M2型巨噬细胞外泌体LncRNA调控慢性胰腺炎中胰腺星状细胞活化的机制研究

M2 polarized macrophage infiltration and pancreatic fibrosis are two major features of chronic pancreatitis. Exosomes-mediated intercellular signaling is an important mechanism of chronic inflammation-induced fibrosis. Recent studies have shown that M2 macrophage-derived exosomes can transmit RNAs to stellate cells, and participate in fibrotic phenotype activation and maintenance, but there has been no report in chronic pancreatitis. Our previous RNA-seq study found that there have been some same LncRNAs both in whole blood exosomes of patients with chronic pancreatitis and M2 macrophage-derived exosomes. Our pre-experiments also found that M2 exosomes can induce autophagy in pancreatic stellate cells and activate fibrosis pathways. And inhibition of autophagy could reverse stellate cells activation. Based on these results, this study intends to further explore the specific mechanism that M2 macrophage-derived exosomal LncRNA regulate the autophagy and fibrosis of pancreatic stellate cells. It is proposed to trace the intracellular transfer of exosomes and LncRNA between M2 macrophages and pancreatic stellate cells by fluorescence labeling, revealing the mechanism of LncRNA entering into exosomes. Morphological change and markers detection will be used to determine the induction of autophagy and fibrosis by exosomal LncRNA. Target gene analysis and LncRNA functional analysis will help determine the specific mechanism. This study is dedicated to find non-invasive diagnostic markers for chronic pancreatitis and provide new targets for anti-fibrotic therapy.

M2型巨噬细胞浸润和胰腺纤维化是慢性胰腺炎两大特征。外泌体介导的细胞间交流和互作是慢性炎症致纤维化的重要机制。M2型巨噬细胞源性外泌体（简称M2外泌体）可向星状细胞传递核酸分子，表观调控下游通路从而诱导纤维化，但在慢性胰腺炎中尚未有此类报道。我们前期RNA测序显示M2外泌体和慢性胰腺炎病人全血外泌体中存在共同差异表达的LncRNAs；预实验又发现M2外泌体可诱导胰腺星状细胞自噬和活化，而抑制自噬可逆转星状细胞活化。基于此，本课题拟深入研究M2外泌体通过传递LncRNA介导胰腺星状细胞自噬及活化的具体机制。拟通过荧光标记示踪外泌体及LncRNA在M2型巨噬细胞与星状细胞间的传递过程，揭示LncRNA进入外泌体的机制；形态学、标志物检测、拯救实验等明确自噬与星状细胞活化的关系；靶基因分析及LncRNA功能分析阐明具体作用机制。本研究致力于寻找慢性胰腺炎无创诊断标志物，为抗纤维化治疗提供新靶点。

M2型巨噬细胞浸润和胰腺纤维化是慢性胰腺炎两大特征。外泌体介导的细胞间交流和互作是慢性炎症致纤维化的重要机制。M2型巨噬细胞源性外泌体（简称M2外泌体）可向星状细胞传递核酸分子，表观调控下游通路从而诱导纤维化，但在慢性胰腺炎中尚未有此类报道。首先，我们通过高通量测序、生物信息学分析，筛选出可以作为慢性胰腺炎诊断标志物的RNAs，包括LncRNA CTB-12A17.3 (AC093503.2), MTRNR2L1, RAI14, CISH, AL445524.1, NACA2, GABARAP, HIST1H1D, USP9Y, APOB, MTRNR2L13。诊断效能优。其次，发现LncRNA CTB-12A17.3是介导M2型巨噬细胞与胰腺星状细胞相互作用之间的关键媒介，逐步揭示了其对纤维化和自噬通路的激活作用，并且探究了其作为CALM3 antisense对CALM3 mRNA有稳定作用，通过CALM3/Ca2+/TFEB通路激活自噬从而促使胰腺星状细胞活化。最后，在慢性胰腺炎动物模型上，进一步证实了LncRNA CTB-12A17.3对胰腺纤维化的作用。本项目成功构建了慢性胰腺炎患者的外泌体诊断标志物panel，实现该领域零的突破，国际上首次筛选并构建了CP外泌体RNAs的诊断模型，并且系统研究了外泌体LncRNA CTB-12A17.3介导的M2型巨噬细胞对胰腺星状细胞的激活作用，揭示了其潜在的机制，研究成果为开发慢性胰腺炎诊断标志物、纤维化程度评估以及抗纤维化治疗的创新模式和新靶点等提供了重要依据。同时，本项目的成果也为自噬、钙离子、内质网在慢性胰腺炎中的作用和机制提供了一定的证据。该研究具有一定的转化价值及较广阔的临床应用前景。