自噬系统调控中性粒细胞胞外陷阱（NETs）与根尖周骨破坏

The findings of our previous NSFC project imply IL-17+ netrophils participate in the pathogenesis of periapical disease. Neutrophil extracellular nets (NETs) as the novel functional structure of neutrophils, was recently demonstrated to positively regulate the IL-17 expression of neutrophils their own. Of note, autophagy was newly discovered to drive NETs formation, which may play an essential role during the progression of a variety of pathological immune responses, including autoimmunity and inflammatory diseases. Therefore, we proposed a new hypothetic model that NETs controlled by autophagy is critically invovled in the pathology of periapical bone destruction. Thus the thesis is devided into three parts: Part one is in vivo experiment: Autophagy, NETs, and IL-17/RANKL are detected and measured in periapical tissues obtained as biopsy samples during periapical surgery and in experimental periapical lesions of conditional knockout mice,as well as in vivo gene transfer model. Part two is downstream signaling characterization: Downstream molecular tagets of autophagy that directs NETs formation are screened by Affymetrix GeneChip, and then functionally confirmed by lentivirus transfection. Part three is in vitro experiment: 1. The three-dimensioned co-culture system of induced NETs and osteoclasts is performed, then the osteoclastogenic potential is analysed. 2. The three-dimensioned co-culture system of induced NETs and osteoblasts is also established, then morphological and functional assessment of osteoblasts is carried out. Therefore, the present project may both deepen molecular understanding of periapical bone destruction and further figure out the role of neutrophil-mediated immune response in osteolytic diseases.

课题组前一项国家自然科学基金资助项目的研究结果提示IL-17+中性粒细胞在根尖周病发生发展过程中起作用。中性粒细胞胞外陷阱（Neutrophil extracelluar nets,NETs）作为中性粒细胞的新型功能结构，正性调控中性粒细胞自身IL-17表达。新发现自噬系统在NETs形成中发挥着"门控效应"，可能是慢性炎症和自身免疫病发生发展的关键因素。本课题提出自噬系统调控NETs影响根尖周骨破坏的新假设：拟通过人病变组织、条件性基因敲除小鼠以及体内基因转移模型等，研究自噬及NETs在根尖周病中的可能作用；基因芯片筛查自噬系统调控NETs的下游关键靶基因；同时将NETs分别与破骨细胞、成骨细胞建立细胞交互作用立体模型，以探讨其参与根尖周骨破坏的机理。本系列研究对进一步阐明根尖周病骨破坏机制和全面认识中性粒细胞介导的免疫反应在溶骨性疾病中的作用都具有重要意义。

中性粒细胞胞外陷阱（NETs）作为中性粒细胞的新型功能结构，正性调控中性粒细胞自身IL-17表达。新发现自噬系统在NETs形成中发挥着"门控效应"，可能是慢性炎症和自身免疫病发生发展的关键因素。本课题拟通过人病变组织、条件性基因敲除小鼠以及体内基因转移模型等，研究自噬及NETs在根尖周病中的可能作用，探讨其参与根尖周骨破坏的机理。结果发现人根尖周病变组织和小鼠根尖周病动物模型中，自噬相关分子、NETs 和IL-17/RANKL的表达量明显高于正常组织。NETs作用于破骨细胞分化体系后，显著促进破骨细胞的形成。进一步探讨发现IKKβ/NF-κB/自噬信号轴促进破骨细胞形成及炎性骨破坏，而异甘草素通过抑制该信号轴减少破骨细胞的形成及炎性骨吸收。同时临床经典根管修补材料MTA通过抑制自噬信号活化干预破骨细胞形成。本系列研究对进一步阐明根尖周病骨破坏机制和全面认识中性粒细胞介导的免疫反应在溶骨性疾病中的作用都具有重要意义。