牙龈卟啉单胞菌膜泡对牙龈上皮细胞MMPs基因表达的影响

目的:探讨牙龈卟啉单胞菌膜泡对牙龈上皮细胞基质金属蛋白酶(MMPs)基因表达的影响,揭示牙龈卟啉单胞菌在牙周炎中的致病作用.方法:以Real-time RT-PCR法检测牙龈卟啉单胞菌膜泡刺激下牙龈上皮细胞MMP-1和MMP-3的mRNA表达水平.结果:牙龈卟啉单胞菌膜泡显著地上调MMP-1和MMP-3 mRNA表达水平.结论:牙龈卟啉单胞菌诱导牙龈上皮细胞发生细胞炎症反应,可能是牙周炎发生、发展的重要因素.     

阳离子诱导牙龈卟啉单胞菌FtsZ的聚合

在细菌细胞分裂过程中 ,一系列的相关蛋白质参与这一过程 ,其中FtsZ(filamentoustemperature sensitiveproteinZ)通过在未来细菌细胞分裂的中点处聚合成FtsZ环发挥着极其重要的作用。在本实验中 ,我们探讨了牙龈卟啉单胞菌(Porphyromonasgingivalis,Pg)的FtsZ(PgFtsZ)的体外聚合特性 ,以期为研制和开发干扰Pg菌细胞分裂的抗菌剂奠定实验基础。1.材料和方法 :(1)PgFtsZ的C末端缺失变异型质粒 pYW 1和 pYW 4在以前的实验中构建。(2 )PgFtsZ的N末端缺失变异型质粒的构建 :使用相同的downstream引物和两个不同的upstream引物 ,通过PC…     

牙龈卟啉单胞菌侵入牙龈上皮细胞的分子机制

牙龈卟啉单胞菌是慢性牙周炎的主要致病菌,牙龈上皮细胞是宿主牙周组织防御机制的第一道天然屏障。牙龈卟啉单胞菌可通过特异性粘附素与牙龈上皮细胞相应配体结合,激活该细胞内多种信号传导途径,最终内化于牙龈上皮细胞。内化后可通过某些细胞因子的合成和分泌减少以及抑制细胞凋亡等机制造成该细胞功能紊乱。本文从分子水平对牙龈卟啉单胞菌侵入牙龈上皮细胞的机制作一综述,以进一步阐明牙龈卟啉单胞菌的致病机理。     

N末端的缺失对牙龈卟啉单胞菌FtsZ体外聚合的影响

目的:探讨N末端的缺失对牙龈卟啉单胞菌FtsZ体外聚合的影响.方法:将质粒pEZ1(PgFtsZ,携带野生型牙龈卟啉单胞菌FtsZ的基因)、pYWN1(ZΔN01, 携带PgFtsZ 的N末端去除了43个保守性氨基酸残基的缺失变异型PgFtsZ的基因)和pYWN2(ZΔN02, 携带PgFtsZ 的N末端去除了205个保守性氨基酸残基的缺失变异型PgFtsZ的基因)分别导入Escherichia coli BL21(DE3)pLysS中表达,通过IPTG诱导表达目的蛋白质,6 mol/L尿素变性及HiTrap SP层析柱纯化目的蛋白质,最后通过沉淀法检测10 mmol/L的MgCl2、CaCl2和MnCl2对PgFtsZ、ZΔN01和ZΔN02体外聚合的影响.结果:10 mmol/L的MgCl2、CaCl2和MnCl2均能诱导PgFtsZ和ZΔN01出现聚合反应,然而10 mmol /L的MgCl2和CaCl2没能引起ZΔN02的体外聚合,而仅有10 mmol /L的MnCl2能诱导ZΔN02出现聚合反应,但效果不如PgFtsZ和ZΔN01明显.结论:PgFtsZ的N末端的43个氨基酸残基对Mg2+、Ca2+和Mn2+诱导其体外聚合反应影响不大,而后的162个氨基酸残基在PgFtsZ的体外聚合过程中起着关键的作用.     

黄芩对牙龈卟啉单胞菌生长、形态影响的体外实验

目的：探讨黄芩在牙髓、根尖周病治疗中的基础理论。方法：采用试管两倍稀释法测定黄芩对牙龈卟啉单胞菌的最低抑菌浓度（MIC）；用扫描电镜观察低于MIC的3个浓度的黄芩对牙龈卟啉单胞菌形态的影响。结果：黄芩对牙龈卟啉单胞菌MIC值为1mg/mL；黄芩改变了牙龈卟啉单胞菌菌体的正常形态。结论：黄芩对牙龈卟啉单胞菌的生长、形态有抑制作用。     

牙龈卟啉单胞菌红细胞凝集素A黏附和入侵人牙龈上皮细胞的研究

目的:检测牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)膜表面红细胞凝集素A(hemagglutinin A,HagA)黏附和入侵人牙龈上皮细胞的功能。方法:构建P.g381hagA基因的变异菌株,连接hagA基因到pYA292质粒上,并克隆到无毒性的沙门杆菌x4072菌中,通过比较变异菌株、野生菌株和表达hagA基因的沙门杆菌对人牙龈上皮细胞的黏附和入侵功能,检测HagA在此过程中的作用。结果:P.g381hagA基因变异菌株和野生菌株在对细胞的黏附和入侵过程中没有显著差别,但沙门杆菌x4072HagA表达菌株对细胞的黏附性和入侵性比对照组分别提高了3倍和4倍。结论:HagA参与了P.g381黏附和入侵牙龈上皮细胞的过程。     

牙龈卟啉单胞菌研究进展

牙周炎是常见的口腔疾病之一,是中国成年人失牙的主要原因.牙龈卟啉单胞菌在引发牙周组织破坏过程中发挥着重要的作用,成为国内外学者研究的热点之一.本文就牙龈卟啉单胞菌与口腔疾病和其他全身性疾病的关系、牙龈卟啉单胞菌全基因组,牙龈卟啉单胞菌与牙龈上皮细胞的相互作用等研究作一综述,以拓宽牙龈卟啉单胞菌的研究思路,进血为牙周炎的...     

甘草酸对牙龈卟啉单胞菌生长影响的体外研究

目的研究甘草酸对体外培养的牙龈卟啉单胞菌的抗菌活性。方法采用微量液体稀释法测定甘草酸对牙龈卟啉单胞菌的最低抑菌浓度（minimal inhibitory concentration,MIC）和最低杀菌浓度（minimal bactericidalconcentration,MBC）。用不同浓度的甘草酸培养牙龈卟啉单胞菌,分别于培养0、4、8、12、24、48 h测定细菌悬液600 nm波长处的光密度值,绘制牙龈卟啉单胞菌的生长曲线。结果甘草酸对牙龈卟啉单胞菌的MIC和MBC分别为1.57 g/L和12.5 g/L,甘草酸对牙龈卟啉单胞菌生长的抑制作用随药液浓度的增加而增强。结论甘草酸对体外培养的牙龈卟啉单胞菌的生长有抑制作用。     

牙龈卟啉单胞菌prtH基因克隆及其多态性的研究

目的：建立牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g)prtH全基因序列的克隆株，分析5株不同P.g菌株中prtH基因多态性。方法：利用PCR技术获得prtH全基因序列，克隆至载体pGEM-T;设计3对引物，用PCR的方法扩增prtH基因的不同区域；以生物素标记prtH基因序列为特异性探针，用斑点杂交和Southern印迹杂交进一步分析prtH基因的多态性。结果：酶切分析和DNA测序证实重组质粒pGEM-T-prtH的插入片段为prtH基因序列；核酸分子杂交显示菌株W 381和ATCC 33277杂交类型一致，菌株ATCC 49417和菌株14－3－2呈现各自不同的杂交条带，而菌株47A－1中未检测到prtH基因。结论：不同P.g菌株中prtH基因序列存在差别，这种基因多态性提示不同P.g菌株毒力大小可能存在差异。所建立的PCR扩增技术可以敏感地检测prtH基因的存在。     

Interaction of Porphyromonas gingivalis with gingival epithelial cells maintained in culture.

Interactions between Porphyromonas gingivalis and gingival epithelial cells were investigated. Gingival epithelial cells were cultured from surgically removed gingival tissue. Electron microscopy demonstrated adherence of P. gingivalis to the cell membranes and microvilli followed by internalization of the bacteria into the epithelial cell cytoplasm. Saliva from healthy and periodontally diseased patients inhibited P. gingivalis association with the epithelial cells. Attachment to and penetration of gingival epithelial cells by P. gingivalis may be important virulence factors in periodontal disease. Salivary molecules may play a role in modulating these interactions.     

Effects of Porphyromonas gingivalis infection on human gingival epithelial barrier function in vitro

The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form various cellular contacts, including tight junctions, and thus are able to create an epithelial barrier. A measurable indicator of barrier function in vitro is the transepithelial electrical resistance (TER). Porphyromonas gingivalis is recognized as a major aetiologic agent of periodontal disease and exhibits a variety of virulence factors. The aim of the study was to investigate the effect, in vitro, of infection with P. gingivalis on gingival barriers composed of primary and immortalized human keratinocytes. Primary and immortalized human gingival keratinocytes were infected with different strains of P. gingivalis. The impact of the bacterial challenge on the barrier was analysed by measuring the TER. The destructive effects of gingipains were blocked by specific enzyme inhibitors. After an initial increase of about 20-30% in infected wells, the TER decreased to zero. Gingipain inhibitors delayed the destruction of the barrier by 12 ± 4 h. In all cases, the loss of TER was accelerated if the system was infected from the basolateral side. A distinct effect of P. gingivalis on the epithelial barrier function of three-dimensional cultured epithelial cell models was demonstrated, which can partly be attributed to the activity of gingipains.     

Adherence of Porphyromonas gingivalis to gingival epithelial cells: modulation of bacterial protein expression

The protein profiles of Porphyromonas gingivalis (ATCC 33277 and W83) bound to KB gingival epithelial cells were analyzed by SDS-PAGE and immunoblotting. We found that a 51-kDa component was formed in bacteria that adhered to the KB cells, whereas 26- to 29-kDa bands were less intensive, in contrast to the protein profile of free bacteria. P. gingivalis ATCC 33277 incubated with protease-treated KB cells retained the profile of free bacteria. These results demonstrate the specificity of bacterial recognition of eukaryotic membrane components.     

牙龈卟啉单胞菌刺激树突状细胞激活炎症反应

[摘要] 目的 分析树突状细胞在牙龈卟啉单胞菌感染后的差异表达基因,探讨牙周炎的致病机制及为日后研究提供线索。方法 利用高通量测序技术来分析体外诱导分化的树突状细胞在牙龈卟啉单胞菌感染情况下的差异表达基因、基因集及相关功能。 结果 本实验总计发现7305个至少有两倍变化的差异表达基因。基因本体结果显示牙龈卟啉单胞菌感染后树突状细胞的生物过程、细胞成份、分子功能均发生了较大变化。基因集富集分析结果显示肿瘤坏死因子基因集、细胞因子及炎症反应基因集、白介素1及转录因子NF-kB家族基因集、干扰素信号基因集、B细胞受体信号基因集、T细胞受体信号基因集富集明显。结论 树突状细胞在牙龈卟啉单胞菌感染后会导致基因水平的变化,引起机体免疫反应及炎症反应的发生, CXCL及SOCS的水平及作用可作为日后研究的方向。     

牙龈卟啉单胞菌对牙龈上皮细胞焦点黏附成分paxillin和FAK的作用

目的:研究牙龈卟啉单胞菌(P.gingivalis)对牙周组织的破坏机制,探讨菌毛的致病作用.方法:应用Western blot法检测P.gingivalis ATCC 33277及其fimA缺陷突变株对Hela细胞及永生化人牙龈上皮细胞 (immortalized human gingival epithelial cells, IHGE细胞) 的焦点黏附成分——焦点黏附蛋白paxillin和焦点黏附激酶 (focal adhesion kinase,FAK) 蛋白表达的影响.结果:P.gingivalis ATCC 33277野生株和fimA缺陷突变株能够使HeLa细胞和IHGE细胞的paxillin和FAK发生降解,fimA缺陷突变株对paxillin和FAK的降解能力较野生株显著减弱.在IHGE细胞中,paxillin和FAK的降解呈时间依赖性和MOI依赖性.结论:菌毛介导的P.gingivalis的黏附和侵入可能对焦点黏附成分的降解起促进作用,IHGE细胞较Hela细胞更适用于牙周致病菌的研究.     

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

目的 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.方法 P.gingivalis感染CA9-22细胞,在感染24 h后,采用实时荧光定量聚合酶链反应、免疫印迹法和免疫荧光法检测钙结合蛋白1(CALB1)的表达.通过RNA干扰法抑制CALB1表达,BrdU分析检测细胞增...     

Cytotoxicity of Porphyromonas gingivalis toward cultured human gingival fibroblasts

Direct Cytotoxicity of black-pigmented anaerobic rods was studied on the confluent monolayer of human gingival fibroblasts in vitro. Only strains of Porphyromonas gingivalis caused morphological alteration (cell-rounding) and notable depression of viability of fibroblasts. To determine the location of the Cytotoxicity, bacterial surface components, i.e., outer membrane, lipopolysaccharide, fimbriae and outer membrane vesicles were prepared from P. gingivalis and their cytotoxicity was assessed. Among these preparations, only outer membrane vesicles are supposed to have high affinity to human gingival fibroblasts, and the cytotoxicity of outer membrane vesicles was found to be much stronger than that of the other constituents. This cytotoxic factor seemed to consist largely of protein and to be associated with the enzyme activity of outer membrane vesicles. The effects of some protease inhibitors and L-cysteine on the cytotoxicity of outer membrane vesicles suggest that the mechanism of cell-rounding is different from that of cell death.