TLR4在骨髓间充质干细胞治疗内毒素血症器官功能障碍中的调节作用及其机制

Organ dysfunction induced by endotoxemia is mediated mainly through widespread inflammation. It is life-threatening with high mortality.It is thus essential to block inflammation as early as possible. Toll-like receptor 4(TLR4)is one of the key molecules mediating the release of inflammatory cytokines by immune cells in endotoxemia,particularly by macrophages. Bone marrow mesenchymal stem cells(BMSC) are very potent in inhibiting immune and inflammatory response. However, BMSC express high level of TLR4. It remains to be answered that whether activation of TLR4 can influence the potential therapeutic effects of BMSC in ameliorating inflammation and organ dysfunctions in endotoxemia. Thus,we try to answer this question in this study. Firstly,we will explore the effects of ex vivo expanded BMSC on serum level of inflammatory cytokines, as well as on the structure and functions of organs in endotoxemia. Based on these results we will further study the regulatory role of BMSC on phenotype orientation of macrophages. Secondly,we will induce overexpression as well as gene silencing of TLR4 through gene technique,and then investigate the effects of TLR4 activation on BMSC-mediated immune inhibition and on BMSC-mediated treatment of organ dysfunction in endotoxemia. Finally, to clarify the potential mechanisms underlying the regulatory role of TLR4 in inhibitory immune effects of BMSC, we will detect the effects of TLR4 activation on molecules expression level,cell signaling activation and macrophage phenotype,which are involved in BMSC-mediated immune inhibition. With the solution of the above questions, this study can provide systematic and important experimental evidence for better utilisation of BMSC in the treatment of endotoxemia.

内毒素血症所致器官功能障碍主要通过失控的炎性反应介导，致死率高，早期阻断炎症反应非常重要。Toll样受体4（TLR4）是介导内毒素血症中免疫细胞特别是巨噬细胞释放炎性介质的关键分子。骨髓间充质干细胞（BMSC）能有效抑制炎性反应，对炎性相关疾病的治疗有重要价值；但它高表达TLR4。而TLR4活化对BMSC在内毒素血症炎症及器官功能障碍中治疗作用的影响尚待阐明。为此，本研究拟从动物和细胞水平研究BMSC对内毒素血症炎症及组织器官结构功能的影响，确定BMSC的治疗作用并进一步阐明它对巨噬细胞表型的影响；通过基因工程技术诱导TLR4过表达或沉默，探讨TLR4活化对BMSC治疗内毒素血症作用的影响；研究TLR4活化对与BMSC免疫调节相关的分子表达、信号通路活化及巨噬细胞表型分化的影响，最终揭示TLR4调控BMSC免疫抑制功能的机制，为更好地利用BMSC的免疫抑制功能来治疗内毒素血症提供实验依据。

内毒素血症所致器官功能障碍主要通过失控的炎性反应介导，致死率高，早期阻断炎症反应非常重要。Toll样受体4（TLR4）是介导内毒素血症中免疫细胞特别是巨噬细胞释放炎性介质的关键分子。众多研究表明骨髓间充质干细胞（BMSC）能有效抑制炎性反应，对炎性相关疾病的治疗有重要价值；但其表面表达TLR分子。而TLR4活化对BMSC在内毒素血症炎症及器官功能障碍中治疗作用的影响尚待阐明。为此，本研究从动物和细胞水平探讨了BMSC对内毒素血症模型组织病理变化及组织细胞活性的影响，并以TLR4位切入点探讨BMSC对内毒素血症模型治疗结果的分子机制。结果发现，体外培养的大鼠BMSC能在体外抑制小鼠脾细胞增殖。但BMSC输注至内毒素血症小鼠体内后并不能有效改善内毒素血症模型动物肝、肺及肾脏等组织的炎性损伤，甚至有加重炎症损伤的趋势；BMSC输注增加模型动物肺脏湿干比（P<0.05），模型体内炎性细胞因子IL-1β水平显著高于未输注动物 （P<0.05）。同时，BMSC输注组腹腔巨噬细胞培养上清诱导血管内皮细胞及心肌细胞凋亡率增高。进一步研究发现，BMSC表达一定水平的TLR4，LPS刺激后细胞上TLR4表达增高并活化增强。LPS诱导TLR4活化能促进BMSC迁移，增加BMSC内促炎因子（如IL1β）的表达、降低抗炎因子IL10的表达，促进巨噬细胞向促炎型分化，但能增强BMSC抑制脾细胞增殖的能力。内毒素血症模型血清具有相似作用，能促进BMSC迁移，抑制BMSC增殖、阻滞细胞周期，促进炎性因子IL-6、IL-1、MCP1等的表达。进一步研究表明，BMSC上TLR4信号通路与Notch信号通路间存在交叉对话：TLR4活化通过非NF-κb通路促进Notch信号通路的活化，Notch通路在一定程度上调控TLR4/NF-κb的活化，调节BMSC细胞因子的表达。本研究的结果提示内毒素血症炎性环境能诱导BMSC向促炎表型转化，这对更好地利用BMSC在抗炎疾病中的应用提供了参考。如，在BMSC用于感染性炎性病变的治疗中，需要充分考虑TLRs等分子对BMSC免疫调节作用的影响。