iPSC-MSCs来源的外泌体对脓毒症肺损伤的治疗作用及机制研究

The fundamental measure to prevent sepsis-induced lung injury is to inhibit overactivation of alveolar macrophages (AM) for reducing lung inflammatory response. Recent studies have shown that in mesenchymal stem cell (MSC) exosome (Exo) is an important carrier with biological functions to reduce inflammation. Thus, researchers have been exploring for the application of MSC-Exos to treat the sepsis-induced lung injury. However, due to the limited number of autologous MSC-Exos we plan to use induced pluripotent stem cell (iPSC) as the source of exosomes due to its higher proliferation capacity. In our initial study we innovatively found that iPSC-MSC-Exos could reduce inflammatory factors released from AM stimulated by LPS. Furthermore, with an exploratory application of miRNA chip assay we found the expression of miRNAs relevant to the inflammation in the iPSC-MSC-Exos. Therefore, we hypothesize that iPSC-MSC-Exos can transfer miRNAs to alleviate the sepsis-induced lung injury. Based on our preliminary data and findings, the proposed project is to understand the underlying mechanisms of biological functions and relevant pathways of reducing the sepsis-induced lung injury by iPSC-MSC-EXos and to prioritize key miRNAs of regulating the AM inflammatory response in iPSC-MSC-EXos as well as to illustrate the molecular mechanisms of their regulation in the lung inflammatory response. We anticipate that the proposed research will provide a new way for the effective treatment of the sepsis-induced lung injury.

抑制肺泡巨噬细胞（AM）过度激活，降低肺内炎症反应是防止脓毒症肺损伤根本措施。研究表明外泌体（Exo）是间充质干细胞（MSC）发挥作用的重要载体，有减轻炎症的生物学功能。有研究者正探索将MSC-Exos用于脓毒症肺损伤治疗，但自体MSC-Exos数量有限，故我们利用诱导多能干细胞（iPSC）增值旺盛的特点将其作为外泌体来源细胞，在前期研究中创新性发现iPSC-MSC-Exos可减少LPS刺激AM释放炎症因子，并探索性采用miRNA芯片发现iPSC-MSC-Exos内炎症相关miRNAs表达。我们假设iPSC-MSC-Exos可通过转运miRNAs减轻脓毒症肺损伤。本项目拟在前期基础上，明确iPSC-MSC-Exos减轻脓毒症肺损伤的生物学功能及作用途径；筛选出iPSC-MSC-Exos中调控AM炎症反应的关键miRNAs，明确其调控肺内炎症反应的分子机制，为脓毒症肺损伤治疗提供新手段。

外泌体作为细胞间信息交流的桥梁，在炎症反应过程中发挥重要的调节作用。人诱导多能干细胞来源的间充质干细胞分泌的外泌体（iPSC-MSC-Exo）因具备干细胞特性、自体来源丰富且无免疫原性的优点，并能有效避免致瘤、伦理等问题，已在免疫和组织修复等各大领域广泛研究，然而有关iPSC-MSC-Exo对脓毒症肺损伤的调控作用及机制仍未明确。本项目利用第二代高通量测序技术建立iPSC-MSC-Exo干预肺泡巨噬细胞（AM）炎症反应模型后的miRNA及mRNA差异表达谱，结合生物信息学分析筛选出关键miRNA，探讨iPSC-MSC-Exo对脓毒症肺损伤肺内炎症反应的作用及机制，结果发现：（1）iPSC-MSC-Exo可被AM摄取，抑制LPS诱导的AM炎症反应，减轻肺组织损伤。（2）miRNA差异表达谱显示miR-125b-5p上调明显且与炎症反应密切相关，同时miR-125b-5p在iPSC-MSC-Exo内表达丰度高。（3）在AM中过表达或沉默miR-125b-5p后，炎症因子和TRAF-6表达相应的降低或增加；双荧光素酶报告基因结果表明miR-125b-5p与TRAF-6的3’UTR区域存在靶向结合。（4）沉默iPSC-MSC-Exo内的miR-125b-5p后，iPSC-MSC-Exo对脓毒症肺损伤的保护作用受到显著削弱。基于以上结果，我们得出结论，iPSC-MSC-Exo 能够降低炎症反应，减轻脓毒症肺损伤，其机制是通过miR-125b-5p 靶向 TRAF-6，抑制AM NF-κB炎症信号通路的激活，从而减轻肺内炎症反应。本研究为脓毒症肺损伤的治疗提供了新思路。