具核梭杆菌调控牙龈卟啉单胞菌感染对口腔鳞状细胞癌KB细胞周期和细胞因子分泌的研究

目的: 通过研究牙龈卟啉单胞菌(Porphyromonasgingivalis, P. gingivalis)和具核梭杆菌(Fusobacteriumnucleatum, F. nucleatum)感染对KB细胞的细胞周期和炎症因子分泌的调控,初步探讨牙周细菌感染对口腔鳞状细胞癌进展和炎症反应的影响。方法: 建立P. gingivalis和F. nucleatum单独或联合感染KB细胞模型,透射电镜观察细菌感染细胞情况,流式细胞术检测细胞周期变化,酶联免疫法检测IL-6和IL-8蛋白分泌水平。结果: P. gingivalis联合F. nucleatum感染KB细胞8 h抑制细胞周期进程,感染24 h后加速细胞周期进程;P. gingivalis联合F. nucleatum感染促进KB细胞分泌IL-6和IL-8,并且有时间和F. nucleatum浓度依赖性。结论: 牙周致病菌之间的联合作用更能促进口腔鳞状细胞癌的发展及免疫炎症反应。     

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。     

牙龈卟啉单胞菌唾液酸酶基因缺失影响其感染上皮细胞后炎症相关microRNA的表达

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P. gingivalis)唾液酸酶基因(PG0352)缺失是否影响其感染上皮细胞后对上皮细胞炎症相关小RNA的调控。方法:厌氧培养P. gingivalis W83及其唾液酸酶基因突变株(△PG0352),以MOI为100∶1的比例分别与永生化牙龈上皮细胞epi4进行共培养,PCR array的方法检测88个与炎症相关的小RNA的表达。结果:△PG0352组epi4的hsa-miR-9、hsa-miR-23a、hsa-miR-23b和hsa-miR-410的表达分别是P. gingivalis W83组的4.3387、3.3978、4.7930和4.3137倍。结论:与P. gingivalis W83野生株相比,唾液酸酶基因缺陷株对牙龈上皮细胞炎症相关小RNA的表达有不同的调控作用。     

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。     

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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.     

牙龈卟啉单胞菌感染宿主细胞后对其凋亡调控研究进展

牙龈卟啉单胞菌（Porphyromonas gingivalis）是证据充分的重要的牙周致病菌之一,参与牙周炎的发生、发展及牙周组织的破坏过程。以往研究表明,牙周感染与全身健康及系统疾病均有着密切关系。P. gingivalis感染多种宿主细胞后,通过干扰相关信号传导及蛋白表达,对宿主细胞凋亡进行调控。现就P. gingivalis感染宿主细胞后对其凋亡调控研究进展做一综述。     

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。     

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.     

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.     

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.