Objective: This work aims to determine the effect of cytosolic bacteria on the expression of cyclic GMP-AMP synthase (cGAS) in human periodontal ligament cells (hPDLCs) and gingival tissues.
Methods: The ability of Porphyromonas gingivalis (P. gingivalis) to invade hPDLCs was detected using laser scanning confocal microscope assay at a multiplicity of infection of 10. P. gingivalis-infected cells were sorted by fluorescence-activated cell sorting (FACS). Then, quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) and Western blot were used to detect cGAS expression in infected cells. Finally, the location and expression of cGAS in inflammatory and normal gingival tissues were investigated by immunohistochemistry.
Results: P. gingivalis actively invaded hPDLCs. Moreover, cGAS expression significantly increased in P. gingivalis-infected cells. Although cGAS was expressed in the epithelial and subepithelial cells of both inflamed and normal gingival tissues, cGAS expression significantly increased in inflamed gingival tissues.
Conclusions: Cytosolic bacteria can upregulate cGAS expression in infected cells. These data suggest that cGAS may act as pattern-recognition receptors and participate in recognizing cytosolic nucleic acid pathogen-associated molecular patterns. .
目的 研究侵入牙周组织细胞内的细菌对细胞表达环鸟苷-腺苷合成酶（cGAS）的影响。方法 将SYTO9标记的牙龈卟啉单胞菌（P. gingivalis）以感染复数（MOI）为10与人牙周膜细胞（hPDLCs）共培养24 h后，使用激光共聚焦显微镜观察P. gingivalis对hPDLCs的侵袭情况，并应用荧光标记活细胞筛选（FACS）分选出被P. gingivalis入侵的hPDLCs，利用实时定量逆转录聚合酶链反应（qRT-PCR）和Western blot对hPDLCs中cGAS的表达变化进行检测。此外还利用免疫组织化学技术对细菌感染性牙龈组织和正常牙龈组织中cGAS表达定位和表达水平变化进行分析。结果 激光共聚焦显微镜下观察，P. gingivalis和hPDLCs共培养24 h后，几乎所有的细胞内都可观察到绿色荧光；被P. gingivalis感染的hPDLCs中cGAS表达水平明显上调；在牙龈组织中cGAS主要在上皮细胞和上皮下组织中的炎性细胞中表达；且细菌感染性的牙龈组织中cGAS的表达水平明显高于正常牙龈组织。结论 侵入hPDLCs内的活体P. gingivalis，可以明显上调细胞中cGAS的表达；且在细菌感染的炎性牙龈组织中cGAS的表达也明显升高。该结果提示cGAS可能参与了牙周组织细胞中细菌来源的病原dsDNA的识别。.
Keywords: DNA sensor; Porphyromonas gingivalis; cyclic GMP-AMP synthase; human periodontal ligament cells.