线粒体DNA-TLR9信号通路在脑死亡致供体肺炎症反应中的作用研究

Lung transplantation remains the ultimate treatment option for patients with end-stage pulmonary disorders.Brain-death (BD)-induceda series of pathophysiological changes may result in pulmonary inflammation, thereby affecting the long-term survival of recipients after transplantation. However, the mechanism by which BD-induced pulmonary inflammation remains unknown. Our preliminary datademonstrated thatthe protein level of cytokines and chemokines were increased in the lung tissue from BD donor, and visible inflammatory consolidation and different degree of inflammatory cells infiltration were also observed in BD donor lung. Furthermore, we found that the mRNA level of Toll-like receptor 9 (TLR9) was most significantly elevated among all the measured Toll-like receptors. Combined those data with literature, we propose that mitochondrial DNA released following BD may bind to TLR9 in alveolar macrophage and activate the TLR9-mediated downstream signaling pathway, lead to inflammatory mediators release and chemotaxis neutrophils into pulmonary tissue, thus resulting in lung inflammation and injury. In the present study, we firstly detect 1) the inflammation in lung tissue; 2) the level of mtDNA in plasma from living donor and BD donor; 3) the mRNA level of TLR9 in lung tissue from both donor. After identifyingcorrelation between mtDNA-TLR9 and lung inflammation, we then investigate the role of mtDNA-TLR9 in BD-induced donor lung inflammation by using alveolar macrophage, mouse BD model and lung transplantation model. Hopefully, our study will provide evidence that therapeutic intervention to modulatemtDNA-TLR9 signaling may ameliorate BD-induced lung inflammation, thus improving the long-term survival of recipients after lung transplantation.

肺移植是目前治疗终末期肺病的唯一有效方法。脑死亡(BD)后的病理生理改变可致肺部炎症发生，影响移植受体的长期生存，但BD致供肺炎症反应的机理尚不明确。前期研究发现，脑死亡供体(DBD)供肺存在炎性实变，组织间隙多种细胞因子升高，且有不同程度炎性细胞浸润。研究还发现Toll样受体9(TLR9)在DBD肺组织中升高尤为显著。结合文献报道我们推测：BD后组织释放的线粒体DNA(mtDNA)与肺泡巨噬细胞(AM)内TLR9受体结合，激活下游信号通路，引起炎症介质释放，同时通过趋化因子促进中性粒细胞迁移至肺内，导致肺部炎症反应和肺组织损伤发生。本研究拟通过对临床样本的分析，确定mtDNA-TLR9和肺部炎症的关系，进一步采用AM及小鼠BD和肺移植模型，探讨mtDNA-TLR9在BD致肺部炎症中的作用，以期为临床上采取治疗措施提高肺移植后移植物及受体的长期生存提供理论依据。

肺移植已成为治疗多种终末期肺疾病的唯一有效方法，也是公认的改善终末期肺疾病患者生存质量和延长寿命的重要手段。随着脑死亡（DBD）供肺成为我国肺移植的唯一合法供肺来源，供肺质量问题已成为肺移植取得长足进步需要逾越的最大障碍。如何改善DBD供肺质量，是目前肺移植领域亟待需要解决的问题。对DBD供体肺部炎症反应机理的研究将为采取临床治疗措施改善供肺的质量和提高肺移植成功率提供重要依据。本项目主要研究结果显示：1）与正常肺组织（NC）相比，DBD供体血浆中和供肺组织间隙炎性因子水平均升高；2）与NC相比，DBD供体血浆中线粒体DNA（mtDNA）含量升高；3）与司法肺相比，DBD供肺组织中TLR2和TLR9表达水平升高；4）体外给予巨噬细胞mtDNA处理后，DBD mtDNA诱导TLR9表达，同时炎症因子释放增多；干扰TLR9表达后，DBD mtDNA引起的炎症因子释放明显减少；5）给予野生型小鼠尾静脉注射mtDNA，与对照组（给予PBS）相比，正常肺组织mtDNA未引起明显的肺部炎症反应，而DBD mtDNA则引起广泛的肺部炎症反应，同时肺组织TLR9的表达水平升高，磷酸化Iκ-B表达水平明显降低。我们的研究结果提示，脑死亡后损伤的线粒体释放mtDNA入血，mtDNA随血液循环至肺脏与TLR9受体结合，激活TLR9下游信号通路，导致肺部炎症反应和肺组织损伤的发生。本研究探讨mtDNA-TLR9在脑死亡致肺部炎症反应中的作用及可能机制，以期为临床上采取治疗措施，提高脑死亡供体肺的质量，进而提升肺移植后移植物以及受体的长期生存率提供理论依据。