Department of Operative Dentistry and Endodontics, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, 56 Lingyuan Xi Road, Guangzhou 510055, Guangdong, China; Guangdong Province Key Laboratory of Stomatology, No. 74, 2nd Zhongshan Road, Guangzhou 510080, Guangdong, China.
Discipline of Endodontology, Faculty of Dentistry, The University of Hong Kong, Pok Fu Lam, Hong Kong SAR, China.
Exp Cell Res. 2018 Jan 1;362(1):152-158. doi: 10.1016/j.yexcr.2017.11.012. Epub 2017 Nov 10.
Persistent apical periodontitis (PAP) is characterized by refractory inflammation and progressive bone destruction. Enterococcus faecalis infection is considered an important etiological factor for the development of PAP, although the exact mechanisms remain unknown. This study aimed at investigating the role of E. faecalis in cell proliferation, inflammatory reactions and osteoclast differentiation of macrophages using an in vitro infection model of osteoclast precursor RAW264.7 cells. A cell viability assay of cultured RAW264.7 cells exposed to live E. faecalis at a multiplicity of infection of 100 for 2h, indicated that the infection exhibited no cytotoxic effect. Transmission electron microscopy images revealed no apoptotic changes but a rise of metabolic activity and phagocytic features in the infected RAW264.7 cells. Confocal laser scanning microscopic and flow cytometric analysis indicated that the phagocytosis of RAW264.7 cells was activated by E. faecalis infection. Furthermore, quantitative real-time PCR assays demonstrated that the expression of inflammatory cytokines was remarkably elevated in infected RAW264.7 cells. Differentiation of infected RAW264.7 cells into osteoclasts was remarkably attenuated, and expression of osteoclast marker genes as well as fusogenic genes significantly dropped. In summary, E. faecalis appears to attenuate osteoclastic differentiation of RAW264.7 precursor cells, rather stimulates them to function as macrophages.
持续性根尖周炎（PAP）的特征为难治性炎症和进行性骨破坏。粪肠球菌感染被认为是 PAP 发展的一个重要病因，但确切机制尚不清楚。本研究旨在通过体外感染破骨细胞前体细胞 RAW264.7 的模型，研究粪肠球菌在巨噬细胞增殖、炎症反应和破骨细胞分化中的作用。对培养的 RAW264.7 细胞进行细胞活力测定，用感染复数为 100 的活粪肠球菌感染 2 小时，结果表明感染没有细胞毒性作用。透射电子显微镜图像显示没有凋亡变化，但感染的 RAW264.7 细胞的代谢活性和吞噬特征增加。共聚焦激光扫描显微镜和流式细胞术分析表明，粪肠球菌感染激活了 RAW264.7 细胞的吞噬作用。此外，实时定量 PCR 检测表明，感染 RAW264.7 细胞的炎症细胞因子表达显著升高。感染 RAW264.7 细胞分化为破骨细胞明显减弱，破骨细胞标记基因和融合基因的表达显著下降。总之，粪肠球菌似乎减弱了 RAW264.7 前体细胞的破骨细胞分化，而是刺激它们作为巨噬细胞发挥作用。
