细菌感染影响牙周组织细胞表达环鸟苷-腺苷合成酶的实验研究

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。     

牙龈卟啉单胞菌合成环二腺苷酸的高效液相色谱-串联质谱法定性分析

目的 定性检测牙龈卟啉单胞菌（P. gingivalis）是否能产生细菌信号分子环二腺苷酸（c-di-AMP），为探索其在P. gingivalis生命代谢以及牙周炎免疫中的作用奠定基础。方法 以P. gingivalis标准菌株ATCC33277为实验菌株，抽提细菌内核酸物质作为样品，配置c-di-AMP标准品，通过高效液相色谱-串联质谱法（HPLC-MS/MS）和高效液相色谱法（HPLC）对样品进行验证。结果 HPLC-MS/MS检出限按照信噪比（S/N）3∶1计算，c-di-AMP标准品出峰的保留时间为7.49 min，P. gingivalis提取物样品在保留时间为8.82 min时有目标峰出现（大于3 S/N）。HPLC检测结果表明，P. gingivalis核酸提取物样品及c-di-AMP标准品均在15.7 min处出现目标峰，且二者的紫外吸收光谱相同。结论 牙龈卟啉单胞菌核酸提取物中含有c-di-AMP，牙龈卟啉单胞菌可以合成产生c-di-AMP。     

牙龈卟啉单胞菌上调钙结合蛋白1促进牙龈上皮细胞增殖

探究钙结合蛋白1在牙龈卟啉单胞菌（P. gingivalis）影响牙龈上皮细胞增殖和凋亡中的作用。     

牙龈卟啉单胞菌与细胞自噬相关性的研究进展

细胞自噬是真核细胞的溶酶体依赖的代谢机制之一,参与宿主抵御病原菌感染的过程。牙龈卟啉单胞菌(P.gingivalis)能够侵入血管内皮细胞并利用自噬逃避宿主清除、持续并传播感染。本文就P.gingivalis与细胞自噬相关性研究的进展作一综述。     

牙龈卟啉单胞菌与细胞自噬相关性的研究进展

细胞自噬是真核细胞的溶酶体依赖的代谢机制之一，参与宿主抵御病原菌感染的过程。牙龈卟啉单胞菌（P.gingivdis）能够侵入血管内皮细胞并利用自噬逃避宿主清除、持续并传播感染。本文就P.gingivdis以括与细胞自噬相关性研究的进展作一综述。     

microRNA-146a对牙龈卟啉单胞菌脂多糖刺激下淋巴细胞分泌细胞因子的调节作用

探讨microRNA-146a（miR-146a）对牙龈卟啉单胞菌（P.gingivalis）脂多糖（LPS）刺激下淋巴细胞分泌细胞因子的作用。     

牙龈卟啉单胞菌来源的脂多糖通过X盒结合蛋白1调控脂肪细胞胰岛素信号通路的机制研究

探讨内质网应激（ERS）关键信号分子X盒结合蛋白1（XBP1）在牙周炎致病菌牙龈卟啉单胞菌（P. gingivalis）来源的脂多糖（LPS）刺激下对脂肪细胞胰岛素信号通路的影响。     

二十碳五烯酸对人牙龈成纤维细胞生物学活性及炎症因子表达的影响

目的: 探讨二十碳五烯酸（eicosapentaenoic acid,EPA）对人牙龈成纤维细胞（human gingival fibroblasts,HGFs）生物学活性及炎症因子表达的影响。方法: 分别通过活-死细胞染色、免疫荧光染色、流式细胞术观察EPA对HGFs细胞活性、形态、细胞周期的影响,采用牙龈卟啉单胞菌（Porphyromonas gingivalis,P. gingivalis）脂多糖（lipopolysaccharides,LPS）或热灭活P. gingivalis刺激HGFs,分别通过实时定量PCR 和ELISA观察EPA对白细胞介素6（interleukin-6,IL-6）、IL-8、IL-1β基因和蛋白表达的作用;进一步采用实时定量PCR 和Western免疫印迹法,观察EPA对血红素氧合酶1（heme oxygenase-1,HO-1）基因和蛋白表达的作用。采用SPSS 22.0软件包对数据进行统计学分析。结果: 200 μmol/L EPA可抑制HGFs的细胞活性;100 μmol/L EPA不影响HGFs的细胞活性、形态,对细胞周期也无显著影响（P>0.05）。EPA浓度依赖性抑制P. gingivalis LPS及热灭活P. gingivalis诱导HGFs表达的IL-6 mRNA、蛋白以及IL-1β mRNA（P<0.05）;EPA可呈浓度依赖性诱导HGFs表达HO-1 mRNA（P<0.05）,并上调其蛋白表达。结论: EPA在不影响HGFs生物学活性的前提下,显著抑制细胞的炎症因子表达,可能与其对HO-1的诱导作用有关,提示EPA在牙周炎防治中具有潜在应用价值。     

外源性ATP对牙龈成纤维细胞NLRP3炎症小体活化的影响

目的: 观察外源性三磷酸腺苷（ATP）对牙龈卟啉单胞菌(P.gingivalis)和热灭活牙龈卟啉单胞菌(HP.gingivalis)感染的人牙龈成纤维细胞NLRP3 炎症小体（inflammasome）的活化以及下游因子IL-1β分泌的影响。方法: 组织块法体外培养人牙龈成纤维细胞（hGFs）获取原代细胞,经5 mmol/L ATP预处理,用100 MOI P.gingivalis、100 MOI HP.gingivalis体外刺激hGFs,实时定量PCR检测NLRP3、ASC、Caspase-1、IL-1β的基因表达;Western免疫印迹技术检测细胞内NLRP3、Caspase-1和IL-1β蛋白的表达;ELISA法检测白细胞介素1β（IL-1β）的分泌。采用Graphpad prism 6软件包对数据进行t检验或单因素方差分析。结果: 与对照组相比,P.gingivalis 下调NLRP3 、ASC mRNA,上调IL-1β基因表达,下调NLRP3 、IL-1β胞内蛋白水平。HP.gingivalis 诱导NLRP3 、IL-1β、ASC基因和胞内蛋白的表达,P.gingivalis或者 HP.gingivalis单独刺激对Caspase-1 mRNA水平及IL-1β分泌均无影响。ATP/P.gingivalis或ATP/HP.gingivalis 共刺激均明显上调NLRP3、ASC、Caspase-1和IL-1β基因及胞内蛋白水平,增加上清液中IL-1β的分泌水平。结论: 外源性ATP可调控牙周主要致病菌P.gingivalis感染的人牙龈成纤维细胞 NLRP3炎症小体的激活,介导炎症因子IL-1β的成熟与分泌。     

不同龈下楔状缺损充填材料对牙龈卟啉单胞菌生物膜形成的影响

目的：探讨常用龈下楔状缺损充填材料对牙龈卟啉单胞菌（P.gingivalis）生物膜形成的影响。方法：在48颗离体前磨牙颊侧牙颈部制备同一规格V类洞,将其随机分为A、B、C 3组,每组16颗,分别采用通用纳米固体树脂、通用纳米流体树脂、玻璃离子充填、抛光后,使用Isomet 4000 精密切割机制成包含全部充填材料的试件（近远中径5 mm、龈径3 mm、颊舌向深度 2 mm）。将上述3组试件置于P.gingivalis菌液中培养24 h,采用结晶紫染色法观察P.gingivalis的黏附量,使用激光共聚焦显微镜(confocal laser scanning microscopy, CLSM）观察P.gingivalis生物膜的形成情况。采用SPSS 22.0 软件包对所得数据进行Kruskal-Wallis 秩和检验。结果：结晶紫染色实验显示,玻璃离子表面黏附P.gingivalis的量显著少于通用纳米固体树脂与通用纳米流体树脂（P<0.05）。CLSM扫描可见,3组间P.gingivalis生物膜内的活菌比例及生物膜厚度无显著差异（P>0.05）;玻璃离子表面P.gingivalis生物膜稀疏,呈小团块状,未聚集成片;通用纳米固体树脂组的P.gingivalis生物膜则呈大块片状,立体结构明显。结论：与通用纳米树脂材料相比,玻璃离子表面更不利于P.gingivalis生物膜的形成。对牙周健康而言,玻璃离子更适用于龈下楔状缺损的充填治疗。     

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??Porphyromonas gingivalis?? a gram-negative obligate anaerobic bacterium??has been proved to be one of the main periodontal pathogens?? participating in development of periodontitis and process of periodontal tissue destruction. Previous studies have demonstrated the close relationship between periodontal infection and systemic health and diseases. P. gingivalis infection on host cells mediated cell apoptosis by interfering with cell signal transduction and expression of related proteins. This article reviews research progress of P. gingivalis infection on mediation of apoptosis of host cells.     

牙龈蛋白酶及其致病作用

牙龈卟啉单胞菌是引起和加重牙周炎的重要致病菌,其分泌的牙龈蛋白酶是其主要的毒力因子之一。本文就牙龈卟啉单胞菌牙龈蛋白酶以及牙龈蛋白酶对细菌生长和黏附的影响、对组织的破坏作用、对宿主防御机制的作用等研究进展作一综述。     

Tumor necrosis factor modulates fibroblast apoptosis, PMN recruitment, and osteoclast formation in response to P. gingivalis infection.

P. gingivalis is an important oral pathogen, which has been closely linked to periodontal disease as well as lesions of endodontic origin. Both infections are associated with a decrease in fibroblast numbers, formation of an inflammatory infiltrate, and bone resorption. The goal of this study was to investigate the role that the host response plays in the capacity of P. gingivalis to stimulate fibroblast apoptosis, PMN recruitment, and osteoclastogenesis. This was accomplished by the use of an in vivo calvarial model in mice with targeted deletion of TNF receptors p55 and p75 and matched wild-type mice. The results indicate that P. gingivalis induces fibroblast apoptosis in vivo and establish for the first time that this involves the stimulation of a host response. Moreover, bacteria-stimulated PMN recruitment and osteoclastogenesis were also dependent upon the host response. The results suggest that much of the damage caused by P. gingivalis infection, including fibroblast apoptosis, at least under some circumstances, results from stimulation of the host response rather than the direct effect of bacterial products. Furthermore, this may represent a more general mechanism by which bacterial challenge induces apoptosis of matrix-producing cells through the induction of TNF.     

Porphyromonas gingivalis lipopolysaccharide: an unusual pattern recognition receptor ligand for the innate host defense system

Lipopolysaccharide (LPS) is a key inflammatory mediator. Due to its ability to potently activate host inflammatory and innate defense responses, it has been proposed to function as an important molecule that alerts the host of potential bacterial infection. However, although highly conserved, LPS contains important structural differences among different bacterial species that can significantly alter host responses. For example, LPS obtained from Porphyromonas gingivalis, an etiologic agent for periodontitis, causes a highly unusual host innate host response. It is an agonist for human monocytes and an antagonist for human endothelial cells. Correspondingly, although it activates p38 MAP kinase in human monocytes, P. gingivalis LPS does not activate p38 nor ERK MAP kinase in endothelial cells. In fact, P. gingivalis LPS is an effective inhibitor of Escherichia coli LPS induced p38 phosphorylation. These data show that P. gingivalis LPS modulates host defenses in endothelial cells by interfering with MAP kinase activation. In addition, P. gingivalis LPS is unusual in that it engages TLR-2 but not TLR-4 when examined in stably transfected CHO cell lines. We propose that, since LPS is a key ligand for the human innate host defense system, these unusual properties of P. gingivalis LPS are associated with the bacterium's role in the pathogenesis of periodontitis.     

Invasion of Porphyromonas gingivalis into human gingival fibroblasts in vitro

Porphyromonas gingivalis, one of the important periodontal pathogens, exhibits many virulence properties. Among these, the adhesion to and invasion into host tissues are crucial for the initiation and progression of periodontal diseases. While evidence indicating the ability of this organism to adhere to and invade into epithelial cells as well as endothelial cells has accumulated, that involving the gingival fibroblasts is very limited. Therefore, this study aimed to determine the ability of P. gingivalis to invade primary cultures of human gingival fibroblasts using the antibiotic protection assay. In addition, interactions between P. gingivalis and the gingival fibroblasts were investigated using electron microscopy. The results demonstrated that P. gingivalis 381 could invade human gingival fibroblasts with an invasion efficiency of 0.17%. Using the scanning electron microscopic study, numerous filopodia were seen on the surfaces of gingival fibroblasts after P. gingivalis adhesion. The transmission electron microscopy revealed the presence of an intracellular bacterium. After 90 min incubation, the bacterium was found in the cytoplasm of the gingival fibroblasts, without membrane surrounding. Some fibroblasts contained a number of vacuoles and dilated rough endoplasmic reticulum even when bacteria were not found intracellularly. Thus, the invasion of this organism into the gingival fibroblasts may play a direct role in the destruction of the periodontal tissues and may also relate to the difficulties of eradicating the bacteria from periodontitis lesions.     

Porphyromonas gingivalis-epithelial cell interactions in periodontitis

Emerging data on the consequences of the interactions between invasive oral bacteria and host cells have provided new insights into the pathogenesis of periodontal disease. Indeed, modulation of the mucosal epithelial barrier by pathogenic bacteria appears to be a critical step in the initiation and progression of periodontal disease. Periodontopathogens such as Porphyromonas gingivalis have developed different strategies to perturb the structural and functional integrity of the gingival epithelium. P. gingivalis adheres to, invades, and replicates within human epithelial cells. Adhesion of P. gingivalis to host cells is multimodal and involves the interaction of bacterial cell-surface adhesins with receptors expressed on the surfaces of epithelial cells. Internalization of P. gingivalis within host cells is rapid and requires both bacterial contact-dependent components and host-induced signaling pathways. P. gingivalis also subverts host responses to bacterial challenges by inactivating immune cells and molecules and by activating host processes leading to tissue destruction. The adaptive ability of these pathogens that allows them to survive within host cells and degrade periodontal tissue constituents may contribute to the initiation and progression of periodontitis. In this paper, we review current knowledge on the molecular cross-talk between P. gingivalis and gingival epithelial cells in the development of periodontitis.     

Effects of dipeptide bestatin on Porphyromonas gingivalis and epithelial cells.

BACKGROUND: Dipeptide bestatin has been previously reported to selectively inhibit the growth of Porphyromonas gingivalis. The aims of this study were to investigate the mechanism of action of bestatin and to evaluate its effect on epithelial cells. METHODS: The inhibitory effect of bestatin on P. gingivalis was tested in vitro (culture medium) and in vivo (guinea pig model). Radiolabeled compounds were used to investigate the effect of bestatin on the uptake of amino acids and peptides. The cytotoxic effect of bestatin was evaluated using a keratinocyte cell line. RESULTS: The growth inhibition of P. gingivalis by bestatin was concentration-dependent. Even at high concentrations, compounds possessing a chemical structure or an aminopeptidase inhibitor activity related to bestatin had no effect on growth of P. gingivalis. When injected in the presence of P. gingivalis, bestatin was able to prevent the development of a necrotic abscess in a guinea pig model. Data were obtained suggesting that bestatin does not act on proteinases of P. gingivalis. Rather, bestatin was found to inhibit the intracellular uptake of radioactivity from 14C-labeled amino acids or heat-denatured type I collagen. This was not observed with a spontaneous mutant of P. gingivalis, whose growth was not affected by bestatin. In the second part of the study, bestatin was found to have no effect on epithelial cell viability in culture at concentrations effective on P. gingivalis. In addition, bestatin did not show effects on epithelial cell migration or production of gelatinases. CONCLUSIONS: This study suggests that bestatin selectively inhibits growth of P. gingivalis by affecting the intracellular uptake of amino acids and peptides, which serve as energy and nitrogen sources for this bacterial species. Bestatin has no cytotoxicity and may represent a therapeutic molecule for local treatment of P. gingivalis-associated periodontitis.     

Porphyromonas gingivalis survives within KB cells and modulates inflammatory response

BACKGROUND/AIMS: The purpose of the study was to investigate the intracellular survival of Porphyromonas gingivalis as a possible mechanism for maintaining periodontitis. METHODS: P. gingivalis strains, the strain ATCC 33277 and seven clinical isolates, were co-cultured with KB cells. The number of intracellular bacteria was determined up to 3 days after infection. In addition, the numbers of KB cells per well, the concentrations of the cytokines interleukin-1beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor-alpha (TNF-alpha) and the arginine-specific amidolytic activity were measured. The 16S rRNA of P. gingivalis and the mRNA expression of IL-1beta, IL-6, IL-8, TNF-alpha and rgpA were also determined. RESULTS: All the P. gingivalis strains studied were able to survive within KB cells. In contrast to the reduced values of colony-forming units at day 3, equal and higher levels of 16S rRNA were seen in comparison to day 0. Arginine-specific amidolytic activity declined in all samples during infection. Expression of mRNA for rgpA was not found after infection of KB cells by P. gingivalis strains. IL-8 was detectable in all samples 2 days after infection with P. gingivalis strains. Principal components analysis underlined a correlation between the arginine-specific amidolytic activity 1 h after infection and both the released IL-8 and the mRNA expression of IL-8. Associations were found between the cultivable numbers of intracellular P. gingivalis and the mRNAs of IL-1, IL-6 and TNF-alpha at the day of infection. CONCLUSION: The results indicate survival of P. gingivalis within epithelial cells, possibly in a non-cultivable stage. Invasion into cells modulates the virulence properties of P. gingivalis as well as the inflammatory response of the cells.