槲皮素对牙龈卟啉单胞菌脂多糖刺激的人牙龈成纤维细胞生物学行为的影响研究

目的研究槲皮素对牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharide,P.g LPS)刺激的人牙龈成纤维细胞(human gingival fibroblasts,HGFs)生物学行为的影响。方法采用DNA片段凝胶电泳、CCK-8法、细胞划痕法观察不同浓度槲皮素(10、20、50、100μmol/L)对体外培养HGFs的毒性作用,以及对HGFs增殖与迁移的影响。采用P.g LPS刺激HGFs来建立体外炎症刺激模型,通过流式细胞术、细胞活性氧(reactive oxygen species,ROS)检测、酶联免疫吸附试验(ELISA)和实时荧光定量PCR(qRT-PCR)进一步观察槲皮素对HGFs凋亡、ROS的产生及肿瘤坏死因子-α(tumor necrosis factorα,TNF-α)和前列腺素E2(prostaglandin E2,PGE2)表达的影响。结果槲皮素对HGFs无毒性作用,且对HGFs的增殖无影响(P>0.05)。各组细胞迁移率总的比较,差异有统计学意义(F=9.973,P<0.05),在处理48 h后,20μmol/L槲皮素处理组细胞迁移率大于10、50μmol/L槲皮素处理组以及对照组,差异均有统计学意义(均P<0.05)。槲皮素对P.g LPS刺激的HGFs凋亡具有抑制作用,且其能够抑制并预防P.g LPS刺激的HGFs中ROS相对产生量增加现象(均P<0.05)。槲皮素处理显著抑制了P.g LPS诱导的TNF-α表达增加现象(P<0.05),而槲皮素处理组PGE2的表达水平与对照组比较,差异无统计学意义(P>0.05)。结论浓度为20μmol/L的槲皮素能够促进体外培养HGFs的迁移,且具有抗氧化、抗炎保护作用。     

牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的研究

目的研究牙龈卟啉单胞菌活菌细胞内感染牙髓成纤维细胞的体外模型。方法将牙龈卟啉单胞菌和牙髓细胞以100∶1比例共同孵育0.5、1.0、2.0 h,倒置显微镜观察牙髓细胞形态。加入双抗和甲硝唑杀死胞外细菌,牙髓细胞用蒸馏水裂解后厌氧培养细胞裂解液,观察活细菌是否进入牙髓细胞胞内。将牙龈卟啉单胞菌和牙髓细胞以多重感染比（MOI）100、50共同孵育1.0 h,采用MTT法检测感染牙龈卟啉单胞菌后牙髓细胞存活率。结果倒置显微镜下显示,被胞内感染的牙髓细胞培养2.0 h未见胞膜破裂,形态完整。采用双抗能彻底杀灭胞外培养基中的细菌而对胞内细菌无影响,共同孵育1.0 h和2.0 h活细菌能进入牙髓细胞。MTT法显示细菌感染后牙髓细胞仍有一定存活率,其存活率分别是MOI 100组74.43%、MOI 50组99.07%。结论成功建立了牙龈卟啉单胞菌对牙髓成纤维细胞胞内感染的模型。     

五倍子水提取物对牙龈卟啉菌膜泡抑制牙周膜成纤维细胞生物活性的影响

目的:探讨五倍子水提取物对牙龈卟啉菌(Porphyromonas gingivalisPg)膜泡(extracellular vesicles ECV)抑制牙周膜成纤维细胞生物活性的影响。方法:采用体外培养的人牙周膜成纤维细胞,用3H-TdR掺入方法,检测ECV对人牙周膜成纤维细胞DNA合成的影响,以及中药五倍子提取物对ECV这一生物活性的阻断作用。结果:细胞培养物中加入50μg/mLECV时,人牙周膜成纤维细胞DNA的合成受到明显抑制,与空白对照组相比P<0.01。当同时加入各浓度五倍子水提取物时其DNA合成量明显增加,与ECV组相比均(P<0.05),且随五倍子水提取物浓度升高,DNA的合成量也随之增高,并呈一定浓度依赖性。结论:适宜浓度的五倍子提取物可有效地阻断ECV的这种生物活性作用,从而可推测其对牙周病的发生、发展能起到一定的阻断作用。     

牙龈卟啉单胞菌超声提取物对人牙周膜细胞活性的影响

目的探讨牙龈卟啉单胞菌超声提取物对人牙周膜细胞活性的影响。方法组织块法体外培养人牙周膜细胞（hPDLC）,以6．25、12．5、25、50mg·L^-1质量浓度的超声提取物作用于hPDLC24h,阴性对照组为无超声提取物作用的hPDLC;用12．5mg·L^-1质量浓度的超声提取物分别作用于hPDLC24、48、72、96h,阴性对照组亦培养24、48、72、96h。以甲噻唑四唑氮比色比较试验组与对照组hPDLC的活性变化,用SPSS13．0软件包对数据行单因素方差分析和独立样本t检验。结果当作用于hPDLC的超声提取物质量浓度为6．25～50mg·L^-1时,经过24h,试验组hPDLC的活性低于阴性对照组;当超声提取物质量浓度为12．5mg·L^-1时,在24-96h内,试验组hPDLC的活性低于阴性对照组。结论牙龈卟啉单胞菌可导致体外培养的hPDLC的活性降低,且作用呈质量浓度和时间依赖性。     

microRNA-223在牙龈卟啉单胞菌诱导牙龈成纤维细胞炎症调控的研究

目的 探讨microRNA-223在牙龈卟啉单胞菌脂多糖(Porphyromonas gingivalis lipopolysaccharides, P.g-LPS)诱导牙龈成纤维细胞(gingival fibroblasts,GFs)炎症过程中对相关炎症因子表达水平的调控作用。 方法 采用慢病毒转染、干扰GFs中的microRNA-223的表达,在最适P.g-LPS刺激浓度(800 μg/L)分别刺激过表达、抑制以及正常表达microRNA-223的GFs,采用实时聚合酶联反应（Real-time quantitative polymerase chain reaction,qPCR）检测TNF-α、IL-1β、IL-6的mRNA表达水平变化,酶联免疫吸附试验(enzyme linked immunosorbent assay, ELISA)检测其蛋白水平的变化。 结果 LPS刺激GFs产生炎症反应时,细胞内microRNA-223以及相关促炎因子TNF-α、IL-1β、IL-6的mRNA和蛋白表达水平较正常细胞中的表达量明显上调。当细胞内microRNA-223上调时,促炎因子的mRNA和蛋白表达水平也会上调(P<0.001);当细胞内microRNA-223下调时,促炎因子TNF-α、IL-1β的蛋白水平会显著下降(P<0.001)。 结论 当GFs受P.gingivalis-LPS刺激发生炎症时,microRNA-223的表达量增多,上调促炎因子TNF-α、IL-1β、IL-6,进一步加重组织细胞的炎症。     

不同fimA基因型牙龈卟啉单胞菌对牙龈成纤维细胞表达白细胞介素-8的影响

目的:研究不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis, Pg)对人牙龈成纤维细胞表达IL- 8的影响,探讨fimA基因型与Pg致病力差异之间的关系.方法: Pg ATCC 33277(Ⅰ型),WCSP115(Ⅱ型),WCSP1.5(Ⅲ型),W83(Ⅳ型)分别与牙龈成纤维细胞在标准条件下共同孵育,于孵育后1、3、6、12 h收集细胞和上清,应用实时荧光定量RT- PCR和ELISA法分别检测牙龈成纤维细胞IL- 8 mRNA和蛋白的动态表达.结果:与对照组比较,各fimA型Pg对牙龈成纤维细胞IL- 8 mRNA和蛋白的表达均有明显的促进作用(P<0.01);其中ⅡfimA型组IL- 8 mRNA和蛋白的表达量明显高于其它fimA型组,不同时间点IL- 8 mRNA相对表达量分别为20.42±2.21～103.01±12.50,蛋白分泌水平为(137.46±4.97～323.24±13.74) pg/ml;而Ⅲ fimA型Pg组的表达水平弱于其它fimA型组,IL- 8 mRNA相对表达量为3.61±0.39～12.88±1.56,蛋白分泌水平为(44.83±3.68～189.19±87.34) pg/ml, 差异有统计学意义(P<0.05).结论:Pg可促进牙龈成纤维细胞IL- 8 mRNA和蛋白的表达,且各fimA型Pg的促进作用有所差异.提示: fimA基因型的不同可能是Pg的致病力差异的基因基础.     

人牙龈成纤维细胞和牙周膜成纤维细胞的培养和生物学特征

目的：探讨采用不同方法建立体外培养人牙龈成纤维细胞和人牙周膜成纤维细胞并对其生物学特性作初步探讨。方法：分别采用消化培养法和组织块培养人的牙龈和牙周膜成纤维细胞。通过倒置显微镜活体观察，以及光镜、透射电镜、免疫组化和生长曲线等方法对其生物学特性作初步观察。结果：消化培养法成功率低于组织块培养法。光镜下和透射电镜下观察两种细胞在形态和结构上相似。免疫组化染色证实此两种细胞均来源于中胚层的纤维母细胞。生长曲线表明牙龈成纤维细胞的倍增时间比牙周膜成纤维细胞稍短，而牙周膜成纤维细胞的增殖活性比牙龈成纤维细胞高。结论：组织块培养法适用于此二种细胞的培养。细胞生物学特征方面有许多相似形，提示这两种细胞来于同一个细胞群。     

牙龈卟啉单胞菌脂多糖引起牙周炎症的机制和特点

革兰阴性厌氧菌牙龈卟啉单胞菌和牙周炎密切相关，脂多糖是革兰阴性菌外膜中的主要结构成分，具有广泛的生物学活性，被认为是革兰阴性菌的主要毒力因子之一。本文就牙龈卟啉单胞菌脂多糖的化学组成和结构，所启动炎症信号通路及其起始受体，引起牙周炎症的机制和特点作一综述。     

TLR4在人牙周膜成纤维细胞中的表达研究

目的：探讨TLR4在体外培养的人牙周膜成纤维细胞中的表达情况。方法：体外分离培养人牙周膜成纤维细胞,采用免疫荧光染色检测TLR4蛋白在该细胞中的表达;用半定量RT—PCR法检测TLR4基因的表达及0．1ng／mL牙龈卟啉菌脂多糖刺激24h后细胞内TLR4基因表达水平的改变。结果：免疫荧光检测显示体外培养的人牙周膜细胞中有TLR4表达,0．1ng／mL牙龈卟啉菌脂多糖刺激24h,其TLRd基因表达水平显著性增高（P〈0．05）。结论：TLR4在牙周膜成纤维细胞中有表达,牙龈卟啉菌脂多糖成分可刺激TLR4基因上调,提示与该细胞参与牙周免疫应答有关。     

牙龈卟啉单胞菌脂多糖引起牙周炎症的机制和特点

革兰阴性厌氧菌牙龈卟啉单胞菌和牙周炎密切相关,脂多糖是革兰阴性菌外膜中的主要结构成分,具有广泛的生物学活性,被认为是革兰阴性菌的主要毒力因子之一。本文就牙龈卟啉单胞菌脂多糖的化学组成和结构,所启动炎症信号通路及其起始受体,引起牙周炎症的机制和特点作一综述。     

Porphyromonas gingivalis lipopolysaccharide modulates the responsiveness of human periodontal ligament fibroblasts to platelet-derived growth factor

Lipopolysaccharides (LPS) prepared from periodontopathic bacteria have been known to induce various biological responses which may lead to periodontal tissue breakdown. The purpose of this study was to determine if Porphyromonas gingivalis LPS could affect cellular functions of human periodontal ligament fibroblasts (HPLF). We showed here the responsiveness of cultured HPLF to platelet-derived growth factor (PDGF)-BB, a growth factor for mesenchymal cells, in the presence of P. gingivalis LPS. DNA synthesis of HPLF was enhanced in a dose-dependent manner when LPS were co-incubated for 48 h; thereafter, it decreased to the baseline level within 24 h incubation. The stimulating effect of PDGF-BB was further enhanced by the pretreatment of HPLF with LPS (10 μg/ml) for 48 h. The binding assay of [¹²⁵I] PDGF-BB and the flow cytometric assay using rabbit antiserum to human PDGF receptor (PDGF-R) β-type indicated that this enhancement was due to the increase of the number of PDGF-R β-type on HPLF. Immunoprecipitation using antiserum to human PDGF-R β-type also showed that the synthesis of PDGF-R β-type was augmented in the LPS-treated HPLF. These results indicate that P. gingivalis LPS stimulate cellular proliferation and responsiveness to PDGF-BB of cultured HPLF. These cellular reactions may be mediated by PDGF-BB binding, followed by increased synthesis of the receptor protein.     

Enhanced collagen phagocytosis by rat molar periodontal fibroblasts after topical application of lipopolysaccharide – Ultrastructural observations and morphometric analysis

To investigate the effect of lipopolysaccharide (LPS) on phagocytic activity of collagen fibrils by periodontal fibroblasts, we studied rat molar gingival connective tissue and periodontal ligament under light and electron microscopy after topical application of LPS (5 mg/ml in physiological salt solution (PS)) on the gingival sulcus. Phagocytic activity of collagen fibrils by fibroblasts was evaluated by counting the number of collagen-containing vacuoles inside fibroblasts that were present within a defined area (1200 microns2). Values obtained from fibroblasts in the subepithelial connective tissue, the region near the alveolar crest, and the middle region of periodontal tissue were compared. Periodontal ligament fibroblasts showed increased phagocytosis of the collagen fibrils from 3 hours to 1 day after topical LPS application, but no differences were observed in the gingival tissue. The intracytoplasmic vacuoles containing collagen fibrils were of various sizes and shapes, showing positive for acid phosphatase and/or alkaline phosphatase reaction. Collagen phagocytic activity of the fibroblasts in the middle region of the periodontal ligament also increased after PS treatment. However, this was significantly less than that observed in LPS-treated animals (p less than 0.01). This study indicates that LPS may enhance the degradation of collagen by stimulating the phagocytic activity of the periodontal ligament fibroblasts.     

Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts

We hypothesized that bacterial viability and strain characteristics of Porphyromonas gingivalis could affect the induction of pro-inflammatory mediator secretion by human gingival fibroblast cultures. Both killed and viable P. gingivalis elicited production of prostaglandin E2, interleukin-1 beta (IL-1 beta), IL-6 and IL-8, although killed P. gingivalis induced generally higher levels, particularly IL-6 and IL-8, compared with the viable bacteria. P. gingivalis strains, which exhibited wild-type levels of trypsin-like protease activity, stimulated human gingival fibroblasts to secrete increased levels of prostaglandin E2 and IL-1 beta, although minimal levels of IL-6 and IL-8 were noted in supernatants from the gingival fibroblast cells. P. gingivalis strains BEI and NG4B19, which have either decreased or undetectable levels of trypsin-like protease, respectively, induced significantly greater IL-6 and IL-8 levels in gingival fibroblast cultures compared with the other strains. The ability of antibody to P. gingivalis to alter human gingival fibroblast production of pro-inflammatory mediators was tested using nonhuman primate antisera. Both immune and nonimmune sera altered the P. gingivalis-generated pattern of mediators from the gingival fibroblasts. We conclude that: (i) viable and killed P. gingivalis were capable of inducing various pro-inflammatory cytokines from human gingival fibroblasts; (ii) strain differences in cytokine induction were noted, and the expression of a trypsin-like protease activity was related to decreased extracellular levels of IL-6 and IL-8; and (iii) the presence of serum, particularly with specific antibody to P. gingivalis, significantly altered human gingival fibroblast cytokine production compared with P. gingivalis alone.     

Human periodontal ligament cells exhibit no endotoxin tolerance upon stimulation with Porphyromonas gingivalis lipopolysaccharide

Background/Objectives

Endotoxin tolerance is characterized by a state of hyporesponsiveness after confrontation with endotoxins such as lipopolysaccharides (LPS) at low concentrations. The aim of this study was to investigate, whether pretreatment with Porphyromonas gingivalis leads to endotoxin tolerance induction and possible alterations in toll‐like receptor (TLR) 2‐ and 4‐induced response in human periodontal ligament cells (hPDLCs).

Material and Methods

Primary hPDLCs were pretreated with P. gingivalis (0.1 or 0.3 μg/mL) LPS for 24 hours and afterwards treated with one of the following stimuli: P. gingivalis LPS (1 μg/mL); TLR4 agonist Escherichia coli LPS (0.1 μg/mL; 1 μg/mL); TLR2 agonist Pam3CSK4 (0.1 μg/mL; 1 μg/mL). The protein expression of interleukin (IL)‐6, IL‐8 and monocyte chemotactic protein‐1 was analyzed with quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay. Gene expression levels of TLR2 and TLR4 were determined by quantitative polymerase chain reaction.

Results

Pretreatment of cells with low concentrations of P. gingivalis LPS did not result in lower production of IL‐6, IL‐8 and monocyte chemotactic protein‐1 compared to control group. In some cases, pretreated cells exhibited lower gene expression levels of TLR2 and TLR4 compared to non‐pretreated cells.

Conclusion

The results of this study implicate that hPDLCs do not develop endotoxin tolerance. Furthermore, the amplitude of the inflammatory response shows no significant dependency on TLR2 and TLR4 expression levels.