小鼠调节性树突状细胞分泌的外泌体诱导免疫耐受的实验研究

Immune Tolerance Induced by Exosomes Derived from Regulatory Dendritic Cells of Mice

本研究探讨小鼠调节性树突状细胞(rDC)分泌的外泌体(regulatory exosomes,rDex)诱导免疫耐受的作用,并与正常未成熟树突状细胞的外泌体(immature exosomes,iDex)诱导免疫耐受的作用进行比较。取C57BL/6(H-2b)小鼠骨髓细胞诱导未成熟树突状细胞(iDC),TGF-β1联合IL-10诱导调节性树突状细胞(rDC),流式细胞术检测两种DC的表型。采用超速离心结合膜超滤的方法分别提取rDex和iDex。建立小鼠皮肤移植模型,以C57BL/6小鼠为供者,以BALB/c(H-2d)小鼠为受者,按对受者处理的不同实验分3组,iDex组术前7、3天受者经尾静脉注射10μg供者的iDex,rDex组术前7、3天受者经尾静脉注射10μg供者的rDex,另设PBS对照组,观察移植皮肤存活情况。通过单向混合淋巴细胞反应(MLR)观察供者及非亲缘异基因供者DBA/2对同种异基因小鼠T细胞增殖情况。结果表明:TGF-β1、IL-10可下调DC表面共刺激分子CD80、CD86、CD40的表达。移植皮片平均存活时间(MST),在对照组为7.8天,iDex组为10.7天,rDex组为18.8天;iDex组的移植皮片MST明显长于对照组(p(0.05);rDex组的移植皮片MST明显长于iDex组(p(0.01)。MLR结果证明iDex组和rDex组B/C小鼠均对C57小鼠的脾细胞产生特异性耐受,尤其是rDex组;对非亲缘异基因DBA供者的脾细胞两组仍表现出强烈的免疫应答。结论:iDex和rDex均有诱导免疫耐受的作用,且rDex作用较iDex作用好。

The study was aimed to explore the roles of exosomes derived from regulatory dendritic cells of mice in the induction of immune tolerance. Immature DC （ iDC ） from mouse bone marrow cells and regulatory DCs （ rDC ） were induced by treating iDC with TGF-β1 and IL-10, The phenotype of regulatory DCs and normal DCs were assayed by flow cytometry, Exosomes from immature DCs （iDex） and regulatory DCs （rDex） were isolated by ultracentrifugation and ultra_filtration. A skin transplantation model was established with the recipients BALB/c mice and the donor C57BL/6 mice. Recipients were divided into PBS control group, iDex group （ injection 10 μg iDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation） , rDex group （ injection 10 μg rDex of donor C57BL/6 mice via tail vein at days 7 and 3 before skin transplantation）. The capacity of the donor mice and the unrelated allogeneic donor mice to stimulate allogeneic T lymphocyte proliferation was examined by mixed lymphocyte culture （MLR）. The results showed that TGF-β1 and IL-10 could down-regulate the expressions of costimulatory molecules,including CD80,CD86 and CD40. The graft mean survival time （MST） in control group, iDex group and rDex group was 7.8, 10.7 and 18.8 days, respectively. There was significant difference in MST between iDex group and control group （p 〈 0.05）, and between rDex group and iDex group （p 〈 0.01 ）. The results of MLR assays indicated donor-specific hyporeactivity especially in rDex group, while the tolerant B/C mice were still immunocompetent to unrelated allogeneic DBA mouse. It is concluded that injection iDex or rDex of donor mice via via vein before skin transplantation induces immunotolerance, and the effect of rDex is more significant.