BMSCs来源的外泌体依赖膜相关Hsp70对烟雾吸入性损伤的保护作用及其机制研究

Stem cells consist of some shortcomings，such as low survival rate, difficult saving and tumorigenesis. It has been demonstrated that exosomes secreted by stem cells could protect injured tissues and solve those problems, but the mechanism is still unknown. Exosomes membrane associated heat shock protein 70 (Hsp70) could protect the damaged tissues through reducing cell apoptosis, which may be part of the therapeutic mechanisms. Inspired by this, on the basis of bone marrow mesenchymal stem cells (BMSCs) transplantation for therapy of smoke inhalation injury in the past, we attempt to use exosomes derived from BMSCs to treat inhalation lung injury in rat by investigation of survival rate and apoptosis ratio, inflammatory factors and histopathological changes of the lung tissue and cells, as well as other methods in vitro and vivo, thus comprehensively evaluating the therapeutic effect of exosomes derived BMSCs for the treatment of smoke inhalation injuries. We will also detect that whether the membrane associated Hsp70 derived from exosomes as a "switch" binds with the toll like receptor-4 (TLR-4) and whether the exosomes protect lung tissue and resist apoptosis much more effectively. Furthermore we will detect whether the membrane associated Hsp70 has an effect on the TRL-4/Trif signaling pathway independent of Myd88.Finally the above research will lay a theoretical and experimental foundation for the therapeutic effect and mechanism of exosomes in the treatment of smoke inhalation lung injury, which has vital significance in the cell-free therapy.

干细胞具有移植存活率低、保存不易和易致瘤等缺点。现已证实，干细胞分泌的外泌体可以保护损伤组织并解决上述问题，但机制不明。外泌体膜表面含有热休克蛋白70（Hsp70），后者可以通过减少细胞凋亡保护损伤组织，推测可能是作用机制之一。项目组在前期将骨髓间充质干细胞（BMSCs）成功移植烟雾吸入性损伤动物模型的基础上，采用BMSCs来源的外泌体进行体内、体外实验，观察其对烟雾吸入性损伤大鼠存活率，肺组织细胞凋亡率、炎症因子和病理等变化，综合评价外泌体对烟雾吸入性损伤的治疗效果；在体外检测外泌体膜表面Hsp70是否作为一种 “开关”与肺细胞表面TLR-4受体结合以更好的发挥保护肺细胞、抵抗凋亡的作用，同时检测膜表面Hsp70对细胞TRL-4/Trif非依赖Myd88信号途径各蛋白水平的变化，揭示其可能的保护机制。该项研究为外泌体治疗吸入性损伤的临床应用奠定理论和实验基础，在无细胞治疗中具有重要意义。

烟雾吸入性损伤是重症烧伤中一种病死率高、预后差的疾病，主要由于吸入烟雾中的有毒物质导致肺部损伤，引起肺上皮细胞损伤、肺毛细血管屏障破坏，通透性增加、炎性细胞浸润。其机制可能是氧化应激反应过度导致低氧血症以及炎症因子的大量释放。我们前期应用间充质干细胞治疗烧伤烟雾吸入性损伤，发现有显著抑制肺部炎症、减轻肺水肿的作用，因此进一步检验干细胞分泌的外泌体是否可以发挥相同的作用、及其可能的作用机制。本研究从体内、外实验探索大鼠骨髓间充质干细胞来源的外泌体(BMSCs-EXO)在烟雾吸入性损伤中的治疗作用，通过检测BMSCs-EXO治疗后肺细胞的凋亡率、肺组织病理切片、免疫组织化学、炎症因子表达、肺通透性和血气分析等，探究其对烟雾吸入性损伤的治疗效果和肺保护作用，并以Western blot、免疫荧光等方法研究其可能的保护修复机制。实验结果显示: 1.成功从原代大鼠骨髓间充质干细胞中提取到外泌体，并通过NTA检测、透射电镜分析、Western blot验证所提取物符合国际上对外泌体的认定标准；2.体内外研究结果表明，BMSCs-EXO处理的大鼠肺组织损伤明显减轻，肺组织纹理、肺泡结构更清晰，肺间隔增厚及肺内出血等情况明显改善，肺干湿比结果提示肺血管通透性明显改善，肺泡灌洗液中蛋白含量明显降低，TUNEL提示肺组织凋亡明显减轻，ELISA和qRT-PCR结果提示促炎因子表达下降，BMSCs-EXO可以通过抑制炎症和细胞凋亡来减轻烟雾吸入所致的肺损伤；3.BMSCs-EXO通过调节 HMGB1/NF-κB 信号通路发挥肺保护作用；4.BMSCs-EXO可以诱导肺泡巨噬细胞极化，来介导烟雾吸入后的肺部炎症。免疫荧光结果提示M1、M2型巨噬细胞标志蛋白比例发生变化，qRT-PCR结果提示M1、M2型代表性炎症因子水平变化。因此我们认为，BMSCs-EXO在烟雾吸入性损伤治疗中，可以减轻炎症、抑制凋亡，发挥肺保护作用。