外源性糖对口腔细菌产生过氧化氢能力的影响

目的：研究不同外源性糖对口腔细菌产生过氧化氢能力的影响。方法：采用酚红还原－微量板法测定１５株口腔细菌在４种外源性糖作用下，细菌孵育液上清中的过氧化氢含量。结果：口腔不同细菌利用葡萄糖、蔗糖、麦芽糖、乳糖产生过氧化氢的能力有很大差异，血链球菌ＡＴＣＣ１０５５６、ＡＴＣＣ１０５５７、唾液链球菌ＨＨＴ等在蔗糖和麦芽糖的作用下，其产生的过氧化氢量高于葡萄糖和乳糖，而变形链球菌ＪＨ１４５、具核梭杆菌ＷＤＣ９７－１１除乳糖略低外，其它３种糖则大致相同。牙龈紫质单胞菌、粘性放线菌、中间普氏菌、伴放线放线杆菌在各实验组中均不产生过氧化氢。结论：口腔细菌产生过氧化氢的能力受外源性糖的影响，各种细菌对外源性糖反应不一，即使是同一种细菌也因菌株不同而有很大差异，提示这些细菌在糖分解或糖酵解中的途径可能不同。     

血链球菌产生过氧化氢能力的差异

目的：初步研究血链球菌产生过氧化氢的能力，筛选过氧化氢高产菌株。方法：从牙周健康人群口腔中收集血链球菌，采用酚红—辣根过氧化物酶微量板法测定细菌产生过氧化氢的量，并测定细菌的蛋白含量以定量观察血链球菌产生过氧化氢的能力。结果：每微克菌蛋白的血链球菌Ⅱ型(又称为口腔链球菌，Streptococus oralis,S.oralis）产生过氧化氢的量高于血链球菌I型(Streptococcus sanguis,S.sanguis)(P＜0．05)；有氧培养时血链球菌比厌氧培养产生更多的过氧化氢(P＜0．05)；和国际标准株S．oralis ATCC10557相比，S．oralis临床分离株l～12能稳定地产生较高的过氧化氢。结论：血链球菌在有氧和厌氧情况下都能产生过氧化氢，有氧时产生过氧化氢的能力更强；S．oralis产生过氧化氢的能力高于S．sanguis，同一种细菌的不同株之间产生过氧化氢的能力有很大差异。血链球菌产生过氧化氢的机制需进一步研究。     

牙周可疑致病菌对血液链球菌产生过氧化氢的影响

目的：了解牙周可疑致病菌对血液链球菌产生过氧化氢的影响，探讨血液链球菌产生的过氧化氢在牙周微生态环境中的作用。方法：采用酚红还原－微量反应板法检测粘性放线菌（Ａｖ）和４种牙周可疑致病菌即牙龈紫质单胞菌（Ｐｇ）、具核梭杆菌（Ｆｎ）、中间普氏菌（Ｐｉ）、伴放线放线杆菌（Ａａ）与血液链球菌混合孵育后上清中的过氧化氢含量变化。结果：在不加外源性糖作为底物时，仅Ｆｎ组有高于对照组的过氧化氢，Ｐｇ、Ｐｉ、Ａａ组均未能测出。在有葡萄糖、蔗糖、麦芽糖、乳糖存在时，Ｐｉ、Ａａ组无过氧化氢积累，Ｐｇ、Ａｖ、Ｆｎ组能检出的过氧化氢含量也低于对照组。结论：本实验中Ａｖ和其它４种牙周可疑致病菌均可导致血液链球菌产生过氧化氢的量下降，提示在牙周微生态环境中，这些细菌可能利用血液链球菌产生的过氧化氢或影响过氧化氢的产生。     

牙周可颖致病菌对血液链球菌产生过氧化氢的影响

目的：了解牙周可颖致病菌对血液链球菌产生过氧化氢的影响，探讨血液链球菌产生的过氧化氢在牙周微生态环境中的应用。方法：采用酚红还原－微量反应板法检测粘性放线菌（Ａｖ）与血液链球菌混合孵育的上清中的过氧化氢含量变化。结果：在不加外源性糖作为底物时，仅Ｆｎ组有高于对照组的过氧化氢，Ｐｇ、Ｐｉ、Ａａ组均未能测出。在有葡萄糖、蔗糖、麦芽糖、乳糖存在时，Ｐｉ、Ａａ组无氧化氢积累，Ｐｇ、Ａｖ、Ｆｎ组能检出的过氧     

口腔环境因素对血型链球菌和变形链球菌生长的影响：II.氧的影响

口腔的链球菌族属于微需氧菌，易受到氧代谢产物的抑制作用，作者采用连续培养方法，观察血型链球菌（以下简称为血链菌）３４和变形链球菌（以下简称变链菌）Ｉｎｇｂｒｉｔｔ（ｃ）在有氧情况下的生长状态，得出血链菌３４的耐氧能力高于变链菌Ｉｎｇｂｒｉｔｔ（ｃ）的结果。这一结果与血链菌为牙菌斑的先锋定殖菌的现象相一致。而变链菌的耐氧能力差使其生长，以及致龋作用对牙菌斑的内环境有一定的依赖性。     

口腔血链球菌与牙周病

血链球菌是口腔正常菌群组份之一，随着牙齿的萌生早期定植牙面，通过植物血凝素样附着素，非植物血凝素样附着素与唾液中受体特异结合，粘附于获得性膜上，以疏水作用维持其稳定，可与多种细菌发生聚集反应，在龈上，龈下菌斑形成中起重要作用，血链球菌通过产生过氧化氢和血链素拮抗牙周病的可疑致病菌，是牙周主要有益菌。     

铍对口腔链球菌细胞膜元素含量及过氧化氢产量的影响

目的:研究铍离子(Be2+)对口腔链球菌细胞膜元素含量及抗菌性能的影响,探讨镍铬合金修复体对牙周损伤的微生物学机制.方法:含有不同浓度(5mg/L、10mg/L、20mg/L和40mg/L)Be2+的口腔链球菌培养液厌氧培养24h.X线能谱仪分析细胞膜元素含量变化,ABTS-HRP法检测细菌产生H2O2的能力.采用SPSS11.0软件包对数据进行单因素方差分析.结果:口腔链球菌细胞膜钙元素含量减少,钠元素先升高后降低,磷元素升高.Be2+浓度为40mg/L时,口腔链球菌H2O2产量显著下降(P＜0.05).结论:Be2+会改变口腔链球菌细胞膜元素含量,降低H2O2产量,从而导致修复体周围正常微生态环境失衡,引起牙周疾病.     

口腔链球菌的新分类

链球菌是口腔最常见的细菌，在口腔正常菌群中所占的比例最大，从口腔中所有部位都可分离得到。在牙菌斑和龈沟的细菌中，链球菌约占30％。舌背和唾液中近一半的细菌是链球菌。近年来对口腔链球菌的研究日益广泛，对其认识也不断地深入，目前口腔链球菌已包括唾液链球菌群，咽颊炎链球菌群，变形链球菌群，轻型链球菌群，牛链球菌群，化脓性逻球菌群以及5个尚未分群的菌种。本文就口腔链球菌的新分类以及小生境主要在口腔的链球菌作一简要的综述。     

口腔环境因素对血型链球菌和变形链球菌生长的影响：I.粘蛋白的影响

采用连续培养方法，以粘蛋白作为化学限定培养基中的限定性因子，观察血型链球菌（以下简称血链菌）和变形链球菌（以下简称变链菌）Ｉｎｇｂｒｉｔｔ（ｃ）的生长状态。结果显示变链菌不能生长于以粘蛋白作为唯一营养源的培养基中，而血链菌可以生长；两菌混合培养时的生长量均有所提高，表明两菌有协同降解粘蛋白作用。     

口腔环境因素对血型链球菌和变形链球菌生长的影响 Ⅰ．粘蛋白的影响

采用连续培养方法，以粘蛋白作为化学限定培养基中的限定性因子，观察血型链球菌（以下简称血链菌）和变形链球菌（以下称简称变链菌）Ｉｎｇｂｒｉｔｔ（ｃ）的生长状态。结果显示变链菌不能生长于以粘蛋白作为唯一营养源的培养基中，而血链菌可以生长；两菌混合培养时的生长量均有所提高，表明两菌有协同降解粘蛋白作用。     

Identification and characterization of a protease from Streptococcus oralis C104

Streptococcus oralis is among the earliest colonizers of the tooth surface during plaque formation. As such, its enzymatic activities may influence ecologic succession on the tooth surface. In the current study, we used zymograms and preparative polyacrylamide gel electrophoresis to identify and purify a protease from S. oralis ( sanguis ) C104. Proteases from 5. oralis C104 were detected in cell pellets at 133, 146 and 176 kDa as clear proteolytic bands on gelatin-substrate zymograms. Preparations of the major (146 kDa) protease were obtained by continuous-elution electrophoresis. The protease was active over the pH range of 7 to 9 with optimum activity between pH 8 and 9. Protease activity was inhibited by several serine protease inhibitors including phenylmethylsulfonyl fluoride, di-iso-propyl-phosphofluoridate and aprotinin. The protease showed highest hydrolytic activity against azoalbumin and Bz-Pro-Phe-Arg-NA. Immunofluorescence studies with a polyclonal antiserum to the 146–kDa protease suggest it is present on the cell surface of S. oralis C104. Zymograms of cell pellets from other S. oralis strains as well as S. sanguis and Streptococcus mitis suggest that functionally similar proteases are elaborated by many early colonizers of the tooth surface.     

口腔链球菌丙酮酸氧化酶基因缺陷型变异株的构建

目的:利用口腔链球菌丙酮酸氧化酶基因(sopox) 的克隆序列,重组构建新的不能产生H2O2 的口腔链球菌变异株。方法:口腔链球菌株ATCC10557 经培养后用酚-氯仿法抽提细菌染色体基因组DNA ,经PCR 扩增sopox 基因,用BamHI 进行限制酶切;参照Chris 方法进行电转化,挑选阳性菌落测定其上清液中H2O2 的含量;将细菌传3～4 代后再次重复上述检测。结果:转化子经筛选后得到1 株阳性菌落,测定上清液中H2O2 含量,第1 次检测表明变异株产生H2O2 的量仅有所下降(介于阳性对照ATCC 10557 和阴性对照大肠杆菌JM109 之间) ,经3～4 次传代后变异株中上清液H2O2 量已经明显低于阴性对照。结论:成功构建了口腔链球菌丙酮酸氧化酶基因缺陷型变异株。     

葡萄糖浓度影响寡发酵链球菌抑制变形链球菌作用的研究

目的 研究不同葡萄糖浓度对寡发酵链球菌(Streptococcus oligofermentans,So)与变形链球菌(Streptococcus mutans,Sm)之间相互作用的影响,及对So产过氧化氢能力的影响.方法通过平板培养法观察在不同葡萄糖浓度下So与Sm之间的相互作用;运用4-氨基安替吡啉-辣根过氧化物酶法测定不同葡萄糖浓度下So过氧化氢的初始产生速率和产量.结果环境葡萄糖浓度为0、10、50 mmol/L时均可见So对Sm有抑制作用;Sm受抑制区面积占菌膜面积比值:同时接种So和Sm时,无糖环境比值为0.202±0.005,10、50 mmol/L葡萄糖环境分别为0.467±0.025和0.468±0.028;先接种So再接种Sm时,无糖环境比值为0.394 ±0.004,10 mmol/L葡萄糖环境为0.811 ±0.075,50 mmol/L葡萄糖环境为0.816 ±0.007.葡萄糖浓度为10、50 mmol/L时So对Sm的抑制作用均较无糖环境下显著(P＜0.05),但两种浓度抑制作用差异无统计学意义(P＞0.05).葡萄糖浓度为10、50 mmol/L时So的过氧化氢初始产生速率[(23.573±0.263)、(23.337±0.473) μmol·L-1·min -1]均显著高于无糖环境[(10.513 ±0.516) μmol·L-1·min-1],P ＜0.05.无糖环境下So对数生长期各时段过氧化氢产量高于有糖环境(P＜0.05).1000 mmol/L葡萄糖环境下未见So抑制Sm,亦未能检测到So产生过氧化氢.结论So抑制Sm的能力受糖环境的影响,在10、50 mmol/L葡萄糖环境下,So具有更强的抑制Sm能力.     

Effect of binding of fibrinogen to each bacterium on coaggregation between Porphymmonas gingivalis and Streptococcus oralis

Fibrinogen inhibits the coaggregation between Porphyromonas gingivalis and Streptococcus oralis. In this study, we determined which bacterium interacts with fibrinogen in this inhibitory process. Although preincubation of each bacterium with fibrinogen did not inhibit coaggregation, its activity was completely eliminated by the addition of protease inhibitors such as N -ethylmaleimide (NEM), p -chloromercuriphenyl sulfonate and N α- p -tosyl-L-lysine chloromethyl ketone to the preincubation mixture with fibrinogen and P. gingivalis. However, the inhibition of coaggregation was not found after preincubation of S. oralis with fibrinogen in the presence or absence of the protease inhibitors. Labelled materials were recovered from the extract of P. gingivalis cells incubated with radioiodinated fibrinogen in the presence of NEM but not in the absence of NEM. In the binding experiment, P. gingivalis showed a much higher binding activity to fibrinogen than S. oralis. These findings suggest that fibrinogen and its fragment(s) may mask directly or indirectly the aggregation site with S. oralis on the P. gingivalis cells in its inhibitory process of coaggregation.     

口腔链球菌丙酮酸氧化酶调节基因的克隆和功能分析

目的 :阐明体外扩增得到的口腔链球菌丙酮酸氧化酶基因的上游区序列是否即是调控序列 ,是否具有启动子活性。方法 :将口腔链球菌丙酮酸氧化酶基因调节区PCR扩增产物经HindⅢ酶切 ,克隆至载体PKK2 32 - 8,转化E .coliJM 10 9,通过氯霉素抗性筛选阳性菌落 ,重新增菌后取质粒DNA ,再经酶切鉴定。将重组子点种于不同浓度的氯霉素平板上 ,37℃培养 18h ,观察菌落生长情况 ,测定重组质粒转化子对氯霉素的抗性水平。结果 :筛选得到 1个阳性菌落 ,重组质粒DNA的酶切产物经 10 g/L琼脂糖凝胶电泳 ,表明获得片段的Mr约为 1.3kb ,与预计大小相符 ,证实其为阳性重组克隆。测定重组质粒转化子对氯霉素的抗性水平显示其抗性达 10 2 0 μg/mL。 结论 :本研究已成功克隆口腔链球菌丙酮酸氧化酶调节基因。     

Streptococcus oralis coaggregation receptor polysaccharides induce inflammatory responses in human aortic endothelial cells

Streptococcus oralis, belonging to the oral viridans group streptococci, has been detected in human cardiovascular lesions including infective endocarditis and atheromatous plaques. The organism has coaggregation receptor polysaccharides (RPS) on the cell wall, which function as receptors for surface adhesins on other members of the oral biofilm community. The present study examined the capacity of S. oralis RPS to induce inflammatory responses in human aortic endothelial cells (HAECs). Purified RPS was used to stimulate HAECs, and the induction of cytokines, adhesion molecules and Toll-like receptors (TLRs) was examined. Involvement of RPS in HAEC invasion by S. oralis was also examined. RPS-stimulated HAECs produced more cytokines (interleukin-6, interleukin-8 and monocyte chemoattractant protein-1) and intercellular adhesion molecule-1 than non-stimulated HAECs. The messenger RNA (mRNA) expression of cytokines and adhesion molecules in RPS-stimulated HAECs increased markedly compared with that in non-stimulated HAECs. Upregulation of TLR-2 mRNA expression was demonstrated in RPS-stimulated HAECs. Moreover, TLR-2 mRNA expression and cytokine production were reduced by the incubation of HAECs with inhibitors against p38 mitogen-activated protein kinase and nuclear factor-κB. An RPS-defective mutant of S. oralis showed greater invasion into HAECs than an RPS-possessing strain. However, HAECs invaded by the RPS-defective mutant produced less cytokines than HAECs invaded by the RPS-possessing strain, indicating that RPS can stimulate HAECs intracellularly. These results suggest that S. oralis RPS may be an important contributor to the pathogenesis of cardiovascular diseases such as infective endocarditis and atherosclerosis.     

Lack of association between Streptococcus oralis and recurrent aphthous stomatitis

In the present study, the potential involvement of Streptococcus oralis in the aetiology of recurrent aphthous stomatitis (RAS) was investigated using the polymerase chain reaction (PCR). Biopsies from 28 RAS patients were analysed, in addition to 20 oral lichen planus (OLP) and 13 normal biopsies that were used as controls. PCR was carried out using a primer pair that targets the D-alanine:D-alanine ligase gene and detects DNA from both S. oralis and the closely related species Streptococcus mitis. Discrimination between these two species was achieved by digestion of PCR products with the restriction endonucleases HaeIII and HindIII, which both give distinct restriction profiles for each species. S. oralis DNA was detected in 8 of 28 (29%) RAS samples, 10 of 20 (50%) OLP samples and 6 of 13 (46%) normal samples. These results suggest that S. oralis is not of primary aetiological significance in RAS.