慢性牙周炎及其Keystone致病菌、龈下菌斑微生物群落调控TCRβCDR3受体库与阿尔茨海默病的关系及相关机制

With the prevalence of Alzheimer's disease(AD) ascending worldwide, we do not clear the causes and pathogenesis of AD, and therefore be at a loss what to do in its treatment. AD affects the quality of life in the aged severely, it's urgent to clarify the etiology of AD and identify its modifiable risk factors. Accumulating evidence show that inflammation is involved in the pathogenesis of AD,but it remains unknown which peripheral inflammatory and infectious conditions play a role and at which stage of AD development. Periodontitis is a most common, chronic, peripheral, polymicrobial infection characterized by local and systemic inflammations. Periodontitis is more prevalent in adulthood and progresses and exacerbates in elderly. Thus, we have reason to hypothesize that periodontal inflammation and periodontal pathogenic microbiota, especially the keystone pathogen, P.gingivalis, maybe contribute to the pathogenesis of AD. This study will analyze the composition and feature of periodontal sub-gingival microbiota and the characteristics of TCRβ CDR3 repertoire in patients with Periodontitis and/or AD using Illumina Solexa high-throughput sequencing. With APP/PS1 double transgenic AD mouse model, we will observe and analyze the effects of periodontal inflammation and P.gingivalis-LPS on learning and memory ability of mice, and on the levels of pro-inflammatory cytokines in serum and brain tissues, and the formation of amyloid β plaque in the hippocampal region of brain. Based on these studies, we will try to elucidate the relationship between periodontitis and AD, and to provide evidences and theoretical basis for individual risk assessment of AD development and early intervention of AD.

阿尔茨海默病(AD)患病率日益升高，但至今对其病因知之甚少、治疗则几乎束手无策，严重影响老年人生命质量，尽早阐明其病因、寻找其可修饰的致病因子系当务之急。研究发现，炎症参与了AD发病机制。但究竟何种外周炎症与感染？又是如何发挥致病作用等?目前均不得而知。慢性牙周炎系最常见的外周、慢性、持续性多细菌感染，造成局部乃至全身炎症，且常中年后高发、病损逐渐严重。因此，有理由认为牙周炎及其以P. gingivalis为keystone的致病菌群可能通过某些机制影响AD发生发展。故本研究拟通过高通量测序技术分析牙周炎与AD患者龈下微生物群落结构与特征、TCRβ CDR3组库特征，牙周炎症及P.gingivalis-LPS对APP/PS1双转基因鼠AD模型学习记忆功能、血清及脑组织炎症因子以及海马区脑组织老年斑Aβ沉积的影响，以期厘清牙周炎与AD的关系，为AD风险预测与早期干预提供理论依据。

牙周炎与阿尔茨海默病（AD）密切相关，但二者相关的生物学基础与机制等尚不清楚。本研究通过：（1）分析牙周炎及其关键致病菌 P. gingivalis/P.gingivalis-LPS对AD模型小鼠、SD大鼠AD样病理发生发展的影响；（2）分析牙周健康、中重度慢性牙周炎及中重度慢性牙周炎伴AD患者临床牙周状况、龈下微生物组成的差异；（3）收集中重度牙周炎患者、AD伴中重度牙周炎患者组龈下菌斑及外周血中TCRβ链CDR3区组库特征，并对各组间菌群、组库的差异及其与AD的关系进行相关性分析；（4）分析胰岛素抵抗和胰岛素信号通路在实验性牙周炎加重AD模型大鼠认知功能障碍及主要病理特征中的可能作用；（5）分析重度牙周炎伴AD临床患者及大鼠模型中口腔及肠道微生物群落构成特点及对学习记忆功能的影响。结果发现:（1）实验性牙周炎可加重鼠脑皮质与海马区淀粉样蛋白Aβ的沉积以及神经炎症反应等 AD 样典型病理损害，加剧学习记忆功能的减退；（2）伴有 AD 的牙周炎患者具有更差的牙周健康状况，中重度牙周炎患者龈下菌斑微生物群落中4菌属（纤毛菌属、Delftia、真杆菌属、Saccharibacteria_genera_incertae_sedis）和4菌种（龈炎普雷沃菌、纤细弯曲菌、变黑普雷沃菌、Prevotella maculosa）的增加可能与AD的发生、发展相关；（3）中重度牙周炎患者、AD伴中重度牙周炎患者TCRβ链 CDR3 组库中 V-J 配对基因的取用存在差异，AD伴中重度牙周炎患者组存在高度保守的氨基酸序列，提示两组 TCRβ链 CDR3 组库特征可能具有一定差异性，同时在两组中发现了差异性龈下菌群物种，推测两组中龈下微生物构成及差异性物种可能会影响外周 TCRβ链 CDR3 组库；（4）实验性牙周炎可加重STZ所致AD模型大鼠的特征性病理变化，其机制可能与牙周炎症下调Akt/GSK-3β信号通路的传导并促进IR程度有关。（5）健康者、中重度牙周炎患者、AD伴中重度牙周炎患者三组各自具有其独特的优势菌群，且牙周炎状态下肠道益生菌数量减少而促炎细菌增加；体内实验性牙周炎可导致大鼠学习记忆能力损害，并加重AD大鼠学习记忆功能损害及AD样病理改变，这些变化可能与牙周炎影响肠道菌群进而影响肠脑轴有关。以上实验结果表明牙周炎在AD的发生和发展过程中发挥了关键作用。