₅单抗腹腔注射，第7天检测CD₄⁺CD₂₅⁺Foxp₃⁺调节性T细胞的比例；并于第7天行C57小鼠的心脏移植，观察移植物存活。门静脉输注后，给予CD₂₅单抗（或同型对照抗体）腹腔注射，ELISPOT检测外周血淋巴细胞分泌IL-2，IL-4，IL-10及IFN-γ的能力；TUNEL染色检测肝内CD₄⁺T细胞CD₈⁺T细胞的凋亡比例。结果：门静脉输注同种抗原后，CD₄⁺CD₂₅⁺T细胞的比例明显升高，术后第7天达到高峰，之后逐渐下降，至第21天仍然维持在高于正常小鼠的水平。CD₄⁺CD₂₅⁺Foxp₃⁺T占CD₃⁺T比例表现出同样的趋势。CD₄⁺CD₂₅⁺T在体外可以明显抑制C57细胞所刺激的Balb/c小鼠淋巴细胞增殖，并呈现出浓度依赖性。CD₂₅单抗腹腔注射可以清除体内70％CD₄⁺CD₂₅⁺T细胞，门静脉输注小鼠给予CD₂₅单抗腹腔注射后，后续的心脏移植物存活时间明显缩短，伴随着CD₄⁺CD₂₅⁺Foxp₃⁺T占CD₃⁺T细胞的比例明显下降。ELISPOT检测发现，门静脉输注后分泌IL-4的细胞比例明显增多，给予CD₂₅单抗清除CD₄⁺CD₂₅⁺T细胞后，分泌IL-4的细胞比例下降，伴随分泌IL-10，IL-2及IFN-γ的细胞比例增多。门静脉输注后，CD₄⁺T细胞CD₈⁺T细胞的凋亡比例明显增加，给予CD₂₅单抗清除CD₄⁺CD₂₅⁺T细胞后，CD₄⁺T，特别是CD₈⁺T细胞的凋亡比例明显降低。结论：门静脉输注同种抗原后，血液、肝脏及脾脏中CD₄⁺CD₂₅⁺Foxp₃⁺T明显增加，升高的调节性T细胞在耐受的诱导中起重要作用。使用CD₂₅单抗降低CD₄⁺CD₂₅⁺T细胞水平后，诱导了后续移植器官的排斥反应，并且伴随着IL-4分泌细胞的减少和T细胞凋亡的降低。第三部分Th17细胞参与同种异体排斥反应的发生目的：探讨Th17细胞在同种异体排斥反应中的作用。方法：建立C57→Balb/c皮肤移植模型，与术后第7天取皮肤移植物，免疫组化检测皮肤移植物中IL-17的表达，以自体皮肤移植为对照。流式分选CD₄⁺T细胞，使用IL-6，TGF-β诱导CD₄⁺T细胞向Th17分化，并观察IL-1β，TNF-α，IL-23在Th17分化中的作用。建立C57→Balb/c裸鼠的皮肤移植模型，过继输注不同浓度的Th17细胞，观察对排斥反应发生的影响。结果：免疫组化可见，同种异体移植物中腺体结构破坏，有大量Th17细胞浸润，多集中在真皮层。IL-1β，TNF-α可明显增加IL-6和TGF-β诱导的CD₄⁺T细胞向Th17分化，而IL-23在二次刺激中可使CD₄⁺T细胞向Th17分化进一步增强。C57→Balb/c裸鼠的皮肤移植手术近期不被排斥，给予体外诱导的Th17细胞后皮肤移植物的存活时间明显缩短，且与输注的Th17细胞浓度呈负相关。结论：IL-6和TGF-β是Th17所必须的细胞因子，IL-1β，TNF-α，IL-23在维持Th17增殖和存活中起重要作用。Th17参与了同种排斥反应的发生。第四部分门静脉耐受过程中CD₄⁺CD₂₅⁺Foxp₃⁺T细胞和Th17的相互作用目的：探讨门静脉耐受中CD₄⁺CD₂₅⁺Foxp₃⁺T和Th17细胞的相互作用。方法：C57脾细胞Balb/c门静脉输注后第7天行C57小鼠皮肤移植，以Balb/c小鼠同基因输注为对照，移植后第7天取皮肤移植物，Wetern-Blot检测移植物中IL-17及Foxp3的表达。体外诱导CD₄⁺CD₂₅⁺T细胞，在na（i|¨）ve CD₄⁺CD₂₅⁺T和induced CD₄⁺CD₂₅⁺T培养体系中分别加入IL-6，观察对CD₄⁺CD₂₅⁺T细胞分化的影响。在Th17细胞培养体系中加入IL-2（或IL-2+TGF-β），观察对Th17分化的影响。建立同种门静脉输注模型，并输注IL-6或Th17观察对后续移植皮肤存活的影响。结果：同种门静脉输注与同基因门静脉输注相比，后续移植物中Foxp3的表达明显升高，而IL-17的表达明显降低。体外实验证实IL-6可诱导CD₄⁺CD₂₅⁺T细胞向Th17转化，且活化的CD₄⁺CD₂₅⁺T细胞接受IL-6的刺激后更易于向Th17转化。IL-2和TGF-β共同存在的条件下，Th17可向CD₄⁺CD₂₅⁺T细胞转化。过继输注Th17或IL-6均可打破门静脉耐受，后续移植的皮肤移植物明显缩短。结论：门静脉输注可以使移植物中Treg的比例明显升高，而Th17的比例明显降低。Treg和Th17在体外实验中可以互相转化，分化方向取决于细胞因子环境。过继输注IL-6和Th17可以打破已建立的门静脉耐受。更多还原

【Abstract】 PartⅠThe roles of different subsets of T lymphocytes in portal veininjection induced tolerance.Objective: To analyze the role of different subsets of T lymphocytes in transferring toleranceinduced by portal vein injection.Methods: C57BL/6 （B6） mice were used as donors. 7 days after portal vein injection ofmitomycin-treated B6 splenocytes into BALB/c mice, the splenocytes of the recipient wereisolate. Mitomycin-treated B6 splenocytes were used as stimulator, PVI splenocytes or normalBALB/c splenocytes were used as reactor. And lymphocytes proliferations were conducted byAlamar Blue. CD4⁺, CD8⁺T lymphocytes were then sorted from PVI mice by FACSAria. Thesorted CD4⁺ （CD8⁺） T lymphocytes were then transfused into another BALB/c mice followedby heterotopic implantation of a B6 heart allograft. A C3H heart allograft and normal BALB/cwere used as controls. The rejection and survival of the allograft were observed. And 7 daysafter transplantation, the grafts were collected for HE stain.Results: Portal vein injection of alloantigen can prolong the subsequently transplantedallograft survival, when transfusion with CD4⁺T lymphocytes isolated from PVI mice, thesubsequent transplanted B6 allograft was survival longer than C3H allograft. Similar results were not observed from BALB/c with transfusion of CD8⁺T lymphocytes from PVI mice orCD₄⁺ T cells from normal Balb/c mice.Conclusion: This finding indicates that portal vein injection of alloantigen can induceantigen-specific tolerance. CD₄⁺T lymphocytes, rather than CD₈⁺T lymphocytes, play animportant role in transferring tolerance induced by portal vein injection and this tolerance isantigen-specific.PartⅡIncreased CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cells in toleranceinduced by portal vein injectionObjective: To assess the proportion and function CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cells inportal vein injection induced tolerance.Methods: Mitomycin-treated C57BL/6 （B6） splenocytes were injected into Balb/c mice viathe portal vein. The proportions of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg cells were determined in the blood,liver, and spleen in the 7, 14 or 21 days after injection. CD₄⁺CD₂₅⁺ Treg cells were isolatedfrom PVI mice and added into B6 stimulated Balb/c mixed lymphocytes culture at differentconcentrations. The proliferations of Balb/c lymphocytes were tested by Alamar Blue.Anti-CD₂₅ mAb were used to delete CD₄⁺CD₂₅⁺Foxp₃⁺ Treg in mice. B6 or C3H heartallografts were implanted into anti-CD25 mAb-treated PVI and isotype antibody PVI mice,and graft survivals were compared. The percentages of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg, cytokineprofiles, and ratios of apoptosis were determined in anti-CD25 mAb-treated PVI anduntreated PVI mice.Results: Portal vein injection of allogeneic cells induced antigen-specific tolerance andincreased the percentage of CD₄⁺CD₂₅⁺Foxp₃⁺ Treg. The CD₄⁺CD₂₅⁺ Treg isolated from PVImice can inhibit the lymphocytes proliferation in a dose-dependent manner. Depletion ofCD₄⁺CD₂₅⁺ T cells prevented the induction of tolerance and decreased the percentage ofCD₄⁺CD₂₅⁺Foxp₃⁺ Treg in the CD₃⁺ T cell pool, and thus was associated with decreasedproduction of IL-4 and apoptosis of T cells. Conclusion: Increased CD₄⁺CD₂₅⁺Foxp₃⁺ Treg play an important role in portal vein toleranceinduction, at least partly via increasing the production of IL-4 and promoting apoptosis of Tcells.PartⅢTh17 cells were involved in acute allograft rejectionObjective: To analyze the role of Th17 cells in acute allograft rejection.Methods: B6 skin allografts were implanted into Balb/c mice. Seven days postoperation, skinallografts were collected to assess the expression of IL-17 by immunohistochemistry. CD₄⁺Tcells were isolated from normal Balb/c, and were induced by IL-6 and TGF-β. Then IL-1β,TNF-αand IL-23 were added to the culture to analysis their roles in differentiation of Th17.In order to test Th17 cells in rejection induction, B6 skin allografts were transplanted toBalb/c nude mice, and in vitro induced Th17 cells were injection into recipients at differentconcentrations. Skin allograft survival was conducted.Results: There were a lot of Th17 cells infiltrated into rejected skin allograft, whereas nearlyno Th17 cell was infiltrated into sygeneous grafts. IL-6 and TGF-βcan induce CD₄⁺Tdifferentiation into Th17 cells and IL-1βand TNF-αcan enhanced the differentiation. IL-23can augment the differentiation in second stimulation. Balb/c nude mouse is thymus deficient.It can not induce acute rejection of allograft. When Th17 cells were adoptively transfused intoBalb/c nude, the C57 allografts were rejected immediately.Conclusions: IL-6 and TGF-βare responsible for differentiation of Th17 cells, while IL-11β,TNF-αand IL-23 can enhanced the differentiation. Th17 cells are, at least partly, involved inacute rejection. PartⅣThe interaction of CD₄⁺CD₂₅⁺Foxp₃⁺ regulatory T cellsand Th17 cells in portal vein toleranceObjective: To explore the interaction of CD₄⁺CD₂₅⁺Foxp₃⁺T and Th17 cells in portal veininjection induced tolerance.Methods: Seven days after portal vein injection of B6 splenocytes, Balb/c mice received B6skin allograft transplantation. The allografts were collected for detection of IL-17 and Foxp3expression 7 days after transplantation, and sygeneous portal vein injection was used ascontrol. IL-2 and TGF-βwere added to culture of CD₄⁺CD₂₅^- T cells, and induced CD₄⁺CD₂₅⁺T cells were isolated from the culture. Then IL-6 was added into na（i|¨）ve CD₄⁺CD₂₅⁺T orinduced CD₄⁺CD₂₅⁺T culture to evaluate the differentiation bias of CD₄⁺CD₂₅⁺T cells. IL-2,with or without TGF-β, was added into Th17 culture to evaluate the differentiation bias ofTh17 cells. IL-6 and in vitro induced Th17 cells were transfused into PVI mice. And the skinallografts survival was conduced.Results: Compared to sygeneous antigen, portal vein injection of alloantigen induced highexpression of Foxp3 and low level of IL-17 in subsequently transplanted skin allografts. IL-6can cause trans-differentiation of Treg into Th17 cells. And IL-2 together with TGF-βcancause trans-differentiation of Th17 into Treg cells. The injection of IL-6, Th17 can cause therejection of B6 heart allograft transplanted into PVI mice.Conclusions: Portal vein injection of alloantigen can increase the accumulation of Treg cellsand reduced the infiltration of Th17 cells. In vitro, Treg and Th17 cells can differentiated intoeach other depending on the cytokine in the microenviroment. Adoptive transfused of IL-6and Th17 can reverse the tolerance induced by portal vein injection of alloantigen.更多还原