【作者】 刁畅；

【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,为研究术后排斥反应提供实验手段。方法采用60 mg/Kg链尿佐菌素（STZ）一次性腹腔注射建立Wistar大鼠Ⅰ型糖尿病模型。在原有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。对移植成功的受鼠3次/周监测血糖,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STZ诱导糖尿病模型成功率高,对机体其它脏器毒性小,动物模型稳定。熟练操作技术,重视细小环节,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,可用于排斥反应的研究。目的探讨供体脾细胞门静脉输注联合抗CD154单克隆抗体（anti CD154mAb）的非清髓性骨髓移植预处理方案在异基因大鼠胰十二指肠移植（PDT）免疫耐受诱导中的作用及可能机制。方法一次性腹腔推注链尿佐菌素60 mg/Kg建立Wistar（Rt1^u）大鼠Ⅰ型糖尿病模型。以Lewis（Rt1¹）大鼠为供体,糖尿病Wistar大鼠为受体,SD大鼠为无关第三品系。受体随机分为3组,每组13只:移植组,行Lewis→Wistar PDT,移植术前、后不进行任何处理;单抗组,PDT术后腹腔注射3mg anti CD154 mAb;耐受组,按耐受诱导方案进行。方案为:第0天,经受鼠门静脉输注供体来源的脾细胞2×10⁸,2h后腹腔注射3mg anti CD154mAb;第3天,经受鼠阴茎背静脉输注供体来源的骨髓细胞1×10⁸;第20天,行PDT。术后第7天,各组随机取6只行血糖水平测定,用酶联免疫吸附法（ELISA）检测血清IL-10和IFN-γ水平;取材移植物行病理检查,TUNEL-POD法检测原位细胞凋亡以及吲哚胺-2,3-双加氧酶（IDO）免疫组化分析;流式细胞术对受鼠脾、胸腺Rt1¹⁺水平进行嵌合体测定;同时行单向混合淋巴细胞反应（MLR）,大鼠重组白细胞介素2（IL-2）逆转实验和体内过继转移实验。各组剩余的7只用于移植物功能存活时间观察。结果PDT术后第7天,耐受组血清IL-10为（188.42±23.15）pg/mL,显著高于移植组和单抗组;IFN-γ浓度为（202.46±24.07）pg/mL,显著低于其余两组;Th1/Th2细胞因子平衡向Th2偏移。病理学检查发现,移植组与单抗组移植物病变均呈典型的急性排斥反应,组织学评分分别为（4.67±0.52）分和（4.00±0.63）分,差异无显著性（P=0.104）;而耐受组移植物排斥反应以Ⅱ～Ⅲ级为主,评分为（2.67±0.82）分,病变明显轻于前两组（P＜0.01）;十二指肠排斥反应程度与胰腺基本一致。术后三组移植物均可检测到细胞凋亡,而耐受组细胞凋亡数显著少于其余两组（P＜0.01）。免疫组化结果提示,各组移植物IDO表达均无显著性差异（P＞05）,胰腺腺泡细胞、十二指肠黏膜上皮细胞及淋巴细胞均呈强阳性表达,IDO表达部位为胞浆。嵌合体检测,只有耐受组大鼠的脾脏、胸腺内可检测到Rt1¹⁺嵌合细胞,嵌合水平分别为（31.56±6.57）%和（22.04±8.12）%。单向MLR实验中,耐受组对Lewis大鼠脾细胞的刺激指数百分率（SI%）为（26.41±5.16）%,显著低于移植组和单抗组（P＜0.01）;而单抗组对Lewis和SD供体脾细胞的刺激均有一定的抑制作用。外源性IL-2可完全或部分逆转上述两个MLR体系中的抑制效应。体内过继转移实验结果显示耐受组大鼠体内存在调节/抑制性T细胞。移植组和单抗组均不能延长移植物功能平均存活时间（MIST）,而耐受组可将MST延长到（21.83±1.11）天。结论单独使用anti CD154 mAb的单抗组不能有效建立嵌合体、诱导免疫耐受,在此基础上联合供体脾、骨髓细胞,可在糖尿病大鼠体内建立异基因混合性嵌合体并在含有两个高免疫原性器官的PDT中诱导机体特异性免疫耐受,减轻移植物排斥反应程度,延长移植物功能MST。诱导方案发挥致免疫耐受作用不依赖于IDO活性表达,联合两种方法可能具有协同作用。免疫耐受形成的可能机制包括外周、中枢性嵌合体的建立、Th1/Th2细胞因子平衡偏离、通过抑制细胞过度凋亡参与T细胞克隆清除、克隆无能以及调节/抑制等外周耐受机制。更多还原

【Abstract】 Objective To establish the model of allogeneic whole pancreaticoduodenal transplantation(WPDT)in rats.To provide a tool for research grafts rejection and to explore the key points of this operation.Methods Wistar-Furth rats with type 1 diabetes mellitus were induced by intraperitoneal administration of streptozotocin(STZ)at a single dose of 60mg/Kg.On the basis of our primal model, 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 pathohistological 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 The successful rate of diabetes induced by STZ is high.The animal model of diabetes is stable,STZ is a safe drug.Skilled technique 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.So,it could be applied to research the theoretical problems involved in grafts rejection. Objective To explore the role and possible mechanisms of spleen cells transfusion via portal vein and anti CD154 monoclonal antibody(mAb)combined with bone marrow transplantation in non-myeloablative preconditioning regimen for induction of immune tolerance in rats with allogeneic pancreaticoduodenal transplantation(PDT).Methods Wistar-Furth(Wistar,Rt1~u)rats with type 1 diabetes mellitus were induced by intraperitoneal administration of streptozotocin at a single dose of 60mg/Kg.We adopt Lewis(Rt1~1)rats as donor,Wistar rats with diabetes as recipient and Sprague-Dawley(SD)rats as the third party donor.Recipient Wistar rats were randomly divided into 3 groups,13 rats in each group.In group transplantation, Wistar rats were performed on PDT alone.In group mAb,Wistar rats were injected 3 mg AH.F5(hamster anti-rat CD154 mAb)via peritonium after PDT.In group tolerance,Wistar rats were performed according to preconditioning regimen.The regimen was portal vein injection of Lewis rats 2×10~8 spleen cells on day 0, intraperitoneal injection 3 mg AH.F5 2 hours later,in combination with intravenous iniection of Lewis rats 1×10~8 bone marrow cells on day 3,PDT on day 20.On day 7 after PDT,the blood glucose of 6 rats from each group were measured.Then enzyme linked immunosorbent assay(ELISA)was applied to determine the concentration of the serum interleukin(IL)-10 and interferon(IFN)-γ.And gafts were collected to observe pathohistological changes and apoptosis in suit with TUNEL and the expression of indoleamine-2,3-dioxygenase(IDO).positive cells by anti-IDO mAb immunochemistry methods.Rt1~1 positive Chimersim in recipient spleen and thymus were followed by flow cytometric(FACS)analysis.Tolerance was assessed by the ratio of stimulation index(SI%)of one-way mixed lymphocyte reaction(MLR).Rat recombined IL-2 reverse assay of one-way MLR and adoptive transfer assay were performed at the same time.Remaining 7 rats of each group were observed the survival time of grafts function.Results The concentration of serum IL-10 and IFN-γwas respectively(188.42±23.15)pg/mL and(202.46±24.07)pg/mL in group tolerance on day 7 posttransplantation,which was significant difference from groups transplantation and mAb.The balance of Th1/Th2 alter to Th2.The pathohistological changes in groups transplantation and mAb were typical acute rejection.The scores of histologic grade were(4.67±0.52)and(4.0±0.63),respectively.There was no significant difference between them(P=0.104).The histologic grade of acute rejection in group tolerance wasⅡ～Ⅲ,score was(2.67±0.82),which was milder than others group(P<0.01).The degree of histologic changes of duodenum was consistent with pancreas.Apoptosis was observed in three groups.But the amount of apoptosis in group tolerance was fewer than others group(P<0.01).The expression of IDO in tissue was no significant difference among groups(P>0.05).The positive staining cells were pancreatic acinal cells and duodenal mucosa endothelial cells and lymphocyte. The positive siteslocated on cytoplasm.The results of chimerism assay revealed chimerism was only detected in group tolerance.The levels were(31.56±6.57)%in spleen and(22.04±8.12)%in thymus.In one-way MLR assay,SI%of group tolerance was(26.41±5.16)%,which was significant lower than other groups (P<0.01).While spleen cells of group mAb could inhibit the stimulus originated by spleen cells of Lewis and SD rats.Exogenous IL-2 could completely or partly reverse immue hyporesponsiveness to allogeneic antibody.In vivo adoptive transfer assay, regulatory and suppressive activity in the spleen cells of rats in group tolerance was observed.The grafts functional survival time of group tolerance was prolonged to (21.83±1.11)days.Conclusion Administration of anti CD 154 mAb alone can not effectively establish chimerism and induce immune tolerance.Combined with transfusion of donor-derived spleen cells and bone marrow cells on the basis of anti CD154 mAb can establish allogeneic mixed chimerism on diabetes rats and successfully induce donor-specific tolerance in PDT which consisted of two immtmogenic organs.Furthermore,the regimen can relieve the degree of rejection and prolong grafts functional survival time.Increased IDO activity in grafts have the effect of anti-injection.However,the preconditioning regimen for induction or maintenance of chimerism and tolerance are not independent of IDO activity.Thus, increasing of IDO activity combined with this regimen have synergistic effect. Multiple mechanisms,including peripheral and central chimersim formation,altering the balance in Th1/Th2,T cells clone deletion by suppression of excessive apoptosis, clonal anergy and regulation/suppression are involved in the tolerance.更多还原