牙周致病菌信号分子AI-2诱导Th亚群细胞在慢性牙周炎微环境表达的机制研究

Periodontitis is one of the most common and serious oral diseases. Oral micro-organisms, host immune and local environment contribute to the pathogenesis of periodontitis, however, the mechanism is still obscure. It is well known that the periodontal bacteria induce immunity response via forming dental plaque biofilm. However, Autoinducer-2 secreted by bacteria can regulate the formation of dental plaque biofilm and mediate the periodontal micro-environment. Our previous study found that Th cell subsets might be synchronically associated with the development of chronic periodontitis. On the basis of our previous study, we plan to conduct a case-control study to further reveal the relationship between periodontal pathogens and Th cell subsets. Peripheral blood, subgingival dental plaque, gingival crevicular fluid and gingival tissue will be collected from both periodontitis patients and healthy controls, and be tested by flow cytometry, immunohistochemistry and RT-PCR. Furthermore, we will establish in vitro plaque biofilm and mouse spleen mononuclear cells co-culture models to investigate the effects of AI-2 on Th cell differentiation and biofilm growing via FACS, scaning electron microscope, crystal violet staining and V. harveyi bioluminescence assay. By revealing the role of AI-2 production in the differentiation of Th cell subsets, our study may enrich the interaction mechanism between periodontal bacteria and host immune cells. We hope it may provide new ideas for the treatment and intervention of periodontitis.

已知牙周炎发生是口腔致病微生物、宿主免疫和环境因素组成生态系统，共同作用的结果，但作用机制尚未阐明。牙周致病菌以菌斑生物膜的方式感染机体，引发免疫反应。而细菌通过分泌自诱导分子（Autoinducer-2）能够调控生物膜形成，改变牙周局部微环境。在课题组前期发现Th细胞亚群与慢性牙周炎发病相关的研究基础上，本项目拟从临床病例对照研究入手，选择慢性牙周炎和牙周健康人群，收集外周血、龈下菌斑、龈沟液和牙龈组织，采用流式细胞技术，免疫组化，RT-PCR，明确牙周致病菌与Th细胞亚群的关系。体外建立菌斑生物膜和制备鼠源的淋巴细胞研究模型，通过流式细胞、扫描电镜、结晶紫法、发光试验探讨不同活性AI-2对菌斑生物膜、Th细胞诱导分化的影响。由此，揭示牙周致病菌信号分子在诱导牙周相关Th细胞亚群中的作用，有助于阐述牙周炎致病菌与宿主免疫细胞之间相互作用机制，为治疗和早期干预牙周炎提供新的思路。

牙周炎菌斑因素与宿主免疫细胞的关系及致病机制目前尚未阐明。细菌分泌自诱导分子（Autoinducer-2）调控生物膜形成，改变牙周局部微环境尚未可知。本研究从临床病例研究角度，及利用流行病学采集牙周炎菌斑样本，探讨牙周致病菌与Th细胞亚群的关系。通过流式细胞技术，ELISA, 和RT-PCR等方法，我们发现慢性牙周炎患者外周血单个核细胞（peripheral blood mononuclear cell,PBMC）CD4+ T淋巴细胞中Th1、Th2和Th17表达上升，发挥促炎作用。调节性Ｔ细胞（regulatory T, Treg）介导的免疫应答减弱， 与Th17细胞在有效抑制其他免疫细胞活化、增殖，在控制免疫反应和维持机体免疫平衡方面发挥重要作用。致病菌Pg局部定植与Th17细胞表达存在相关关系。淋巴细胞特征性细胞因子IL-17、IFN-γ及Th17 细胞与牙周炎关系不明确。发病相关因素分析显示，牙周致病菌牙龈卟啉单胞菌和伴放线聚集杆菌定量分布因素可能与牙周临床指数、牙周炎分度有关。年龄、性别可能影响龈下菌斑中Pg和Aa定量水平。我们以牙周致病菌红色复合体和橙色复合体的代表性细菌P.gingivalis和F.nucleatum构建体外生物膜模型，扫描电镜观察微观形态学结构，采用酶标仪检测报告菌株V.harveyi产生的信号值，间接评估单独及共培养形成生物膜过程中产生的AI-2信号强度。结果显示AI-2信号强度F.nucleatum组高于共培养组高于P.gingival组。此外，课题组大胆创新，积极思考增加了“"新疆多民族自然人群队列建设研究"项目人群的牙周炎资料分析，提出负性情绪、单一肉类食物、葵花籽油过量摄入等膳食结构可能与 CP 的进展存在关联。牙周炎伴冠心病患者存在Th17/Treg比例失衡，且向Th17细胞偏移。类似在2型糖尿病伴有牙周炎患者中Th17 /Treg 失衡也发挥重要作用。上述研究将菌斑微生物和免疫细胞联系起来，并深入了解牙周致病菌通过自身分泌信号分子影响菌斑生物膜的形成。为牙周炎发病与宿主免疫之间的机制研究做了有益的工作，具有较高的学术价值及创新性。