供肝NK细胞对肝移植免疫反应中NF-κB 活性和IL-15表达的影响

 目的：观察大鼠移植肝组织内和外周血白细胞介素15（IL-15）的表达水平及脾组织内核因子κB(NF-κB)的表达活性变化，探讨供肝NK细胞减轻肝移植术后急性排斥反应的机制。方法：Lewis大鼠为肝移植供体，BN大鼠为受体，改良“二袖套”法建立大鼠肝移植模型。应用［60Co］射线照射清除供体免疫细胞和经门静脉回输供肝NK细胞等技术，实验设立3组。A组: 急性排斥组；B组: 单纯［60Co］照射排斥组；C组: ［60Co］照射+供肝NK细胞门静脉回输排斥组。移植术后第1、3、7天收集受鼠外周血清，ELISA法检测IL-15分泌水平，同时切取移植肝行病理学检查，Western blotting法检测移植肝组织IL-15蛋白的表达，凝胶电泳迁移实验（EMSA）检测受鼠脾组织NF-κB表达活性。另外每组各设亚组观察移植后大鼠自然生存时间。结果：B组术后大鼠自然存活时间较A、C组明显缩短，术后发生急性排斥反应程度较A、C组严重；术后第3、7天，3组受鼠外周血IL-15水平均较第1天时显著升高；移植术后第3、7天，B组大鼠血清IL-15 浓度均显著高于A、C组；术后第3、7天，移植肝组织中B组IL-15表达也显著高于A、C组，受鼠脾组织中B组NF-κB表达活性亦显著高于A、C组。结论：IL-15可能作为肝移植急性排斥反应的监测指标,对急性排斥反应的早期诊断和移植物的预后估计具有临床指导意义；供肝NK细胞可能通过下调NF-κB的表达活性来调节IL-15的水平，进而减轻肝移植术后急性排斥反应。

NK cells from donor liver inhibit NF-κB activity and IL-15 expression after liver transplantation in rats

AIM:To investigate the mechanism that donor liver natural killer (NK) cells alleviate acute rejection after liver transplantation by observing the secretion level of interleukin 15 (IL-15) in peripheral blood, the protein expression of IL-15 in transplanted liver tissues and the activity of NF-κB in spleen tissues in rat acute liver graft rejection model. METHODS:An acute rejection model of liver transplantation in rats was established by the modified two-cuff method, in which Lewis rats were used as donors and BN rats as recipients. The donor leukocytes were depleted by whole body irradiation of ［60Co］ source and the donor liver immunity was reconstituted by transfusion of liver NK cells from the same type of donor (donor type liver NK cells, dtlNKs) via portal vein immediately after grafting the irradiated liver. The rats were divided into the following groups: group A, acute rejection group; group B, BN rats receiving the liver of Lewis rats with ［60Co］ irradiation; group C, BN rats receiving the liver of ［60Co］-irradiated Lewis rats and treated with dtlNKs via the portal vein. The recipients were sacrificed at 1 d, 3 d and 7 d after transplantation. IL-15 level in peripheral blood was detected by ELISA. The expression of IL-15 in the liver grafts was determined by Western blotting. NF-κB activity in the spleen tissues of recipient rats was identified by electrophoretic mobility shift assay. The survival quality and living time in crude survival subgroup were observed. RESULTS:Acute rejection in group B was severer than that in group A and group C. The rats in group B showed significantly shorter average survival time compared with group A and group C. At 3 d and 7 d after transplantation, the IL-15 content in peripheral blood was significantly higher in group B than that in group A and group C. The expression of IL-15 in transplanted liver tissues was significantly higher in group B than that in group A and group C. The activity of NF-κB in the spleen tissues was higher in group B. CONCLUSION: IL-15 might be a significant indicator for monitoring acute rejection after liver transplantation. The donor liver NK cells modulate the immunity of liver transplantation by inhibiting the expression of IL-15 via the suppression of NF-κB activity.