摘要
目的 应用雷帕霉素体外预处理小鼠树突细胞（DC）,制备耐受树突细胞（Tol-DC）,并建立同种异基因小鼠皮肤移植模型,Tol-DC过继性输注到小鼠体内,观察移植皮片存活时间及Treg/Th17细胞表达,探讨其诱导移植免疫耐受的可能机制.方法 在小鼠骨髓来源的DC前体培养过程中加入粒细胞-巨噬细胞集落刺激因子（GM-CSF）,白细胞介素4（IL-4）和雷帕霉素,用流式细胞仪检测各组DC表面CD11c、CD40和CD80的表达;MTT比色法行体外混合淋巴细胞反应（MLR）观察每组对同种T细胞的刺激增殖能力;同时建立皮肤移植模型,观察皮肤移植前7d经尾静脉注射Tol-DC的受者移植物存活情况;第9天取其脾脏,检测Treg/Th17细胞变化,取移植皮肤HE染色观察炎性细胞的浸润情况.结果 Tol-DC组显著抑制了外源刺激下DC的成熟过程和表面共刺激分子（CD40、CD80）的表达,并显著抑制其同种T细胞激活能力（P＜0.01）;而且Treg比例升高,Th17下降,小鼠皮肤存活时间延长,炎症反应减轻.结论 应用雷帕霉素能够制备耐受性树突细胞,并诱导小鼠移植免疫耐受,这种作用是通过扩增Treg细胞,抑制Th17细胞而实现的,从而为在临床应用Tol-DC诱导供者特异性免疫耐受提供新的策略.
Objective To investigate the effect of tolerogenic dendritic cells （Tol-DC） generated by Rapamycin （Rapa） on the differentiation of Treg/Thl7 cells and explore the possible mechanism of tolerance induction.Methods DC progenitors from mouse bone marrow were propagated with granulocyte-macrophage colony-stimulating factor （GM-CSF） plus interleukin （IL）-4 stimulation for 6 days in the presence or absence of Rapa （20 ng/ml）.During DC culture,morphology of cell was observed under electron microscope.Cell surface expression of CD11c,CD40 and CD80 was analyzed by flow cytometry.The antigen-presenting function of DC was determined by one-way mixed leukocyte reactions.In vivo,the recipient BALB/c mice receiving transplantation of skin allograft from C57BL/6 mice were divided into control,Rapa,immature DC （imDC） and Tol-DC group.The survival time of the skin allograft was observed and Treg/Th17 cells were analyzed by flow cytometry in each group.Results The immunephenotypic analysis showed that in comparison with those in the control group and the LPS group the expression of the co-stimulatory molecules CD40 and CD80 were significantly lower in the Rapa-group and Rapa ＋ LPS group.The ability to stimulate proliferation of T cells of the same genotype in the Rapa-group was significantly inhibited （P 〈 0.01）.In the in vivo experiment,the mice＆#39; s survival time remarkably prolonged,the percentage of Treg cells was enhanced and Th17 cells was reduced in the mice＆#39;s spleen in Tol-DC group.Conclusions Tol-DC generated by Rapamycin can significantly induce immune tolerance through up-regulate Tregs and down-regulate Th17 cells.The present study highlights the therapeutic potential of preventing allograft rejection using in vitro-generated Tol-DCs,which can be loaded with donor antigen,and potentially used to promote organ transplant tolerance.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2015年第30期2469-2473,共5页
National Medical Journal of China
基金
国家自然科学基金（81050014）
