【作者】 魏晓平；

【导师】 李立； 金克炜；

【作者基本信息】 昆明医学院 ， 外科学， 2008， 博士

【摘要】 目的探讨供体脾细胞及其输注途径以及联合抗CD154单克隆抗体（antiCD154 mAb,克隆号AH.F5）对非清髓性骨髓移植预处理方案建立异基因嵌合体的影响及诱导供体特异性免疫耐受的可能机制.方法Lewis（Rtl^l）大鼠为供体,Wistar（Rtl^u）大鼠为受体,SD大鼠为无关第三品系。按不同预处理方案将Lewis供鼠脾细胞（Sc）、骨髓细胞（BMc）悬液输注给Wistar大鼠。受鼠随机分为4组,每组20只:（1）Sc_外组:第0天（d₀）经受体外周阴茎背静脉输注2×10⁸供鼠Sc;（2）Sc_门组:d₀经受体门静脉输注2×10⁸供鼠Sc;（3）Sc_门+BMc_外组:d₀经受体门静脉输注2×10⁸供鼠Sc,第3天（d₃）再经阴茎背静脉输注1×10⁸供鼠BMc;（4）Se_门+anti CD154mAbq-BMc_外组:d₀经受体门静脉输注2×10⁸供鼠Sc,2h后腹腔注射anti CD154mAb 3mg,d3再经阴茎背静脉输注1×10⁸供鼠BMc。通过酶联免疫吸附法（ELISA）分别检测脾细胞输注术后第20(d₂₀)、40天(d₄₀)Wistar大鼠血清IL-10和IFN-γ水平;通过d₂₀、d₄₀单向混合淋巴细胞反应（MLR）,大鼠重组白细胞介素2（IL-2）逆转实验和d₅₀迟发性超敏反应（DTH）对受鼠耐受程度进行评估;通过流式细胞术对d₂₀、d₄₀受鼠脾、胸腺细胞中Rt1¹⁺嵌合比例进行测定,d₁₀₀对Sc_门+anti CD154 mAb+BMc_外组加做此项检查。结果Sc_外组血清细胞因子水平在两时间点均无变化,与正常对照组比较无显著性差异（P＞0.05）;Sc_门+anti CD154 mAb+BMc_外组血清IL-10、IFN-γ水平在d₂₀分别为（42.01±4.87）pg/mL和（28.97±3.83）pg/mL,d₄₀分别为（40.19±a:5.30）g/mL和（25.89±4.30）pg/mL,其中IL-10浓度明显高于其它各组,差异有统计学意义（P＜0.01）,IFN-γ浓度均处于较低水平,Th1/Th2平衡向Th2偏移;而Scn和Sc_门+BMc_外组细胞因子水平向Th1偏移。单向MLR中Sc_门+anti CD154mAbq-BMc_外组对Lewis供鼠脾细胞的刺激指数（SI）在d₂₀、d₄₀分别为（4.43±0.19）和（4.97±0.25）,均显著低于其余各组（P＜0.01）,加入外源性IL-2后可部分逆转其低反应性,但对第三方SD大鼠来源的脾细胞仍保持强烈增殖活性,SI值与阳性对照体系无显著性差异（P＞0.05）。DTH实验中只有Sc_门+anti CD154 mAb+BMc_外组表现为显著低反应性,其余各组脚掌厚度差值（FI）与正常对照组比较,差异无统计学意义（P＞0.05）。嵌合体检测结果表明Sc_外组在脾和胸腺中均不能形成嵌合体;Sc_门和Sc_门+BMc_外组在d₂₀能建立较低水平嵌合体,于d₄₀很快下降甚至消失;而sc_门+anti CD154 mAb+BMc_外组在d₂₀、d₄₀的脾和胸腺中均可形成稳定的高水平嵌合,并持续超过100天。结论单纯经外周静脉输注2×10⁸供体sc不能诱导嵌合体和免疫耐受的形成;将其输注途径改为门静脉后,可诱导出受鼠轻度的免疫耐受状态,建立短暂（＜20天）、低水平（＜20%）的外周性嵌合体;在此基础上联合输注1×10⁸供体BMc可部分增强耐受状态,延长嵌合体水平至40天,但仍不稳定;而只有加入anti CD154 mAb后才可显著增强BMc植入率,三者具有协同作用。外周嵌合体＞30%、中枢嵌合体＞20%是保证骨髓细胞发挥强大致耐受作用的基础。Sc_门+anti CD154 mAb+BMc_外预处理方案可能与Th1/Th2细胞因子偏移、T细胞克隆无能和克隆清除等多种机制有关,在无大剂量骨髓细胞、骨髓抑制剂和放射线参与下,可获得稳定的高水平异基因混合性嵌合体,成功诱导受体针对供体抗原的特异性免疫耐受。目的为研究胰腺移植术后排斥反应提供实验手段,建立异基因大鼠全胰十二指肠移植（WPDT）模型,并探讨手术过程操作要点。方法在双套管+腹主动脉三通管外接法建模的经验基础之上改进动脉吻合方式,将供受体腹主动脉端侧吻合,供体门静脉与受体左肾静脉袖套吻合,供体十二指肠与受体小肠端侧吻合,成功建立Lewis→Wistar糖尿病大鼠异基因WPDT模型。记录STZ诱导糖尿病大鼠的情况,对移植成功的受鼠3次/周监测随机血糖,并记录存活时间（ST）,术后第7天取材3只大鼠移植物行病理学检查。结果STZ诱导糖尿病大鼠成模率为85.7%,酮症酸中毒（DKA）发生率为5.6%,病死率7.4%。50只糖尿病大鼠行WPDT术式,44例移植成功,术后24h血糖由术前（22.83±4.37）mmol/L降至（6.36±2.18）mmol/L。其中有8例于术后3d内死亡,其余36例受鼠存活时间为6～16d,平均为（10.45±3.3）d,术后4d、7d、10d、13d血糖水平呈进行性升高,术后7～10d为死亡高峰,移植物病理改变呈典型的急性排斥反应.结论熟练显微技术,重视操作细节,是成功建立WPDT模型的关键因素。本法建立的WPDT模型可见到移植胰发挥内分泌功能以及典型的急性排斥反应病理改变,该模型稳定、可靠,适用于排斥反应的研究。更多还原

【Abstract】 Objective To explore the role and possible mechanisms of donor-derived spleen cells（Scs） and it’s transfusion pathway combined with anti CD154 monoclonal antibody（mAb） on bone marrow transplantation in non-myeloablative preconditioning regimen for induction of allogeneic mixed chimerism and donor-specific immune tolerance.Methods We adopt Lewis（Rtl¹） as donor,Wistar-Furth（Wistar,Rtl^u） as recipient and Sprague-Dawley（SD） as the third party donor.Scs and bone marrow cells（BMCs） were harvested from donor Lewis rats.Recipient Wistar rats were randomly divided into 4 groups,20 rats in each group. Group Scs_i.v.,penis dorsal vein（i.v.） injection of Lewis rats 2×10⁸ Scs on day 0 alone. Group Scs_P.V.,portal vein（P.V.） injection of Lewis rats 2×10⁸ Scs on day 0 alone. Group Scs_P.V.+BMCs_i.v.,P.V.injection of Lewis rats 2×10⁸ Scs on day 0,in combination with i.v.injection of Lewis rats 1×10⁸ BMCs on day 3. Group Scs_P.V.+ AH.F5（hamster anti-rat CD154 mAb）+BMCs_i.v.,P.V.injection of Lewis rats 2×10⁸ Scs on day 0,intraperitoneal injection 3 mg AH.F5 2 hours later,in combination with i.v.injection of Lewis rats 1×10⁸ BMCs on day 3.On day 20 and 40, enzyme linked immunosorbent assay（ELISA） to determine the levels of the serum interleukin（IL）-10 and interferon（IFN）-γ,were performed.Tolerance was assessed by stimulation index（SI） of one-way mixed lymphocyte reaction（MLR）,rat recombined IL-2 reverse assay of one-way MLR on day 20,40 and foodpad increment（FI） of delayed type hypersensitivity（DTH） on day 50.Rtl¹ positive chimersim in recipients spleen and thymus were followed by fluorescenceactivited cell sortor（FACS） on day 20,40.Group SCS_P.V.+AH.F5+BMCs_i.v was additionally carried on chimerism detection on day 100.Moreover,tolerance mechanism was investigated.Results The levels of cytokine were invariable in group Scs_i.v. on day 20,40.While,the levels of IL-10 and IFN-γin group Scs_P.V.+AH.F5+BMCs_i.v. were individual（42.01±4.87） pg/mL and（28.97±3.83） pg/mL on day 20,（40.19±5.30） pg/mL and（25.89±4.30） pg/mL on day 40.UP-regulation of IL-10 was significant higher than other groups, down-regulation of IFN-γwas significant difference from groups Scs_P.V. and Scs_P.V.+BMCs_i.v. The balance of Th1/Th2 alter to Th2 in group SCS_P.V.+AH.F5+BMCs_i.v.,to Th1 in groups Scs_P.V. and Scs_P.V.+BMCs_i.v. SI values were（4.43±0.19） and（4.97±0.25） in group Scs_P.V.+AH.F5+BMCs_i.v. on day 20,40. There were significant lower than other groups（P<0.01）.IL-2 added to one-way MLR can partly reverse immue hyporesponsiveness to allogeneic antibody. Meanwhile,Scs derived from this group had strong activity of proliferation to Scs from the third party SD rats.In DTH test,only group Scs_P.V.+AH.F5+BMCs_i.v. had suppressive activity.FI values of other groups had no significant difference from normal control group（P>0.05）.The results of Rtl¹⁺ assay reveal that there was no chimersim in group Scs_i.v..While chimersim was transient and low in groups Scs_P.V. and Scs_P.V.+BMCs_i.v. on day 20,and rapidly descend,even disappeared on day 40. Only group SCS_P.V.+AH.F5+BMCs_i.v. had stable and higher level chimerism in spleen and thymus on day 20,40.Furthermore,long-term chimerism（>100 days） was observed in the group.Conclusion I.V.injection of 2×10⁸ Scs alone can’t induce chimerism and tolerance.Transfusion pathway of Scs via P.V.can only induce gentle tolerance and establish short-course（< 20 days） and low level（< 20%） peripheral chimerism.Combined with 1×10⁸ BMCs can partly enhance tolerance and prolong the existence of chimerism to 40 days,but not stable.However,only combined with anti CD 154 mAb can significantly promote BMCs engraftment.High level of chimerism （>30%in peripheral spleen and >20%in central thymus） provide the foundation to BMCs to play a critical role in the induction or maintenance of tolerance.Our studies demonstrate that P.V.injection of Scs and anti CD154 mAb,plus i.v.injection of BMCs have synergistic effect to induce donor-specific tolerance and establish stable allogeneic mixed chimersim,without participation of megadose BMCs, myleosuppressive drugs and irradiation.Multiple mechanisms,including altering the balance in Th1/Th2,T cells clonal anergy and clonal deletion are involved in the tolerance. Objective To provide a tool for research grafts rejection and to explore the key technique of this operation,we establish the model of allogeneic whole pancreaticoduodenal transplantation（WPDT） in rats.Methods Wistar-Furth rats with type 1 diabetes mellitus（DM） were induced by intraperitoneal administration of streptozotocin（STZ） at a single dose of 60mg/Kg.On the basis of our primal model established by dual cuff combined with abdominal aorta connected with Y-tube,we improved the way of arterial anastomosis.End to side anastomosis was performed for abdominal aorta of donors and recipients.The portal vein of the graft was anastomosed with the recipients left renal vein by cuff technique.And side to side anastomosis was made between the graft duodenum and the host jejunum.According to the technique mentioned above,we successfully established WPDT model between Lewis rats as donors and Wistar rats as recipients.To monitor the levels of blood glucose 3 times weekly and record the survival time（ST） of each rats.And grafts of 3 rats were collected to observe pathohistologieal changes on day 7 posttransplantation. Results The successful rate of diabetes rats induced by STZ was 85.7%.The incidence rate of diabetic ketoacidosis was 5.6%and the mortality was 7.4%.44 rats were successfully performed WPDT of 50 rats.The mean levels of blood glucose were decreased from（22.83±4.37） mmol/L preoperative to（6.36±2.18） mmol/L postoperative in 44 rats.Among of them,8 rats died in 3 days postoperative,the ST of residual 36 rats was 6-16 days,the mean of ST was（10.45±3.3） d.Three was a progressive increase of blood glucose on day 4,7,10 and 13.The peak of death appeared on day 7-10.The typical acute rejection in pathological changes was observed on day 7.Conclusion Skilled microsurgical techniques and emphasis on details are important to establish WPDT model.The endocrine function of grafts and typical acute rejection can be observed in this model.It is a stable and reliable model, which might be used in research of the theoretical problems involved in clinical pancreas transplantation rejection.更多还原