封闭负压条件下培养诱导巨噬细胞治疗烧伤脓毒症的作用及机制研究

Burn sepsis is a systemic inflammatory response syndrome induced by infection secondary to burn with high mortality, and poor curative therapy. How to improve the effect of burn sepsis is the problem to solve. Macrophage activation is the basic link of burn injury and sepsis. Vacuum sealing drainage (VSD) can cover the wound and regulate the immune. However, VSD could not cure the burn sepsis clinically. Based on the previous research, macrophages in the VSD drainage fluid could release less TNF- alpha which promotes inflammation, and release more IL-10 which is an anti-inflammatory factor. And we found that the deacetylase silent information regulator -1 (SIRT1) was inhibited in the macrophages of sepsis, while the SIRT1 was upregulated in VSD group. This suggests that VSD has a significant influence on the function of macrophages, and SIRT1 may be an important mediator. But the mechanism of VSD regulates macrophage in burn sepsis was still not clear. Based on this, we proposed to detect the immunological changes of macrophages and observe the efficacy of macrophgages in burn sepsis with the treatment of VSD. We want to identify the specific mechanism of SIRT1 in this process and verify it in a sirt1-knockout mice model. Through the research of the combination of VSD and macrophage biological treatment to improve the curative effect of burn sepsis and to reveal its mechanism, then provide a new method for clinical treatment.

烧伤脓毒症是烧伤后感染诱发的全身失控性炎症反应综合征。如何提高烧伤脓毒症疗效是亟待解决的难题。巨噬细胞的损伤与激活是烧伤脓毒症发病的根本环节。封闭负压引流（VSD）具有创面封闭和免疫调控作用，临床应用广泛。研究发现，VSD治疗后，巨噬细胞释放TNF-α降低，而释放IL-10增多；进而发现，脓毒症巨噬细胞内去乙酰化酶沉默信息调节因子-1（SIRT1）表达抑制，而VSD环境下SIRT1表达上调。这表明VSD对巨噬细胞功能有显著影响，而SIRT1可能是重要介导分子。但VSD环境下培养诱导巨噬细胞对烧伤脓毒症的疗效和作用机制不明。基于此，我们拟检测VSD环境下巨噬细胞的免疫功能变化及其在小鼠烧伤脓毒症中的疗效，探究 SIRT1影响巨噬细胞功能的机制，最后在sirt1基因剔除小鼠烧伤脓毒症模型中验证。通过本研究以期联合VSD和巨噬细胞生物治疗提高烧伤脓毒症的疗效并揭示其机制，为临床治疗提供新方法。

烧伤脓毒症是烧伤后感染诱发的全身失控性炎症反应综合征。封闭负压引流对于改善创面环境，促进创面愈合疗效显著，临床应用广泛。但其引起广泛生物学效应的作用机制一直存在争议。课题组发现烧伤脓毒症时，巨噬细胞内去乙酰化酶沉默信息调节因子-1（SIRT1）表达抑制，封闭负压引流治疗后，SIRT1表达上调，巨噬细胞炎性因子TNF-、IL-1β、IL-6显著降低，表明封闭负压引流疗法对巨噬细胞功能有显著影响，而SIRT1可能是其拮抗烧伤脓毒症的关键分子。课题组利用烧伤脓毒症小鼠模型，及LPS活化的巨噬细胞模型，明确烧伤脓毒症时，活化SIRT1可抑制失控性炎症反应，拮抗烧伤脓毒症的病理进程。进一步基于生物学分析，证实烧伤脓毒症中，miRNA调节SIRT1 对烧伤脓毒症脏器的保护效应，明确了烧伤脓毒症时，SIRT1的表达与miR-199a的水平负相关，通过调节miRNA199a/SIRT1信号轴可明显拮抗烧伤脓毒症介导的脏器损伤。最后，通过构建小鼠烧伤脓毒症创面模型，通过封闭负压引流疗法设置对照进行全转录组测序，从FC 在 2 倍以上，FDR 值＜0.05为标准，共筛选到864个差异表达mRNAs、67 个差异表达的 miRNAs、 37个差异表达的circRNAs、107个差异表达的lncRNAs 。差异表达的基因经过GO、Pathway富集分析、网络互做分析，所有具有显著性差异的基因主要与Toll样受体信号通路、炎症反应、白细胞迁移、白细胞趋化等信号通路相关。这些成果的取得解释了负压治疗的分子机制，可为临床使用封闭负压引流治疗提供理论依据。