过表达Rictor基因的供者Rapa-DCreg抑制小鼠肾移植排斥反应及机制研究

Regulatory dendritic cell (DCreg) therapy is currently one of the most promising strategies to suppress transplant rejection, but the function of drug-induced DCreg is not stable in vivo and the mechanisms are poorly understood. Our research group previously found that Rapamycin (Rapa) could effectively induce DCreg (Rapa-DCreg), however, Rapa only inhibited the downstream of mTORC1 in DCreg and it did not affect the downstream of mTORC2. Whereas, Rictor (a core component of mTORC2) specifically deficiency in DC can accelerate graft rejection. Therefore, this project will overexpress Rictor in Rapa-DCreg to generate regulatory functional stronger and stable “Rictor-high-Rapa-DCreg” through combining of drug induction and gene modification method. Intravenous infusion of Rictor-high-Rapa-DCreg into recipient can inhibit murine kidney transplant rejection. We will explore its mechanisms including detecting the apoptosis of donor DCreg and its cross-dressing with recipient DC by modern molecular biology techniques and monitoring the interactions of donor DCreg with recipient DC and Treg by intravital two-photon imaging. This project will provide a novel induction scheme and important theoretical basis for DCreg inhibiting rejection.

调节性树突状细胞（DCreg）治疗是目前最有潜力的抑制排斥反应策略之一，但药物诱导的DCreg在体内功能不稳定，且机制不详。本课题组前期发现雷帕霉素(Rapa)能有效诱导DCreg(Rapa-DCreg)，但Rapa仅抑制DCreg的mTORC1信号下游，未影响mTORC2信号下游，而DC在敲除 Rictor(mTORC2的核心成份)之后可加速移植排斥反应。故本项目采用药物诱导和基因修饰相结合的方法拟对供者Rapa-DCreg过表达Rictor产生调节功能更强、更稳定的Rictor-high-Rapa-DCreg，静脉输注到受体小鼠体内抑制肾移植排斥反应。运用现代分子生物学手段检测供体 DCreg的凋亡及其与受体DC的cross-dressing，双光子活体成像观察供体DCreg与受体DC和Treg的互动，多角度解析其作用机制。本研究为DCreg抑制排斥反应提供新的诱导方案和重要理论基础。

树突状细胞DC是连接固有免疫与适应性免疫的重要桥梁，在介导免疫反应及疾病发生中具有重要作用。研究表明，免疫代谢参与DC功能的调节，然而其机制并未明确。哺乳动物雷帕霉素靶蛋白mTOR由TORC1和TORC2两类复合体组成，其在固有免疫的调节和代谢再编程中发挥重要作用。雷帕霉素的发现促进了TORC1的相关研究，然而，由于缺乏特异性的TORC2抑制剂，目前对TORC2的作用知之甚少。本研究旨在阐明TORC2的核心成分Rictor调节DC表型及功能的潜在机制，并为临床问题提供新的思路。.能量代谢检测结果显示，与WT DC相比，小鼠骨髓诱导分化的TORC2-/- DC中代谢发生改变，表现为基础糖酵解水平增强，对糖酵解来源ATP依赖性升高。与此同时，TORC2-/- DC中SRC、线粒体含量、mtDNA拷贝数增加，以及线粒体膜电位稳定性增强共同反映了TORC2-/- DC中线粒体功能增强。.与此同时，基因芯片的结果提示了TORC2-/- DC中TORC1信号通路活化水平提高，包括Itgα2b、Ptk2、IL-7R和JAK表达水平增加，p70S6K、4EBP1、CD36激活增强。TORC1通路活化增强最终导致PPAR-γ表达上调，随后调节Srebf1导致脂质储积增加。总之，本研究显示TORC2的核心成分Rictor可能通过限制TORC1调节的代谢活动及线粒体功能而调控DC，该理论结果可指导细胞甚至组织器官TORC2特异性抑制的研发。