miR-let-7i靶向SOCS1通过TLR4信号通路调控树突细胞成熟诱导心脏移植免疫耐受机制

Heart transplantation is an effective treatment for end-stage heart disease，however,the difficulty in immune-tolerance induction and rejection of transplant graft which greatly limit heart transplantation development. In recent years, several evidences showed that miRNA involved in DCs maturation regulation, but little is known about miRNA function involved in the induction heart transplant immune tolerance. Our group focus on the treatment of heart transplantation rejection for many year,and made some progresses about the DCs function in the induction of immune tolerance.We firstly found that, in vitro transfection of miR-let-7i inhibitor can inhibit DCs maturation, and the target is SOCS1. The CD86-DCs separated by immune-magnetic bead could induce Treg proliferation. Though we already have the evidence of the miR-let-7i could regulate DCs function, induce Treg proliferation,the elaborate mechanism is not clear. Therefore, we aims to reveal the mechanism of miR-let-7i regulate the the DCs functions. Our hypothesis is miR-let-7i regulate the functional status of DC via targeting SOCS1-TLR4/MAL-NF-κB signaling pathway and induce Treg proliferation and Th1/Th2 balance.Meanwhile, in order to confirm miR-let-7i inhibitor treated CD86 -DC could induce immune tolerance in vivo, we set up a heart transplantation model, and evaluates the effect of miR-let-7i in transplantation immune tolerance by infusion with the miR let-7i inhhibitor treated CD86-DC. This subject may help to clarify the mechanisms of miR-let-7i regulate DC maturation status and induced heart transplantation immune tolerance, which provide a new perspect of immune tolerance.

移植排斥和难以诱导特异性免疫耐受是心脏移植治疗的主要瓶颈。树突状细胞(DC)，尤其是未成熟树突状细胞可通过诱导和扩增调节性T细胞（Treg）诱导免疫耐受。近年发现miRNA可参与DC分化、成熟及功能的调控，申请者前期体外研究证实miR-let-7i与DC成熟及Treg增殖有关，但其具体机制尚不明确。本研究拟在前期工作基础上，主要探讨DC中miR-let-7i是否可通过靶向SOCS1调控TLR4/MAL-NF-κB信号通路影响DC功能；阐明miR-let-7i对Treg、Th1/Th2间平衡的调控及机制；并在大鼠心脏移植模型中回输由miR-let-7i处理的CD86-DC，探讨是否可诱导移植免疫耐受，从而验证体外研究的结果及机制。本研究首次探讨通过靶向抑制miR-let-7i来调控DC成熟，达到诱导心脏免疫耐受并阐明其机制，该研究将为临床心脏移植的免疫耐受诱导奠定基础并提供新的治疗思路。

本次研究，我们证明了microRNA(miR) let-7i通过 JAK1-STAT3信号通路靶向调节IL-10表达，进而调控树突状细胞（DC）成熟，并且能够延长小鼠心脏移植后生存时间。我们应用逆转录酶聚合酶链反应，酶联免疫吸附试验和双荧光素酶测定法来证实MiR let-7i的作用靶点为IL-10。应用流式细胞仪及免疫组化来检测调节性T（Treg）细胞。Western blot用于检测JAK1，STAT3与磷酸化STAT3（pSTAT3）的表达。构建同种异体心脏移植物模型，用磷酸盐缓冲液，脂多糖（LPS）诱导的成熟树突状细胞（mDC），或miR let-7i抑制剂（inhibitor）处理的DC回输至大鼠体内。记录下移植大鼠生存时间，并对移植物进行组织学研究。分别检测由MiR let-7i inhibitor 处理的mDC和LPS诱导的mDC中IL-10 mRNA的表达水平和IL-10的在培养液中的分泌均增加。荧光素酶活性检测表明，IL-10的转录水平在回输let-7i模拟体（mimic）后减少，而在回输let-7i inhibitor后转录水平增加。回输MiR let-7i inhibitor可抑制DC成熟，然而，使用IL-10 siRNA 预处理使MiR let-7i inhibitor对DC成熟的抑制作用减退。回输let-7i mimic或预先转染IL-10后，成熟DC中JAK1，STAT3，pSTAT3的表达均受到抑制，而与之相比，回输let-7i mimic后JAK1，STAT3，pSTAT3的表达是上调的。我们发现，将miR let-7i inhibitor处理的mDC细胞回输的Lewis大鼠移植物，可以显著延长抑制物的生存时间。