外泌体miR-146a-5p调控巨噬细胞极化在子宫内膜异位症中的作用机制

Endometriosis (EM) is difficult to cure, easy to relapse, and the side effect of drug treatment is great, which seriously affects the quality of life of the patients. Recent studies have shown that the cytogenetic variations and associated immunological abnormalities are the key to the development of EM. In the early stage, the expression of exosomal miR-146a-5p in ectopic endometrial stromal cells was significantly higher than that in eutopic endometrium by miRNAs sequencing and RT-PCR validation. Further analysis by bioinformatics pathway enrichment shows that the molecule participates in the regulation of multiple pathways such as MAPK through the target gene TRAF6, and the latter may participate in the polarization process of macrophages. To further clarify the regulation and mechanism of exosomal miR-146a-5p on EM development, we plan to identify the signal transduction function of exosome by cell co-culture and exosomal tracking. Macrophages were cultured by regulating the concentration of exogenous exosome and miR-146a-5p expression, and macrophage polarization and its molecular pathways were detected by flow cytometry and ChIRP-seq. Finally, we will study the effects of exosomal miR-146a-5p on the progression of endometriosis, immune system by the rat model experiment and clinical samples analysis, further obtain clinical application value. The purpose of this study is to explore the new theory of the development of endometriosis, which is helpful to elaborate the nature of the disease and provide a new strategy for more effective intervention.

子宫内膜异位症（内异症）难根治、易复发、药物治疗副作用大，严重影响患者的生存质量。最近研究表明遗传学基础和与之相关联的免疫学异常是内异症发生发展的关键。本研究前期通过外泌体miRNAs深度测序和RT-PCR初步显示异位内膜间质细胞及其外泌体的miR-146a-5p的表达显著高于在位内膜；生物信息学分析表明该分子通过靶基因TRAF6参与调控MAPK等多条通路，而后者可能参与巨噬细胞极化。本研究拟采用细胞共培养、外泌体跟踪等实验明确外泌体信号传导功能；通过调控外源性外泌体浓度和miR-146a-5p表达干预巨噬细胞培养，采用流式和ChIRP-seq等技术检测巨噬细胞极化及其分子通路；最后通过内异症大鼠模型实验和临床样本检测研究外泌体miR-146a-5p对内异症进展、免疫系统的影响，评估临床应用价值。本研究旨在探索内异症发生发展的新理论，利于深入阐述内异症的发病本质，并为更有效干预提供新策略。

子宫内膜异位症（内异症）难根治、易复发、药物治疗副作用大，严重影响患者的生存质量。最近研究表明遗传学基础和与之相关联的免疫学异常是内异症发生发展的关键。本研究前期已获得内异症间质细胞外泌体的提取与鉴定，通过外泌体miRNAs深度测序，获得外泌体miRNA差异表达谱，并显示异位内膜间质细胞及其外泌体的miR-146a-5p的表达显著高于在位内膜，通过miRNAs生物信息学分析，发现miR-146a-5p/TRAF6可能参与巨噬细胞的极化过程。本研究分别从细胞、动物、临床三水平进行验证。细胞水平上，首先采用细胞共培养、Transwell小室实验、外泌体跟踪实验，发现外泌体在内异症间质细胞和巨噬细胞之间的转运的效果，阐释了内异症间质细胞可通过外泌体作为信号转导渠道，影响巨噬细胞的功能，为后续的巨噬细胞极化研究奠定基础，并客观上获得内异症通过旁分泌途径改变微环境免疫稳态的直接证据。其次，成功采用GW4689外泌体抑制剂和转染miR-146a-5p mimics/siRNA分子培养内异症间质细胞构建内异症外泌体及其miR-146a-5p调控体系，成功构建自变量体系，为后期观察因变量变化（巨噬细胞极化转换）创造条件。随后，通过调控外泌体分泌，观察巨噬细胞表面标志物的表达变化来分析M1/M2的极化偏转，发现抑制外泌体分泌可降低巨噬细胞的M2极化，阐明了外泌体信号可影响微环境中的巨噬细胞的功能转变，从而改变微环境稳态，佐证了内异症发生发展的免疫基础。通过萤光素酶报告基因实验验证miR-146a-5p对靶基因TRAF6的作用，明确了巨噬细胞内miR-146a-5p对TRAF6表达的调控作用，并通过巨噬细胞中磷酸化JNK、磷酸化P38、磷酸化IKBa及磷酸化P65的表达水平影响其极化偏转。动物实验证实了外泌体和miR-146a-5p可促进内异症病灶发展，并在病灶中观察到了M2型巨噬细胞比例增加的现象，说明动物水平与细胞水平的结论一致。临床实验发现了内异症患者的腹腔液与外周血中miR-146a-5p的表达水平显著高于对照组，但小样本实验显示与内异症分期、EFI、复发等指标的相关性不具有显著性差异，需要进一步后期实验数据夯实，其临床意义有待于后续论证。