白细胞介素35抑制小鼠心脏移植排斥反应的研究

Inhibitive effect of IL-35 on cardiac allograft rejection in mice

目的 探讨白细胞介素（IL）35基因转染延长同种心脏移植小鼠存活时间的作用及其机制.方法 分别以BALB/c小鼠和C57BL/6小鼠作为供、受者,建立同种小鼠腹部异位心脏移植模型.实验组（12只）受鼠术后1～3 d经尾静脉注射IL-35质粒载体（50 μg）,同种对照组（12只）受鼠术后1～3 d经尾静脉注射等量空质粒载体.监测两组移植心存活时间,检测受鼠外周血IL-35表达及外周血和脾组织T淋巴细胞亚群的比例,观察移植心脏病理变化.合成目的基因IL-35,通过阳离子脂质体转染法,体外转染HEK293细胞,分析IL-35基因表达情况;以经丝裂霉素处理的Balb/c和C57BL/6小鼠脾细胞作为刺激细胞,未经丝裂霉素处理的C57BL/6小鼠脾细胞作为反应细胞,进行体外单向混合淋巴细胞培养,观察IL-35对培养体系中CD4＋ CD25＋调节性T淋巴细胞（Treg细胞）及CD8＋T淋巴细胞比例的影响.结果 IL-35质粒载体转染的细胞上清液中有IL-35表达;IL-35能够上调体外同种混合淋巴细胞反应体系中CD4＋ CD25＋ Treg细胞的水平并抑制CD8＋T淋巴细胞的增殖.同种对照组和实验组移植心脏中位存活时间分别为7d和16 d,实验组较对照组明显延长（P＜0.01）.与同种对照组相比,实验组CD4＋ CD25＋ Treg细胞比例明显升高（P＜0.01）,CD8＋T淋巴细胞比例明显降低（P＜0.01）;移植心肌炎症细胞浸润的程度明显减轻.结论 IL-35能够诱导同种心脏移植小鼠体内CD4＋ CD25＋ Treg细胞的增殖和抑制CD8＋效应T淋巴细胞的比例,从而明显减轻急性排斥反应,延长移植心存活时间.

Objective To investigate the action mechanism of IL-35 gene transfection ameliorating cardiac allograft rejection and prolonging allograft survival.Methods pEBI3-L-p35-Fc plasmid was amplified by polymerase chain reaction.In vitro plasmid DNA pEBI3-L-p35-Fc or pSec-L-Fc was,respectively,transfected into HEK293 cells using Lipofectamine 3000.At 48 and 72 h after transfection,IL-35 concentration in culture supernatant of transfected HEK293 cells was detected by ELISA.Balb/c and C57BL/6 splenocytes treated with mitomycin （MMC） served as the stimulators,those not treated with MMC as responders,and they were subjected to one-way mixed lymphocyte culture （MLC）.In the presence or absence of IL-35,the percentage of CD4＋ CD25＋ Tregs was detected by flow cytometry.Abdominal heterotopic heart transplantation model was established by using inbred male Balb/c mice as donors and C57BL/6 as recipients respectively.In experimental group,recipients were intravenously administrated with IL-35 plasmid （50μg） on the day 1 to day 3 post-transplantation.The control mice were treated with normal saline.The IL-35 expression in the blood,CD4＋ CD25＋ Tregs proportion in the blood and spleen,and the survival and the histopathologic changes of the cardiac grafts were also observed.Results In vitro the transfected HEK293 cells expressed IL-35.IL-35 enhanced the proliferation of CD4＋ CD25＋ Tregs of MLC in vitro.The median survival time of the cardiac grafts in experimental group （16 days） was significantly longer than in control group （7 days） （P＜0.01）.As compared with control group,CD4＋ CD25＋ Tregs proportion was significantly increased （P＜0.01）,CD8＋ T cells proportion was decreased （P＜0.01） and the proliferation of lymphocytes and monocytes infiltration was inhibited in the experimental group.Conclusion IL-35 could alleviate cardiac allograft rejection and prolong cardiac allograft survival via the induction of proliferation and differentiation of CD4＋ CD25＋ Tregs and inhibition of proliferation of CD8 ＋ effector T cells.