外泌体miR-199a诱导巨噬细胞极化调控胰腺纤维化的机制研究

慢性胰腺炎（CP）特征性病理变化为胰腺纤维化，申请人主持国家自然科学基金项目，明确miR-199a是胰腺星状细胞（PSC）活化导致胰腺纤维化的关键因素，发现miR-199a不仅在细胞内调控PSC活化，而且可通过外泌体途径释放至细胞外。进一步研究发现，活化的PSC释放携带miR-199a的外泌体可在体外诱导巨噬细胞极化，提高外泌体miR-199a表达丰度可增强巨噬细胞M1型极化，提示PSC和巨噬细胞在纤维化过程中可能存在重要交互作用。据此拟通过荧光标记和免疫电镜等验证PSC产生的外泌体miR-199a进入巨噬细胞，实现信号传递；并将包裹miR-199a或抑制剂的外泌体处理PSC与巨噬细胞共培养体系，分析巨噬细胞极化，阐明外泌体miR-199a促纤维化机制；基于外泌体载药免疫原性低和靶向性高的优势，在CP大鼠中通过外泌体包裹miR-199a抑制剂进行纤维化干预治疗，探讨外泌体基因治疗的可行性。

Chronic pancreatitis (CP) is pathologically characterized by pancreatic fibrosis. Supported by funds of National Natural Science Foundation of China, we have identified that miR-199a was a key factor of pancreatic stellate cells (PSC) activation during fibrosis. We found that miR-199a not only intracellularly regulated the activation of PSC, but also can be released out of cells by exosomes. Activated PSCs-derived exosomes carrying miR-199a could induce macrophages polarization in vitro. Moreover, M1 polarization was increased when elevating miR-199a in PSCs. These findings indicate that the interaction between PSCs and macrophages is essential for pancreatic fibrosis. Consequently, we propose the hypothesis that PSCs derived exosomes could deliver miR-199a into the macrophages, and thus trigger or accelerate polarization. In the project, the process of miR-199a exosomes delivered from PSCs to macrophages will be confirmed via the fluorescence labeling and immune electron microscopy methods. We will explore the fibrogenesis mechanisms of exosomal miR-199a in the PSCs and macrophages co-cultured system and in CP rat model. The role of exosomal miR-199a in polarization encouragement and pro-fibrosis will be investigated by knockdown or overexpressed miR-199a in PSC-derived exosome. Based on the advantages of low immunogenicity and high targeting efficiency of drug delivery, exosome-mediated miRNA-199a inhibitor delivery would be applied for fibrosis treatment in CP rat models. Therefore, the study will investigate the feasibility of exosome-based gene therapy, providing theoretical basis for the development of anti-fibrosis drugs.