The induction of donor-specific transplantation tolerance in pancreas transplantation combined with spleen in rat

目的：目的：探讨脾脏和调节性T细胞在胰腺移植中的作用。方法：采用链脲佐菌素（STZ）诱导大鼠糖尿病模型，采用改良Lee法进行脾和/或胰十二指肠移植（S/PTx或PTx）。比较了同系移植物（Lew至Lew）、同种异体移植物（DA至Lew）和同种异体移植物加他克莫司诱导（1 mg/kg/天，持续4d）组的移植物存活率。采用流式细胞术和RT-PCR分别检测外周血（PB）、受体脾（RS）和供体脾（DS）中CD 25 + CD 4 + T细胞比例和Foxp 3表达。第7天取RS、DS和移植胰腺组织行组织学检查。结果：同系移植的S/PTx（n=4）和PTx（n=4）均存活100天以上。S/PTx（n=6）和PTx（n=6）在未加他克莫司的情况下分别于12.3±2.7d和11.7±2.9d出现排斥反应。S/PTx组（n=6）生存时间为22.8±7.9d，PTx组（n=6）生存时间为13.5±3.7d（p=0.0069）。排斥反应S/PTx移植物（n=5）PB、RS和DS中以CD 25 + CD 4 +T细胞为主，2）供者来源的CD 25 + CD 4 +T细胞在7天后消失。存活的S/PTx移植物（n=5）显示：1）供体CD 25 + CD 4 +T细胞在PB、RS和DS中保留超过7天，2）Foxp 3在RS和DS中的表达率在第7天与排斥移植物相比（p=0.028）。结论：联合脾移植和短疗程钙调磷酸酶抑制剂可延长移植胰腺的存活时间。供、受者脾脏中同时存在的供者源性调节性T细胞（Foxp 3和CD 25/4细胞）可导致微嵌合和延长生存期。

Aim : To investigate the role of spleen and regulatory T cell for pancreas transplantation.Method : After induction of diabetes mellitus by streptozotocin, spleen and/or pancreaticoduodenal transplantation (S/PTx or PTx) into the peritoneal cavity was performed using a modification of Lee's method. Graft survival was compared in isograft (Lew to Lew), allograft (DA to Lew) and allograft with tacrolimus induction (1mg/kg/day for 4days) groups. The rate of CD25+CD4+ T cell and the Foxp3 expression in peripheral blood (PB), recipient spleen (RS) and donor spleen (DS) were examined using Flowcytometry and RT-PCR, respectively. The specimen was obtained from RS, DS, and graft pancreas for histological examination on day7.Results : Isotransplanted S/PTx (n=4) and PTx (n=4) were both survived longer than 100days. Both allotransplanted S/PTx (n=6) and PTx (n=6) without tacrolimus rejected the graft on 12.3±2.7d, 11.7±2.9d, respectively. S/PTx (n=6) with tacrolimus survived for 22.8±7.9d, and PTx (n=6) with tacrolimus survived for 13.5±3.7d (p=0.0069). Rejected S/PTx grafts (n=5) showed that 1)CD25+CD4+T cells were dominant in PB, RS and DS, 2) Donor derived CD25+CD4+T cells disappeared after 7days. Survived S/PTx grafts (n=5) showed that 1)donor CD25+CD4+T cells remained in PB, RS and DS over 7days, 2)the rate of Foxp3 expression was in both RS and DS on day7 compared with Rejected graft (p=0.028). There were more lymphocyte infiltration and reduction in white pulp in RS and DS.Conclusion : The pancreas graft survived longer by combined spleen transplantation and a short course of calcineurin inhibitor. Donor derived regulatory T cells (Foxp3 and CD25/4 cells) existing in both recipient and donor spleen would lead micro-chimerism and prolonged survival.