血管生成素1促进肺泡巨噬细胞极性漂移的机制研究

Promoting inflammation resolution is an ideal strategy for the inflammation therapy. The phagocytosis of apoptotic inflammatory cells by phagocytes is the main mechanism of inflammation resolution. Phagocytes are dynamic and heterogeneous cells with different polarizations, including M1 phenotype, M2 phenotype and regulatory phagocytes. M1 macrophages are microbicidal and inflammatory (postinfectious pathogenesis), M2 macrophages are immunomodulators and take part in the inflammation resolution, tissue restoration and repair with anti-inflammatory effect. The polarization shift of macrophages will be of great benefit to accommodate the different stages of inflammation. We have found in earlier research that angiopoietin-1 could accelerate the inflammation resolution in endotoxic acute lung injury in mice by promoting the nuetrophil apoptosis and its phagocytosis by macrophage cells. But the effect of angiopoietin-1 on the polarization shift of macrophage cells and the underlying mechanism are unclear. This item will first explore the effect of angiopoietin-1 on the expression of different macrophage phenotypes associated factors, type-1 and type-2 helper T-cell cytokines in the bronchoalveolar lavage fluid with ELISA kits or polymerase chain reaction method, including IL-1β, TNF-α, L-arginine, IL-10, TGF-β, IFN-γ, IL-4, IL-13. At the same time, we will analyze the F4/80 antigen expression on the macrophage cells with Flow Cytometry method in vivo. Then we observe the modulating effect of angiopoietin-1 on the PI3K signal pathway and whether this pathway takes part in the polarization shift of macrophage cells through specific blocking the PI3K signal pathway with LY294002 in vitro. In this way, we could deeply discover the mechanism of angiopoietin-1 promoting the inflammation resolution in endotoxic acute lung injury in mice and further enrich the theory of inflammation resolution. At the same time, it would pave a solid foundation for further exploration of the effect of angiopoietin-1 on the immune and metabolic modulation in acute lung injury.

促进炎症消退是炎症治疗的理想策略，巨噬细胞吞噬凋亡的炎症细胞是炎症消退的重要方式。巨噬细胞具有多功能性和异质性，巨噬细胞极性从M1向M2漂移可以促进炎症消退和组织修复。我们前期研究发现血管生成素1（Ang1）促进内毒素性肺损伤炎症消退，但其对巨噬细胞极性的影响及机制尚不清楚。本项目拟在我们前期研究的基础上，通过观察在小鼠气管滴入脂多糖后不同时间点Ang1对肺泡巨噬细胞表面F4/80抗原表达和肺泡灌洗液中不同极性的巨噬细胞相关因子表达的影响，证实Ang1具有促进肺泡巨噬细胞极性从M1向M2漂移的作用。再在体外实验中观察Ang1对PI3K信号通路的调节作用，通过LY294002特异性阻断PI3K信号通路揭示该信号通路在Ang1促进肺泡巨噬细胞极性漂移中的作用，深入的阐明Ang1促进内毒素性肺损伤炎症消退的机制，丰富炎症消退理论，同时为进一步研究Ang1在机体免疫调节和代谢调节中的作用奠定基础。

在我们的前期研究中发现，血管生成素1保护内毒素性肺损伤的同时，通过流式分析发现巨噬细胞表面表达的抗原发生了变化，意味着巨噬细胞的极性也在发生变化。我们通过基因芯片分析，准备对巨噬细胞极性变化特异性的标志分子进行筛选，构建巨噬细胞极性模型。对各种标志分子表达进行研究的时候，已经有文章报道血管生成素1可以通过与特异性的TIE2受体结合，促进巨噬细胞向M1分化。我们及时调整了研究方向。在我们基因芯片分析中发现，Areg在M1型巨噬细胞高表达，但其功能尚不清楚。同时M1和M2的标志性分子也缺乏特异性，因此我们通过基因芯片和不同巨噬细胞极性模型的研究，探索不同极化的巨噬细胞的表面特异性分子表达、机制、及其功能。同时，我们通过LPS诱导小鼠急性肺损伤模型，观察Areg的表达，外源性Areg和Areg中和抗体对急性肺损伤小鼠肺损伤评分和炎症因子表达的影响及其作用机制进行研究。在体外研究发现，Areg在M1型巨噬细胞高表达，M2中几乎不表达，其表达受TLR4-MAPK信号通路调节。同时传统的观念认为il-10是巨噬细胞M2活化的标志性分子，通过我们的研究发现，从数值上，M1中表达更高，并且其强度跟促炎因子表达正相关。体内研究发现，LPS诱导的小鼠急性肺损伤模型中，Areg高表达，外源性的Areg保护LPS诱导肺损伤，而Areg中和抗体加重了肺损伤程度。机制研究发现，Areg通过与上皮细胞表面EGFR结合，AKT磷酸化，抑制了上皮细胞的凋亡，保护了肺上皮屏障。.因此，通过我们的研究一方面发现了巨噬细胞M1活化新的标志性分子，另一方面发现巨噬细胞M1型活化，一方面促进炎症反应，同时通过分泌Areg，保护上皮细胞和器官功能起着重要作用，是巨噬细胞和上皮细胞“对话”的新桥梁。

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