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

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.

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。

胰腺纤维化是慢性胰腺炎（CP）的特征性病理变化。前期研究中我们已经证明miR-199a多靶标调节TGF-β信号途径，促进PSC活化，是胰腺纤维化的关键因素；通过尾静脉注射抗miR-199a腺病毒载体可以延缓CP大鼠模型胰腺纤维化进程。进一步研究发现，miR-199a可由活化的PSC通过外泌体途径释放至细胞外，并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。本项目研究进一步利用体外共培养体系和相关动物模型研究明确外泌体miR-199a在PSC与巨噬细胞的交互中的功能及其作用机制，即外泌体miR-199a可通过分别促进PSC活化和巨噬细胞M1极化来协同调控胰腺纤维化进程；并基于外泌体作为药物内源性载体的独特优势，探讨利用PSC外泌体作为药物传递系统包裹miR-199a抑制剂治疗CP的潜在价值。此外，我们还筛选了四十余种具有抗炎、抗纤维化或抗氧化活性的小分子化合物，检测其对胰腺纤维化的影响作用，并发现其中达沙替尼和异甘草素可通过协同抑制PSC活化增殖和巨噬细胞极化发挥抗炎抗纤维化作用，提示对胰腺炎症-纤维化微环境的协同调控是缓解胰腺纤维化的关键。本研究基本完成了预期目标，加深对胰腺纤维化分子机制的认识，为研发抗纤维化治疗提供新的思路，具有较高临床指导和应用价值。