牙龈卟啉单胞菌协同具核梭杆菌诱导牙龈上皮细胞间充质转化的机制研究

Periodontal infection is the consequence of synergistic effect of various bacteria colonized in oral cavity. Porphyromonas gingivalis (Pg) and Fusobacterium nucleatum (Fn) are important pathogens of chronic periodontitis. Pg infection could accelerate proliferation of gingival epithelial cells by activating PI3K/AKT pathway. According to our previous studies, long-term infection of Pg could increase malignant degree of oral epithelial cells. The expression of proMMP-2/9 was increased. Meanwhile, the synergistic infection of Pg and Fn in KB cells could increase the expression of IL-6/8 and decrease the expression of E-cadherin with the weakening of cell junction, which indicated that epithelial cells gained the potency of mesenchymal transition. From the results of gene microarray, the expression of long non-coding RNA CCAT1 was increased. We speculate that the synergistic infection of Pg and Fn may promote cell mesenchymal-like transition in gingival epithelial cells firstly by binding EGFR and activating CCAT1 with the targeted inhibition of miR-218-5p expression. Accordingly, AKT and NF-κB pathway would be activated in order to increase the expression of SNAIL1 and ZEB2. We aim to look for effective blocking ways for mesenchymal transition and distal migration of gingival epithelial cells, and provide new theoretical basis on transformation from oral chronic inflammation to malignant neoplasm.

牙周感染是口腔多种细菌协同作用结果，牙龈卟啉单胞菌（P.gingivalis，Pg）和具核梭杆菌（F.nucleatum，Fn）是慢性牙周炎重要致病菌。Pg感染牙龈上皮细胞激活PI3K/AKT通路，促进细胞增殖。前期研究发现，Pg持续感染口腔上皮细胞，细胞恶性度增强，proMMP-2/9增高，而Pg和Fn协同感染KB细胞可引起IL-6/8升高，E-cadherin降低，细胞间连接减弱，提示感染的上皮细胞具有向间充质细胞转化的潜能。基因芯片筛查发现长链非编码RNAs－CCAT1表达上调。本研究推测Pg和Fn协同感染牙龈上皮细胞后，通过EGFR启动CCAT1，抑制miR-218-5p表达，激活AKT和NF-κB通路，促进SNAIL1表达，并直接上调ZEB2表达，促进牙龈上皮细胞间充质样转化。本研究将为牙龈上皮细胞间充质转化及远隔转移寻找有效的阻断途径，并为口腔慢性炎症向恶性肿瘤转化提供新的依据

牙龈卟啉单胞菌（Porphyromonas gingivalis，P.gingivalis）与具核梭杆菌（Fusobacterium nucleatum，F.nucleatum）是牙周炎的重要致病菌，口腔内主要以混合形成生物膜存在。本课题组研究显示，P.gingivalis长期小剂量感染可促进口腔上皮细胞恶性进展，通过miR-21/PDCD4/AP-1负反馈环路提高cyclinD1表达促进口腔鳞癌细胞增殖。研究发现口腔鳞癌组织中F.nucleatum富集，而F.nucleatum感染与口腔肿瘤发生发展是否相关尚不明确。为阐明P.gingivalis与F.nucleatum之间的相互作用，将P.gingivalis与F.nucleatum混合感染HIOECs后，发现F.nucleatum侵入减少，且两者共同感染时上皮间充质转化相关转录因子SNAI1表达升高，但其他标志分子表达则在F.nucleatum单独感染HIOECs时变化更为显著，提示F.nucleatum促进EMT不依赖于其侵入能力。因此我们重点探讨F.nucleatum促进口腔上皮细胞发生EMT的机制。构建F.nucleatum感染HIOECs与口腔鳞癌细胞SCC-9模型，发现EMT标志物E-cadherin表达减少，而N-cadherin、Vimentin、SNAI1表达增强，且热灭活F.nucleatum、纯化的FadA蛋白可发挥与活菌相似的作用，提示F.nucleatum感染促进口腔上皮细胞EMT，其中毒力因子FadA可能具有重要作用。通过机制研究阐明F.nucleatum感染通过lncRNA MIR4435-2HG/miR-296-5p/Akt2/SNAI1信号通路促进口腔上皮细胞发生EMT，F.nucleatum通过Ku70/p53途径造成DNA损伤促进口腔鳞癌细胞增殖。通过临床研究表明唾液富组蛋白5能够有效抑制P.gingivalis与F.nucleatum共聚和生物膜形成，减少其在龈下定植，为牙周病防治提供新的理论支持。

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