P. gingivalis感染对血管内皮细胞VCAM-1表达的影响

目的:观察牙龈卟啉单胞菌(P. gingivalis)ATCC 33277和W83感染对血管内皮细胞血管细胞黏附分子-1(VCAM-1)转录和翻译的影响.方法:建立体外P. gingivalis感染血管内皮细胞模型,反转录-聚合酶链式反应检测VCAM-1基因表达;蛋白质印迹技术检测VCAM-1膜蛋白表达变化.采用SAS 8.12软件包,多组均数之间的比较采用重复测量资料的方差分析.结果:P. gingivalis感染后4 h即诱导VCAM-1mRNA表达(与对照组比较,P<0.05),8 h达高峰,24 h有持续的高表达;P.gingivalis感染后4 h即诱导VCAM-1蛋白表达(P<0.05),8 h有持续的高表达,24 h表达到达高峰(P<0.05).P.gingivalis W83诱导VCAM-1表达的能力强于ATCC 33277(P<0.05).结论:P.gingivalis感染可引起血管内皮细胞VCAM-1高表达,提示P.gingivalis可能在动脉粥样硬化炎症病理反应中有重要的意义.     

牙龈卟啉单胞菌影响血管内皮细胞增殖和凋亡的实验研究

目的 观察牙龈卟啉单胞菌(Porphyromonas gingivatis,Pg)对血管内皮细胞增殖和凋亡的影响,探讨Pg在动脉粥样硬化发病机制中的作用.方法 建立体外Pg侵入血管内皮细胞模型,甲基噻唑基四唑(MTT)法观察细胞增殖,碘化丙啶染色、流式细胞仪分析细胞周期,Annexin-V-FITC凋亡试剂盒检测细胞凋亡.结果 PgATCC33277侵入后72 h细胞增殖活性降低12.46%,Pg W83侵入后72 h细胞增殖活性降低10.47%(F=786.68,P<0.01);Pg W83侵入后24 h使G1期细胞增加(F=43.23,P<0.01),ATCC 33277侵入后48 h使G1期细胞增加(F=66.72,P<0.01);Pg侵入后24 h即诱导细胞凋亡(F=1074.56,P<0.01).结论 Pg可能通过细胞毒性及诱导凋亡作用,加重血管内皮细胞的局部炎性反应,在动脉粥样硬化炎性病理反应中有重要意义.     

牙龈卟啉单胞菌侵入对人血管内皮细胞分泌MCP-1影响的研究

目的　通过研究牙龈卟啉单胞菌（porphyrmonas gingivalis,P.g）侵入对人脐静脉内皮细胞（human umbilical vein endothelial cell,HUVEC）分泌单核细胞趋化蛋白（MCP-1）的影响,了解P.g对其趋化功能的影响。方法　建立P.g侵入HUVEC的体外模型,采用酶联免疫吸附法（ELISA）研究P.g381和P.g33277菌株侵入HUVEC 6、24 h后的培养上清液中MCP-1浓度。结果　ELISA结果显示当P.g381侵入HUVEC 6、24 h和P.g33277侵入HUVEC 6 h时,HUVEC分泌的MCP-1水平升高（P＜0.01）;P.g33277侵入HUVEC 24 h时,HUVEC分泌的MCP-1水平恢复最初水平（P=0.46）;P.g381诱导HUVEC表达MCP-1的水平高于P.g33277（P＜0.01）。结论　P.g侵入HUVEC后可促进其表达MCP-1,从而上调其趋化功能,在牙周炎与心血管疾病的相关性中可能发挥作用。     

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目的 探讨牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)W83、ATCC33277刺激人牙周膜成纤维细胞(HPDLFs)分泌基质金属蛋白酶-1(MMP-1)、金属蛋白酶组织抑制剂-1(TIMP-1)的变化.方法 本研究于2010年9月至2011年6月在中国医科大学口腔医学院中心实验室进行.将P.gingivalis W83、ATCC33277作用于HPDLFs0、6、12、24、48h后,运用酶联免疫吸附法(ELISA)检测细胞上清液中MMP-1、TIMP-1质量浓度变化,并计算MMP-1/TIMP-1值.结果 P.gingivalis感染HPDLFs的MMP-1和TIMP-1表达均增强,并呈时间依赖性;且MMP-1/TIMP-1值明显高于未感染的对照组(P＜0.05).P.gingivalis W83感染后MMP-1/TIMP-1值明显高于P.gingivalis ATCC33277(P＜ 0.05).结论 P.gingivalis具有促进HPDLFs分泌MMP-1、TIMP-1的作用,且可造成牙周组织破坏;P.gingivalis W83降解细胞外基质的能力高于P.gingivalisATCC33277.     

牙龈卟啉单胞菌外膜囊泡差异表达mRNA的生物信息学分析

目的 探讨牙龈卟啉单胞菌(P.gingivalis)W83与ATCC33277外膜囊泡mRNA表达谱的差异.方法 超速离心法分离W83和ATCC33277来源的外膜囊泡,进行粒径检测、电镜及Western blot鉴定;高通量测序检测W83和ATCC33277外膜囊泡mRNA表达谱,筛选差异表达的mRNA,进行基因本体...     

牙龈卟啉单胞菌感染对人脐静脉内皮细胞表达IL-8影响的研究

目的 研究牙龈卟啉单胞菌(porphyrmonas gingivalis,P.g)感染时人脐静脉内皮细胞(human umbilical vein endothelialcell,HUVEC)表达趋化因子白介素-8(interleukin-8,IL-8)的变化,了解P.g对其趋化功能的影响。方法 建立P.g侵入HUVEC的体外模型,采用酶联免疫吸附法(ELISA)研究P.g381和P.g33277菌株感染HUVEC 6、24 h后的培养上清液中IL-8的浓度。结果 ELISA结果显示当P.g381感染HUVEC 6、24 h和P.g33277感染HUVEC 6、24 h时,HUVEC分泌IL-8的水平明显升高(P<0.01);感染24 h时P.g381促进HUVEC表达IL-8的水平高于P.g33277(P<0.01)。结论 P.g侵入HUVEC后可促进其表达IL-8,从而上调其趋化功能,可能与动脉粥样硬化(atherosclerosis,AS)的发生发展相关。     

两种fimA基因型牙龈卟啉单胞菌刺激下口腔上皮细胞ICAM-1的表达

目的比较2种fimA基因型牙龈卟啉单胞菌（Porphyromonas gingivalis,P．gingivalis）刺激下口腔上皮细胞ICAM-1的表达水平。方法实验组用P．gingivalis ATCC 33277（Ⅰ型菌毛组）,W83（Ⅳ型菌毛组）,47A-1（Ⅳ型菌毛组）分别与KB细胞（ATCC CCL17）共同孵育24h;对照组为未受P.gingivalis刺激的KB细胞。分别在1h、3h、6h和24h收集细胞,运用流式细胞仪检测KB细胞膜上ICAM-1的动态表达。结果P．gingivalis刺激细胞后3h、6h和24h,实验组ICAM-1的表达水平均高于对照组;2种fimA基因型P．gingivalis调节KB细胞表达ICAM-1的方式相似,Ⅳ型菌毛组的调节作用强于Ⅰ型菌毛组。结论P.gingivalis上调口腔上皮细胞表达ICAM-1的水平与其fimA基因型相关,提示P.gingivalis致病性与其fimA基因型相关。     

牙龈卟啉单胞菌rag位点基因分型与细菌毒力的关系研究

目的：探讨牙龈卟啉单胞菌rag位点基因分型与细菌毒性的关系。方法将不同rag基因位点的牙龈卟啉单胞菌W83(rag-1型)、A011/9(rag-2型)、QM220(rag-3型)和ATCC33277(rag-4型)菌株分别刺激中性多形核白细胞,未受刺激的中性多形核白细胞作为对照。采用RT-PCR检测FcγⅢb受体基因,流式细胞术检测中性多形核白细胞凋亡情况,试剂盒检测毒力因子。结果 W83组 FcγⅢb基因相对表达量显著高于其他组( P <0．05),其次为QM220组,显著高于A011/9和ATCC33277组(P<0.05)。流式细胞术检测发现,ATCC33277组细胞凋亡率显著高于其他各组(P<0.05),A011/9组凋亡率显著高于W83组(P<0.05)。各基因分型组LPS、LBP、mCD14、MMP-8 MMP-9、IL-8以及IL-1β均高于对照组(P<0.05)。W83组上述指标显著高于其他各基因型组(P<0.05),QM220组次之,与 A011/9、ATCC33277组比较差异有统计学意义(P <0.05)。 A011/9组在 LPS、LBP、mCD14方面高于ATCC33277组(P<0.05),而MMP-8、MMP-9两者差异无统计学意义(P>0.05)。结论牙龈卟啉单胞菌菌株中rag位点基因分型的细菌毒性rag-1型最强,rag-3型次之,较弱的为rag-2型和rag-4型。     

不同fimA基因型牙龈卟啉单胞菌刺激上皮细胞表达细胞因子的比较

目的比较不同fimA基因型牙龈卟啉单胞菌（Porphyromonas gingivalis,P.gingivalis）刺激下口腔上皮细胞表达细胞因子的水平。方法P.gingivalis ATCC33277（IfimA）,W83（ⅣfimA）,47A-1（ⅣfimA）分别与KB细胞ATCCCCL17共同孵育6h,在3h和6h收集细胞和培养上清液。逆转录-聚合酶链式反应检测KB细胞IL-8,IL-6,IL-1βmRNA的表达,酶联免疫反应检测培养上清液中IL-8,IL-6,IL-1β的变化。结果3h时,ⅣfimA组IL-6蛋白水平低于IfimA组（P〈0.05）,6h时,ⅣfimA组IL-6和IL-8蛋白水平均低于ⅠfimA组（P〈0.05）;IL-8,IL-6mRNA和蛋白水平表达不一致,提示存在转录后水平的调节。3h,6h时,IL-1β对P.gingvalis刺激不敏感,mRNA和蛋白表达水平都很低。结论P.gingivalis fimA基因型与上皮细胞表达细胞因子的水平相关,提示P.gingivalis致病性与其fimA基因型相关。     

不同fimA基因型牙龈卟啉单胞菌刺激口腔上皮细胞白细胞介素-8的表达

目的比较不同fimA基因型牙龈卟啉单胞菌（P.gingivalis）刺激下口腔上皮细胞白细胞介素-8（IL-8）的表达水平,初步探讨P.gingivalis致病性与其fimA基因型的关系。方法P.gingivalis ATCC 33277（Ⅰ型fimA）、W83、47A-1（Ⅳ型fimA）和KB细胞ATCC CCL-17共同孵育24 h,以未受刺激的KB细胞作为对照组,分别在1、3、6、24 h收集细胞和培养上清液。RT-PCR检测KB细胞IL-8 mRNA的动态表达,酶联免疫反应检测培养上清液中IL-8的动态变化。结果2种fimA基因型菌株刺激1 h,KB细胞IL-8 mRNA的表达上调至峰值,高于对照组,3~24 h IL-8 mRNA的表达水平接近对照组;P.gingivalis感染细胞后3~24 h,上清液中的IL-8水平低于对照组,Ⅳ型菌株低于Ⅰ型菌株;IL-8 mRNA及其蛋白表达不完全一致,提示IL-8的表达存在转录后水平的调节。结论fimA基因型与口腔上皮细胞IL-8的表达水平相关,提示P.gingivalis致病性与其fimA基因型相关。     

细菌array-CGH技术用于比较牙龈卟啉单胞菌不同菌株基因组差异的研究

目的:构建牙龈卟啉单胞菌(简称P.gingivalis)全基因组规模的微阵列比较基因组杂交(简称array-CGH)芯片平台,分析不同菌株间基因组的差异,为后续检测临床分离株的毒力基因,进一步阐述牙周炎发病机制提供依据。方法:综合已经全基因组测序的12株P. gingivalis菌的序列信息,设计探针序列,定制芯片。提取P. gingivalis高毒力株W83和低毒力株ATCC 33277的基因组DNA,利用array-CGH检测其全基因组的差异DNA片段,并运用PCR验证结果的准确性。结果:Array-CGH结果显示两组菌株间存在几个连续片段的拷贝数不同。P.gingivalis W83的部分特异片段参与编码毒力致病因子。在P.gingivalis ATCC 33277的特有片段中,部分编码蛋白可增强细菌表明粘附力,使其具有高粘附力。从中挑选12个基因进行PCR差异性验证,证实与芯片结果一致。结论:用array-CGH芯片的方法来分析全基因组拷贝数的变化具有分辨率高,能精确定位异常片段的特性。本文成功构建了array-CGH技术检测P.gingivalis不同毒力株基因差异的平台,为后续比较临床分离株的差异DNA片段,筛查毒力基因,深入阐述牙周炎的发病机制奠定基础。     

牙龈卟啉单胞菌不同毒力株基因差异的比较研究

目的比较牙龈卟啉单胞菌（Porphyromonas gingivalis，Pgingivalis）高毒力株W83与低毒力株标准参考菌ATCC 33277之间的差异基因。方法采用抑制消减杂交技术（SHH）对比牙龈卟啉单胞菌高毒力株W83与低毒力株标准参考菌ATCC 33277的基因差异。以高毒力株W83为被检菌，低毒力株ATCC 33277为参考菌，将提取的基因组DNA用内切酶Rsa Ⅰ酶切，连接特殊设计的接头进行两次消减杂交和PCR扩增，得到消减混合物，与TA克隆载体连接，转化到JM109中，建立消减文库，经PCR筛选鉴定阳性克隆，进而对部分片段进行测序和同源分析。结果经SSH筛选鉴定得到36个片段大小为88～372bp的阳性克隆基因片段。结论从全基因角度研究牙龈卟啉单胞菌高毒力株W83与标准株ATCC 33277之间的分子遗传差异，为牙龈卟啉单胞菌致病基因的筛选及今后牙周病预防、诊治的靶标提供依据。     

Identification of the genes associated with a virulent strain of Porphyromonas gingivalis using the subtractive hybridization technique

Background/aims:  Porphyromonas gingivalis , a major etiological organism implicated in periodontal disease, can be classified into virulent and avirulent strains. Our aim was to identify a gene for the virulence of P .  gingivalis .
Methods:  The subtractive hybridization technique was employed to identify the genes specific to P .  gingivalis W83, a virulent strain. In this study, P. gingivalis W83 was used as the tester strain, and P .  gingivalis ATCC 33277 was the driver strain. The prevalence of W83-specific genes was determined by Southern blot analysis of several P. gingivalis strains.
Results:  We obtained 575 colonies using the subtractive hybridization technique. From among these, 26 DNA fragments were subjected to a homology search using the BLAST program. Compared with strain ATCC 33277, strain W83 contained 12 unique clones. The specificities of the isolated DNA fragments were analyzed among four P. gingivalis strains by Southern blot analysis. Five genes showed specificity for strain W83 compared with strain ATCC 33277. All five genes were also identified in strain W50.
Conclusions:  The subtractive hybridization technique was effective in screening the two strains for specific DNA sequences, some of which might be responsible for determining virulence. The results suggested that several genes specific to strain W83 were associated with its virulence. Further analysis of these DNA fragments will provide important information on the pathogenesis of virulent P .  gingivalis strains.     

Effect of gamma-immunoglobulin on the asaccharolytic growth of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVES: A minimal medium is indispensable for examining the growth properties of the asaccharolytic bacterium, Porphyromonas gingivalis. The purpose of the present study was to improve the widely used KGB medium to support good growth of P. gingivalis. MATERIAL AND METHODS: Growth of P. gingivalis (W50, W83, and ATCC33277) in a minimal medium was monitored by measuring the optical density of the culture during incubation. RESULTS: W50, W83, and ATCC33277 grew poorly with bovine serum albumin as the sole carbon and nitrogen source, and alpha-ketoglutarate had little or no effect on this poor growth. In contrast, FeCl3 improved the growth of W83 and ATCC33277; however, the use of a high concentration of FeCl3 elicited black pigmentation of the cells. Bovine gamma-immunoglobulin greatly recovered the growth defect. None of alpha-ketoglutarate, citrate, or trace metal ions, when used to supplement KGB medium, was required for growth. We determined the optimal conditions for growth, and developed a new simple minimal medium for P. gingivalis (GA medium). Growth of ATCC33277 in GA medium was dependent on gingipains; Arg-gingipains and Lys-gingipain contributed comparably to proliferation of the bacterium. CONCLUSION: These data indicate that GA medium is currently the most reliable minimal medium for examining the growth properties of P. gingivalis.     

Correlation between cell-adherent activity and surface structure in Porphyromonas gingivalis

The cell-adherent ability of 6 strains of Porphyromonas gingivalis (381, ATCC 33277, SU63, KD1, W50 and W83) was compared by using radiolabeled bacterial cells and human gingival fibroblasts (Gin 1), human periodontal ligament fibroblasts (HPLF) and human epithelial cells (Ca9-22) that had been grown on collagen beads. The cell-adherent activity of these organisms varied among strains; P. gingivalis strains 381, ATCC 33277 and SU63 bound to the target cells at a range of 14% to 72%, but the other 3 strains (KD1, W50 and W83) were scarcely bound (0.6% to 3.5%). On the other hand, whole bacterial cells and culture supernatants of all strains showed distinct hemagglutinating activity. The 3 strains showing high cell-adherent activity were hydrophobic and the other strains showing less activity were relatively hydrophilic. Furthermore, a number of peritrichous fimbriae were found on the surface of P. gingivalis strains 381, ATCC 33277 and SU63, which showed high adherent activity, whereas, fimbriae on the other 3 strains showing low adherent ability were barely apparent. Therefore, it was assumed that the cell-adherent activity of P. gingivalis was related to the hydrophobicity of the cell surface, which was related to the number of fimbriae.     

Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway,but not through the p38 MAPK pathway

Zhang D, Zheng H, Zhao J, Lin L, Li C, Liu J, Pan Y. Porphorymonas gingivalis induces intracellular adhesion molecule‐1 expression in endothelial cells through the nuclear factor‐kappaB pathway, but not through the p38 MAPK pathway. J Periodont Res 2011; 46: 31–38. © 2010 John Wiley & Sons A/S Background and Objective: Porphyromonas gingivalis is a major pathogen in the development and progression of periodontal disease. The aim of this study was to investigate whether endothelial intracellular adhesion molecule‐1 (ICAM‐1), an inflammation biomarker for periodontitis, could be modified by infection with either of two strains of P. gingivalis with different virulence capacities: avirulent ATCC 33277 and virulent W83. Material and Methods: We examined the expression of ICAM‐1, IκBα, phospho‐p38 MAPK and nuclear factor‐kappaB (NF‐κB) p65 in an umbilical vein endothelial cell line (ECV‐304) treated with ATCC 33277 and W83, with or without the NF‐κB antagonist MG132 and/or a specific p38 inhibitor (SB203580), by real‐time PCR, western blotting and immunofluorescence. Results: Both strains could induce ICAM‐1 expression; additionally W83 was able to increase ICAM‐1 expression more significantly than ATCC 33277. In P. gingivalis‐infected endothelial cells, both p38 MAPK and NF‐κB signaling pathways were triggered by a rapid increase of p38 MAPK phosphorylation and a more delayed degradation of IκBα, followed by the nuclear translocation of NF‐κB. It was found that ICAM‐1 production in endothelial cells was abrogated by inhibition of the NF‐κB pathway, but not by inhibition of the p38 MAPK pathway, using the inhibitors of the latter two molecules. Conclusion: The induction of ICAM‐1 by infection of umbilical vein endothelial cells with P. gingivalis might be mediated through the NF‐κB pathway, but not by the p38 MAPK pathway.     

Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases

Sun W, Wu J, Lin L, Huang Y, Chen Q, Ji Y. Porphyromonas gingivalis stimulates the release of nitric oxide by inducing expression of inducible nitric oxide synthases and inhibiting endothelial nitric oxide synthases. J Periodont Res 2010; 45: 381–388. © 2010 The Authors. Journal compilation © 2010 Blackwell Munksgaard Background and Objective: The purpose of this study was to examine the ability of Porphyromonas gingivalis to invade human umbilical vein endothelial cells (HUVECs) and to study the effects of P. gingivalis ATCC 33277 on the production of nitric oxide (NO) and on the expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in HUVECs. We attempted to throw light on the pathway of damage to endothelial function induced by P. gingivalis ATCC 33277. Material and Methods: P. gingivalis ATCC 33277 was cultured anaerobically, and HUVECs were treated with P. gingivalis ATCC 33277 at multiplicities of infection of 1:10 or 1:100 for 4, 8, 12 and 24 h. HUVECs were observed using an inverted microscope and transmission electron microscopy. NO production was assayed through measuring the accumulation of nitrite in culture supernatants. Expression of both iNOS and eNOS proteins was investigated through western blotting. Results: It was found that P. gingivalis ATCC 33277 can adhere to HUVECs by fimbriae, invade into HUVECs and exist in the cytoplasm and vacuoles. P. gingivalis ATCC 33277 can induce iNOS and inhibit eNOS expression, and stimulate the release of NO without any additional stimulant. Conclusion: Our study provides evidence that P. gingivalis ATCC 33277 can invade HUVECs, and the ability of P. gingivalis ATCC 33277 to promote the production of NO may be important in endothelial dysfunction, suggesting that P. gingivalis ATCC 33277may be one of the pathogens responsible for atherosclerosis.     

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.