M2c巨噬细胞对阿霉素肾病小鼠调节性T细胞迁移活动的调控作用及机制研究

Keeping the number of regulatory T cells (Tregs) steady and maintaining their dynamic migration in vivo are both essential for kidney protection against chronic kidney disease（CKD）. In our previous stduy, we found out that M2c macrophage (M2cФ) significantly reduced renal inflammation and renal injury by inducing Tregs from CD4+CD25- T cells in vitro and increased the number of Treg in local draining lymph nodes in adriamycin nephrotic mice. We also found out that M2cФ down-regulated the number of exogeneous Tregs in the circulation of adriamycin nephrosis mice but increased the number of Tregs in spleen, draining lymph node and kidney. The regulation and mechanisms of M2cФ on Tregs migration are still elusive. Therefore, we plan to lable the M2cФ and Tregs with fluorescent in vitro, then inject them into adriamycin nephrotic mice. The movement and location of Tregs in adriamycin nephrosis mice will be tested by using cell tracking and fluorescence-activated cell sorting technologies. The role of M2cФ in Tregs migration will be focused on screening chemokines of Tregs by PCR assay and confirmed by ELISA. In sum, our study may further clarify the protectional mechanisms of M2cФ on adriamycin nephrotic.

调节性T细胞（Tregs）数量的稳定及它们在体内的迁移活动是慢性肾病（CKD）重要保护机制。我们前期研究证实巨噬细胞M2c在体外能诱导Tregs生成，并能使阿霉素肾病小鼠（adriamycin nephrosis mice,ANM）肾引流淋巴结Tregs明显增加,较好保护肾损伤；我们进一步发现M2c使ANM循环内外源性Tregs减少的同时增加Tregs在脾脏、肾引流淋巴结及肾脏的数量，提示M2c不仅诱导Tregs增殖，还调控Tregs迁移；但如何调控及相关机制还不清楚。本项目拟体外对M2c和Tregs进行荧光标记后注入ANM体内，采用细胞示踪技术结合荧光显微及FACS技术追踪M2c调控下Tregs在ANM体内的迁移活动；以PCR array筛选M2c调控下Tregs表达差异的趋化因子，并以ELISA技术和疗效实验确认筛选因子在研究中的机制作用，进一步阐明M2c保护AMN肾损害的作用机制。

调节性T细胞（Tregs）数量的稳定及它们在体内的迁移活动是慢性肾病（CKD）重要保护机制。我们前期研究证实M2c巨噬细胞(M2cФ)在体外能诱导Tregs生成，并能使阿霉素肾病小鼠（ANM）肾引流淋巴结Tregs明显增加,较好保护肾损伤；进一步发现M2cФ使ANM循环内外源性Tregs减少的同时增加Tregs在脾脏、肾引流淋巴结及肾脏的数量，提示M2cФ不仅诱导Tregs增殖，还调控Tregs迁移；但如何调控及相关机制还不清楚。本项目体外对M2cФ和Tregs进行荧光标记后注入ANM体内，采用流式细胞分析技术及组织免疫荧光染色技术追踪M2c调控下Tregs在ANM体内的迁移活动；以Rt-PCR检测体外M2cФ和Tregs细胞共培养、以及ANM体内M2cФ调控下Tregs相关趋化因子受体表达水平的差异，并以疗效实验确认筛选因子在研究中的机制作用，以阐明M2c保护AMN肾损害的作用机制。项目研究的结果揭示，外源性Tregs在ANM向肾脏募集，而M2cФ可促进Tregs在ANM体内向炎症部位和免疫器官募集，这种募集活动的增强与M2cФ促进Tregs相关CCR2、CCR4、CCR5、CCR7、CXCR5和CD62L表达水平上调有关，CD62L抑制剂能够抑制M2cФ对Tregs向炎症部位和免疫器官募集活动的影响。这些发现进一步阐明了M2cФ和Tregs协同免疫保护ANM肾损伤的机制，为进一步研究M2cФ和Tregs两种免疫细胞在肾损伤免疫细胞治疗提供实验基础依据。