炎性因子IL-1β调控牙周膜干细胞α7 nAChR表达参与吸烟相关性牙周炎发生发展的机制研究

Periodontitis is a bacteria infected disease; it is the most commonly accepted reason that leads to teeth loss for adults. Smoking is a critical risk factor of periodontitis, however, detailed mechanisms between inflammation and nicotine are unknown. Our research group confirmed that there exists functional expression of α7 nicotinic acetylcholine receptor (α7 nAChR) in periodontal tissues and cells for the first time; through which nicotine deteriorates inflammatory reaction and bone destruction in local tissues. Preliminary experiments further demonstrated that IL-1β could up regulate the expression of α7 nAChR in periodontal ligament stem cells through phosphorylating GSK-3β. Based on these results, we confer that IL-1β might up regulating the expression of α7 nAChR through IL-1R-GSK-3β pathway. The up regulated α7 nAChR then combines with nicotine, for one thing, aggravating the inflammatory reaction forming a positive feedback loop of its destructive effect; for the other, suppressing the osteogenic differentiation while enhancing the osteoclast differentiation, deteriorating the absorption of local bone tissues. This program would proceed from molecular, cell and tissue levels through in vitro and in vivo studies to investigate the effect and related mechanisms of IL-1β up regulating the expression of α7 nAChR in aggravating inflammatory injury and bone destruction in smoking related periodontitis. We aim to provide experimental evidences for explaining the pathological progress of smoking related periodontitis and to provide new thoughts for effective disease prevention.

牙周炎是导致成人牙齿脱落的最常见原因，吸烟是其重要危险因素，但炎症与尼古丁相互作用的机制并不明确。课题组前期首次证明牙周组织及细胞中存在α7亚型烟碱型乙酰胆碱受体（α7 nAChR）的功能性表达，尼古丁正是通过该受体加重局部组织炎性反应及骨质破坏。预实验证实IL-1β能够通过上调牙周膜干细胞中GSK-3β磷酸化水平升高α7 nAChR表达。由此推测炎性环境下，IL-1β可能通过IL-1R-GSK-3β途径导致α7 nAChR表达升高。尼古丁与上调的α7 nAChR结合通过下游信号通路进一步加重炎性反应并造成局部骨组织更加严重的吸收。本课题拟从不同水平出发，通过体内外实验系统探讨炎性因子IL-1β通过上调牙周膜干细胞α7 nAChR表达来加剧吸烟相关性牙周炎发生发展的作用及相关机制，为阐明吸烟相关性牙周炎的病理进程提供实验依据，也为疾病的有效预防提供新思路。

慢性牙周炎是导致成人牙齿脱落的最常见原因，极大影响患者生活质量。吸烟是影响牙周炎发生发展的高危因素,阐明吸烟相关性牙周炎的发病机制，制定针对性防治方案可有效改善目标人群口腔健康水平，具有重要的临床及科学意义。前期相关研究多观察尼古丁通过α7亚型烟碱型乙酰胆碱受体（α7 nicotinic acetylcholine receptor，α7 nAChR）对细胞的影响。且大量临床证据表明，部分吸烟者的牙周炎病情虽重，即表现为更加严重的骨质破坏，但炎症程度同普通牙周炎患者相比并未加重甚至有所减轻。因此，除尼古丁影响牙周组织炎性反应外，牙周炎患者局部炎症微环境是否也能通过某种机制增强尼古丁协同作用下的骨质破坏作用？目前尚无相关研究报道。.本项目采用体外分离的方法培养并鉴定了正常及炎症组织来源PDLSC，观察了炎症微环境对不同来源人牙周膜组织及PDLSC α7 nAChR mRNA及蛋白表达的影响。之后采用实时定量PCR和western blot技术检测了炎症组织来源PDLSC同正常组织来源PDLSC在成骨分化能力和诱导破骨细胞形成能力上的差异。通过shRNA慢病毒颗粒转染的方法成功构建α7 nAChR表达沉默PDLSC细胞模型，进一步观察了在尼古丁参与与否的条件下，沉默α7 nAChR对PDLSC在炎症微环境中成/破骨平衡的影响，从而明确炎症微环境中表达上调的PDLSC α7 nAChR功能作用。最后，检测了不同微环境来源人牙周膜组织和PDLSC GSK-3β/磷酸化GSK-3β蛋白表达。并通过GSK-3β慢病毒激活颗粒转染的方法逆转炎症微环境对PDLSC GSK-3β磷酸化的影响，再次观察PDLSC中α7 nAChR表达，从而明确GSK-3β在炎症微环境调控α7 nAChR表达过程中的作用。通过上述实验明确尼古丁及炎症微环境在吸烟相关性牙周炎发生发展中的协同功能作用及可能机制，为阐明疾病病理进程提供实验依据，也为疾病的有效预防提供新思路。.本项目可得出以下结论：.1.炎症微环境可上调人牙周膜组织及细胞中α7 nAChR表达.2.炎症微环境下表达上调的α7 nAChR并未参与PDLSC成/破骨平衡改变.3.炎症微环境通过α7 nAChR与尼古丁发挥协同效应.4.炎症微环境通过增强GSK-3β磷酸化水平上调PDLSC α7 nAChR表达

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