抗原特异性Treg通过IL-10调控巨噬细胞功能抑制心脏移植慢性排斥反应的机制研究

Chronic rejection response is the major reason that affected long-term graft survival. There are still no effective treatment for chronic rejection. Reports have shown that antigen specific regulatory T cells (aTreg) mitigate or delay the incidence of chronic rejection after solid organ transplantation combined with bone marrow transplantation. However, the related mechanism is still uncertain. Our previous study showed that macrophage played an important role in the pathogenesis of chronic rejection in mouse model of heart transplant. Much number of macrophage infiltration in graft has bad effect on the chronic rejection. Here, we would investigate the mechanism of macrophage infiltration regulated by aTreg in the chronic rejection of mouse heart transplant. We will first induced antigen specific Tregs in vitro. Those Tregs well be infused intravenously into mouse model of heart transplant, and the macrophage phenotype and function will be detected by flow cytometry. Coculture of macrophage and antigen specific Tregs in vitro will be used to confirm the effect of Tregs in vivo. Using IL-10 knockout mice as the donor or recipient of heart transplant or IL-10 knock out aTreg, we will further investigate whether aTreg regulates macrophage function via IL-10 signaling pathway with the help of flow cytometry, immunofluorescence, western or PCR technology. Finally, isolating macrophage from graft of tolerant mice treated by aTreg, we would detect if this phenotypic macrophage can induce "infectious tolerance" and promote the function of aTreg in vivo and in vitro. This project would identify the mechanism of aTreg on regulating macrophage function each other in chronic rejection of heart transplantation. Elucidating this process will provide extensive understanding of the effect of aTreg on transplantation, and also provide theoretical basis for clinical application of aTreg.

移植物慢性排斥反应是影响移植物长期存活的重要因素，目前尚缺乏有效防治策略。既往研究显示抗原特异性调节性T细胞(aTreg)可抑制慢排发生，但其具体机制仍未明确。我们前期研究发现在小鼠心脏慢性排斥模型的移植物中有大量巨噬细胞浸润，明显影响移植物预后。本项目拟观察aTreg调控慢性排斥反应中巨噬细胞功能的作用及机制。在小鼠心脏移植慢排模型中，用aTreg处理后检测慢排病理改变及移植物中调节性巨噬细胞表型；然后，利用IL-10 基因敲除鼠作为移植供、受体，并诱导IL-10 KO aTreg，通过流式、免疫荧光、蛋白及PCR等技术分析aTreg是否通过IL-10调控巨噬细胞功能并抑制慢排；最后，从耐受小鼠移植物中分离巨噬细胞，体内、外观察其是否促进aTreg并诱导"传染性耐受"。本项目将初步探索aTreg抑制移植物慢性排斥反应的作用机制，阐明其与巨噬细胞的相互调控作用，为其临床应用提供理论依据。

移植物发生慢性排斥反应是组织和细胞移植失败的主要原因，抗原特异性Treg细胞(aTreg)可抑制慢性排斥反应发生，但其具体机制仍未明确。阐明慢排作用机理，探索aTreg细胞抑制慢性排斥反应的机制研究具有重要意义。本项目建立心脏移植慢性排斥反应模型，通过Foxp3-GFP knock in 小鼠中分离纯化 Foxp3-GFP+ Treg 细胞体外直接或间接途径诱导aTreg细胞，注射至移植模型小鼠中，观察移植心脏慢排反生情况，并检测巨噬细胞表型及功能，明确aTreg是否诱导调节性巨噬细胞，并观察aTreg诱导的心脏移植耐受小鼠是否具有抗原特异性而排斥第三方小鼠皮肤移植物；应用氯膦酸二钠脂质体清除单核/巨噬细胞处理，明确巨噬细胞在慢排发生过程中的作用，并观察其诱导的调节性巨噬细胞是否能诱导“传染性耐受”；然后，利用IL-10 KO小鼠中分离诱导抗原特异性Treg细胞，以IL-10敲除小鼠作为供、受体行小鼠心脏移植，观察aTreg注射后移植物存活情况，并检测炎症因子产生情况。最后，分离耐受小鼠心脏移植物中的巨噬细胞，体内、外观察其是否促进aTreg 增殖及功能，其机制是否与IL-10或TGF-β有关。本项目可初步阐明抗原特异性Treg细胞通过调控巨噬细胞抑制小鼠心脏移植慢性排斥反应的分子机制，探索 aTreg 细胞与巨噬细胞的相互调控作用，为将来临床上应用aTreg细胞治疗移植物慢性排斥反应奠定理论基础。

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