miR-30b/d调控肝基质细胞诱导的调节性树突状细胞功能机制的研究

The liver is an unique organ in which induction of tolerance may be superior to induction of immunity. There is a lot of experimental and clinical evidence to support this observation. Unfortunately ,the underlying mechanisms are not fully understood. Except NK, NKT and kupffer cell, which can secrete chemokines to trap the activated T cells to death or secrete suppressive cytokines in liver, liver microenvironment induced LRDCs which play an important role in liver tolerance through multiple mechanisms. However, the exact molecular mechanisms for immune suppressive functions of LRDCs are not totally clear. .MicroRNAs (miRNAs) are an abundant class of 22-nucleotide regulatory RNAs and control biological functions of cells, such as proliferation, differentiation, immune response and so on. Our published data showed that several miRNAs contributed to the immune suppressive functions of myeloid-derived suppressive cells(MDSCs), but the role of miRNAs in the functions of LRDCs are still not explored. Our preliminary data showed that the miR-30b/d expression in LRDCs and conventional dendritic cells (cDCs) are different by using miRNAs highthroughput sequencing. In addition, Jagged-2 induced signal pathway involved in the immune functions of regulatory dendritic cells. So, combined its tightly relationship with miR-30b/d in our preliminary data, we hypothesized that miR-30b/d regulate the immune functions of LRDC. In this grant, we will explore roles of miR-30b/d and associated mechanisms in LRDC regulation. With their successful demonstrated, miR-30b/d will rich the basic theory in immunity regulation, and will show their potential usage in the treatment of autoimmune disease, especially in autoimmune hepatitis.

调节性树突状细胞是负向调控免疫应答的重要机制之一，对于机体免疫耐受的形成至关重要。其中，肝脏独特微环境产生的调节性树突状细胞（Liver regulatory dendritic cells, LRDCs）可通过多种机制参与肝脏耐受的形成。但到目前为止，有关调控该调节性树突状细胞功能的分子机制仍不清楚。miRNA作为细胞内重要的功能调控分子，参与多种免疫细胞的分化及功能调节。本研究将在前期研究LRDC分化及功能的基础上，拟通过高通量测序分析、体内外T细胞抑制功能试验等，研究候选miR-30b/d分子在该LRDC免疫调节功能中的作用，及其相应的信号通路机制。对于该调节性树突状细胞负向调控免疫应答分子机制的阐明，不仅将丰富免疫负向调节的分子机制内容，还将为其未来的潜在临床应用提供理论基础。

肝脏内的负向调控免疫细胞对于肝脏免疫耐受的形成至关重要，其中也包括肝基质细胞诱导的树突状细胞，进来有研究表明miRNAs可调控负向免疫细胞的功能，具体机制还需进一步研究。根据前期的研究和生物信息学研究，我们推测miRNA-200b-3p和miR-30b通过抑制靶基因JAG2的表达从而调控肝基质细胞诱导的树突状细胞（LRDCs）的功能。实验结果证明，miR-30b对JAG2没有靶向作用，WB和luciferase reporter实验证明miRNA-200b-3p mimics在转录后水平抑制靶基因JAG2蛋白水平的表达。miRNA-200b-3p mimics能够抑制LRDCs分泌的IL-10, miRNA-200b-3p inhibitor 能够促进LRDCs分泌的IL-10。进一步的实验证明，JAG2 siRNA也能够抑制LRDCs分泌的IL-10，这与我们的预测结果相符，即miRNA-200b-3p通过抑制靶基因JAG2的表达从而调控LRDCs 中IL-10的分泌。下一步的实验验证了miRNA-200b-3p通过抑制靶基因JAG2的表达从而缓解了LRDCs对成熟树突状细胞促进CD4T细胞增值的抑制作用。对于该调节性树突状细胞调控免疫应答分子机制的阐明，不仅将丰富免疫负向调节的分子机制内容，还将为其未来的潜在临床应用提供理论基础。