牙龈卟啉单胞菌感染通过Wnt通路调节牙周膜干细胞的成骨分化

目的 观察牙龈卟啉单胞菌(P.gingivalis)感染通过Wnt通路调节牙周膜干细胞(PDLSCs)成骨分化的作用。 方法 培养原代PDLSCs,分为常规处理的对照组、P.gingivalis感染的P.gingivalis组和P.gingivalis感染并用Wnt3a处理的P.gingivalis+Wnt3a组,成骨诱导后茜素红染色并检测A405值,Western blot检测Wnt通路分子的蛋白表达量,碱性磷酸酶(ALP)试剂盒检测ALP活力,PCR检测成骨标志基因Runt相关转录因子2(Runx2)、骨钙素(OCN)的mRNA表达量。 结果 与对照组比较,P.gingivalis组Wnt3a、βcatenin、pGSK3β的蛋白表达水平(0.33±0.07)、(0.27±0.08)、(0.44±0.09)以及成骨诱导后A405值(0.55±0.08)、ALP活力(20.14±6.54)U/mL和Runx2、OCN的mRNA表达量(0.45±0.09)、(0.51±0.07)均明显减少;与P.gingivalis组比较,P.gingivalis+Wnt3a组成骨诱导后A405值(0.89±0.15)、ALP活力(29.44±5.26)U/mL及Runx2、OCN的mRNA表达量(0.89±0.17)、(0.81±0.18)均明显增加。 结论 P.gingivalis感染能够抑制PDLSCs的成骨分化,抑制Wnt通路是可能的分子机制。     

牙龈卟啉单胞菌感染通过激活NLRP3小体诱导牙周膜细胞炎症反应及凋亡效应

目的 观察牙龈卟啉单胞菌感染通过激活含NLR家族PYRIN域蛋白3(NLRP3)小体诱导人牙周膜细胞(hPDLCs)炎症反应及凋亡的效应。 方法 取健康前磨牙样本并分离培养hPDLCs,分为牙龈卟啉单胞菌感染的感染组和常规处理的对照组,检测细胞中NLRP3小体[NLRP3、凋亡相关斑点样蛋白(ASC)、含半胱氨酸的天冬氨酸蛋白水解酶(Caspase)-1]、凋亡基因[自杀相关因子(Fas)、Fas配体(FasL)、B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关x蛋白(Bax)、Caspase-3]的表达量及培养基中炎症细胞因子[白细胞介素(IL)-1β、IL-18、肿瘤坏死因子-α(TNF-α)]的含量。 结果 感染组hPDLCs中NLRP3、ASC、Caspase-1、Fas、FasL、Bax、Caspase-3的表达量及培养基中IL-1β、IL-18、TNF-α的含量明显高于对照组,细胞中Bcl-2的表达量明显低于对照组。 结论 牙龈卟啉单胞菌感染能够诱导hPDLCs的炎症反应及凋亡且该作用与NLRP3小体的激活有关。     

牙龈卟啉单胞菌相关致病因子的研究进展

牙龈卟啉单胞菌是一种生长于口腔内的革兰阴性厌氧茵.它能够利用脂多糖、荚膜多糖、菌毛、牙龈蛋白酶等一系列致病因子,侵袭局部牙周组织并逃避宿主的免疫防御机制,是诱发牙周炎的重要因素之一,引起了学者们的广泛关注.因此,探讨牙龈卟啉单胞菌的致病因子对于牙周炎的防治具有重要意义.     

牙龈卟啉单胞菌及其细胞外膜泡多克隆抗体的比较研究

分别制备牙龈卟啉单胞菌４７Ａ－ １ 及其膜泡的多克隆抗体,并用间接免疫荧光法对６７ 个临床标本进行检测。结果经统计学检验,两种抗体显著相关,Ｐ＜ ０．０１ ,符合率为８２．１ ％ ;膜泡抗体的检出率高于菌体抗体,Ｐ＜０．０５ ;膜泡抗体相对于菌体抗体的敏感性为９３．５ ％ 。膜泡抗体与牙龈卟啉单胞菌以外的其他口腔细菌无交叉反应,说明膜泡抗体的特异性较强。     

牙龈卟啉单胞菌酪氨酸激酶致病性的分子机制

目的 探讨牙龈卟啉单胞菌（Porphyromonas gingivalis,P. gingivalis）酪氨酸激酶（Ptk1)致病性的分子机制。方法 采用重组PCR技术构建P. gingivalis野生菌株ATCC 33277的Ptk1单基因缺失的突变菌株（ΔPtk1）,通过Real-time PCR技术检测并比较参与调控P. gingivalis（野生型P. gingivalis ATCC 33277与突变型ΔPtk1）细胞外多糖（extracellular polysaccharides,EPS）合成的转录因子SinR的表达情况,同时采用激光共聚焦显微镜观察Ptk1缺失的突变菌株与野生型菌株EPS的形成情况,最后通过ELISA试剂盒检测并比较Ptk1缺失的突变菌株与野生型菌株白细胞介素-1β(IL-1β)表达情况。结果 与野生菌株P. gingivalis ATCC 33277比较,Ptk1单基因缺失的突变菌株转录因子SinR的表达量没有显著变化（t=–1.572,P>0.05）;ELISA检测发现,Ptk1单基因缺失的突变菌株IL-1β的表达量较野生型菌株显著下降,差异有统...     

多肽免疫抑制牙龈卟啉单胞菌引起的小鼠感染及牙槽骨吸收作用的研究

目的探讨牙龈卟啉单胞菌(Porphyromonasgingivalis,P.g)血凝素2(hemagglutinin-2,HA-2)的氯化血红素结合位点多肽作为抗原免疫小鼠,对降低P.g感染后毒性和抑制牙周炎牙槽骨吸收的作用。方法将多肽与牛血清白蛋白(Bovine serum albumin,BSA)偶联作为抗原免疫BALB/c小鼠。P.g国际标准菌株ATCC33277厌氧培养,皮下注射菌液,监测小鼠脓肿的大小及转归,评价多肽对P.g皮下感染的保护作用;口腔接种细菌感染小鼠,检测小鼠牙周炎牙槽骨吸收量,评价其对于P.g引起的牙周炎牙槽骨吸收的保护作用。结果多肽DGFPGDHYAVMISK作为抗原免疫小鼠可减小小鼠皮下接种P.g处形成脓肿的大小,并加快皮下脓肿的愈合;可抑制口腔感染P.g后小鼠牙周炎模型牙槽骨的吸收。结论多肽DGFPGDHYAVMISK免疫小鼠对P.g感染小鼠具有一定的保护作用;可减少小鼠牙周炎模型牙槽骨的吸收。为开发新的牙周病防治方法奠定基础。     

牙龈卟啉单胞菌脂多糖通过TXNIP/Nlrp3炎性通路对小鼠牙周膜成纤维细胞迁移的影响

摘要 目的：研究牙龈卟啉单胞菌脂多糖（Porphyromonas lipopolysaccharide,LPS-PG）对牙周膜成纤维细胞（mouse periodontal Ligament: Normal Fibroblasts,mPDLFs）增殖及迁移的影响,探讨TXNIP/Nlrp3炎性体途径在其中的作用。方法：采用不同浓度的LPS-PG刺激小鼠mPDLFs细胞不同时间,CCK-8法检测细胞增殖抑制率。然后将细胞分为对照组（培养基作用24 h）和LPS-PG组（2 M的LPS-PG作用24 h）,划痕实验检测细胞迁移,ELISA法检测白细胞介素-1β（Interleukin-1β,IL-1β）和高迁移率族蛋白B1（High mobility group protein B1,HMGB1）的水平,Western Blot检测Nod样受体蛋白3（Nod-like receptor pyrin domain3,NLRP3）、凋亡相关斑点样蛋白（Apoptosis-associated speck-like protein,ASC）、活化半胱氨酸蛋白酶（cleaved-caspase-1）和硫氧还蛋白相互作用蛋白（Thioredoxin-interacting protein,TXNIP）的表达,免疫共沉淀检测以上蛋白间的相互作用。结果：与0 M的LPS-PG相比,1 M和2 M的LPS-PG可显著增加mPDLFs的细胞增殖抑制率（P<0.05）。与LPS-PG作用0 h相比,LPS-PG作用12 h、24 h和48 h均可显著增加mPDLFs的细胞增殖抑制率（P<0.05）。LPS-PG组细胞向中间''伤口''迁移的距离及细胞数量远远低于对照组。与对照组相比,LPS-PG组细胞中IL-1β和HMGB1的水平、NLRP3、cleaved-caspase-1和TXNIP蛋白表达均显著增加,且NLRP3和ASC、NLRP3和cleaved-caspase-1、TXNIP和NLRP3的蛋白互作能力显著增强（P<0.05）,而两组ASC蛋白的表达无显著差异（P＞0.05）。结论：LPS-PG可抑制mPDLFs细胞的增殖和迁移,其机制与Nlrp3炎性体的形成与活化有密切的关系。     

20种中药提取物对牙龈卟啉单胞菌抑制作用的体外研究

目的筛选评价20种中药提取物对牙龈卟啉单胞菌抑制作用。方法选择20种有较强抑菌作用及清热解毒作用的中药提取物,采用杯碟法进行体外抑菌试验,比较各中药对牙龈卟啉单胞菌的抑菌环直径和最低抑菌浓度(MIC)。结果水煎剂中黄连、乌梅、五味子、五倍子和黄芩的MIC为0.0488~1.5625mg/mL。油剂中丁香油、香薷油的MIC为0.3906~1.5625mg/mL。结论本实验选择的20种中药提取物中香薷油、黄连、乌梅、五味子、五倍子和黄芩的抑菌敏感度最高。     

牙龈卟啉单胞菌诱导T淋巴细胞活化、凋亡的体外研究

目的检测牙龈卟啉单胞菌对人外周血中T淋巴细胞活化及凋亡的作用,并检测Fas/FasL在牙龈卟啉单胞菌(Porphyrom onas gingivalis,Pg)诱导的T淋巴细胞凋亡中的表达。方法选取10例全身及牙周组织健康受试者,分离外周血中T淋巴细胞,在有/无Pg情况下培养0~96 h,用荧光探针(Annexin V-FITC、PI、CD69)及特殊的单克隆抗体(Fas、FasL)进行标记,并进行流式细胞仪检测。结果 CD69+淋巴细胞+Pg组Annexin V+/PI-细胞百分数在各个时间点上都明显高于T淋巴细胞+Pg组(P0.01)。Fas和FasL的表达量明显上调。用抗Fas单克隆抗体阻滞Fas-FasL相互作用导致T细胞凋亡的明显减少,百分比为(20.56±2.43)%,未加抗体的为(50.41±2.68)%。但残余的细胞凋亡活动与阴性对照相比仍高。结论 Pg能够诱导人外周血中T淋巴细胞活化,并且能够通过活化促进其凋亡,Pg诱导T淋巴细胞凋亡主要通过Fas-FasL途径,并具有时间依赖性。     

兰州地区人群的牙周健康状况和牙龈卟啉单胞菌感染情况分析

目的了解兰州地区人群的牙周健康状况,检测牙周健康者及牙周疾病患者口腔唾液中牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)的阳性率,为兰州地区人群防治牙周疾病提供科学依据。方法收集942例唾液标本,采用常规厌氧培养法,挑取产黑色素菌落进行革兰染色与生化鉴定。结果兰州地区人群牙周疾病患病率为78.45%,男女性别间差异无统计学意义(P=0.655);农村人群(53.45%)高于城市人群(46.55%),差异有统计学意义(P=0.029);各年龄段差异有显著的统计学意义(P=0.001),随年龄增长患病率呈增长趋势。兰州地区人群唾液中Pg总阳性率为71.23%,牙周疾病组(82.22%)显著高于牙周健康组(27.57%),差异有显著的统计学意义(P=0.000)。结论兰州地区人群牙周健康状况与生活环境、年龄、性别有关;在不同的牙周健康状况人群中牙龈卟啉单胞菌的分布不同。     

Porphyromonas gingivalis lipopolysaccharide induces miR-146a without altering the production of inflammatory cytokines

Lipopolysaccharide (LPS) from Porphyromonas gingivalis, an oral Gram-negative bacterium, acts as a virulence factor for periodontal disease. Although P. gingivalis LPS does not induce proinflammatory cytokines as strongly as Escherichia coli LPS, it is still able to exploit negative Toll-like receptor (TLR) regulatory pathways and facilitate pathogen persistence. Recent reports suggest that microRNAs (miRNAs) are also involved in the regulation of TLR signaling. Here, we demonstrate that P. gingivalis LPS strongly induces miRNA-146a expression in THP-1 cells and THP-1-derived macrophages. However, the inhibition or overexpression of miR-146a, through the transfection of a specific inhibitor or precursor, respectively, had little effect on cytokine production in macrophages stimulated with P. gingivalis LPS. Moreover, the expression of interleukin-1 associated-kinase-1 (IRAK-1) and tumor-necrosis factor (TNF) receptor-associated factor-6 (TRAF6), potential target molecules of miR-146a, were not affected by the stimulation with P. gingivalis LPS. Because TLR signaling induces various negative regulators, these results call into question the role of miR-146a in cells stimulated with TLR ligands.     

Effects of non-iron metalloporphyrins on growth and gene expression of Porphyromonas gingivalis

The oral anaerobic bacterium Porphyromonas gingivalis, which is implicated as an important pathogen for chronic periodontitis, requires heme for its growth. Non-iron metalloporphyrins, In-PPIX and Ga-PPIX, were examined for antibacterial effects on P. gingivalis. Both In-PPIX and Ga-PPIX caused retardation of P. gingivalis growth in a dose-dependent fashion. Microarray and qPCR analyses revealed that In-PPIX treatment upregulated the expression of several genes encoding proteins including ClpB and ClpC, which are members of the Clp (caseinolytic protease, Hsp100) family, and aRNR, aRNR-activating protein and thioredoxin reductase, whereas In-PPIX treatment had no effect on the expression of genes encoding proteins involved in heme uptake pathways, Hmu-mediated, Iht-mediated and Tlr-mediated pathways. P. gingivalis ihtA and ihtB mutants were more resistant to In-PPIX than was the wild-type parent, whereas hmuR and tlr mutants did not show such resistance to In-PPIX. The results suggest that In-PPIX is incorporated by the Iht-mediated heme uptake pathway and that it influences protein quality control and nucleotide metabolism and retards growth of P. gingivalis.     

Porphyromonas gingivalis infection and cell death in human aortic endothelial cells

Porphyromonas gingivalis is a periodontal pathogen that promotes a proatherogenic response in endothelial cells. Cell death responses of human aortic endothelial cells to P. gingivalis at various multiplicities of infection (MOI) were investigated by assessment of cell detachment, histone-associated DNA fragmentation, lactate dehydrogenase release and ADP:ATP ratio. Porphyromonas gingivalis at MOI 1:10-1:100 did not have a cytotoxic effect, but induced apoptotic cell death at MOI 1:500 and 1:1000. Monocyte chemoattractant protein-1 production was significantly enhanced by P. gingivalis at MOI 1:100. At higher MOI, at least in vitro, P. gingivalis mediates endothelial apoptosis, thereby potentially amplifying proatherogenic mechanisms in the perturbed vasculature.     

Proinflammatory gene expression in mouse ST2 cell line in response to infection by Porphyromonas gingivalis

Porphyromonas gingivalis is a predominant periodontal pathogen, whose infection causes inflammatory responses in periodontal tissue and alveolar bone resorption. Various virulence factors of this pathogen modulate host innate immune responses. It has been reported that gingipains degrade a wide variety of host cell proteins, and fimbriae are involved in bacterial adhesion to and invasion of host cells. In the present study, we profiled ST2 stromal cell gene expression following infection with the viable P. gingivalis strain ATCC33277 as well as with its gingipain- and fimbriae-deficient mutants, using microarray technology and quantitative real-time polymerase chain reaction. Using a mouse array of about 20,000 genes, we found that infection with the wild strain elicited a significant upregulation (greater than 2-fold) of expression of about 360 genes in ST2 cells, which included the chemokines CCL2, CCL5, and CXCL10, and other proinflammatory proteins such as interleukin-6 (IL-6) and matrix metalloproteinase-13 (MMP-13). Further, infection with the gingipain-deficient mutant elicited a reduced expression of the CXCL10, IL-6 and MMP-13 genes, suggesting that gingipains play an important role in inducing the expression of those genes following P. gingivalis infection. On the other hand, the pattern of global gene expression induced by the fimbriae-deficient mutant was similar to that by the wild strain. These results suggest that P. gingivalis infection induces gene expression of a wide variety of proinflammatory proteins in stromal cells/osteoblasts, and gingipains may be involved in inducing several of the proinflammatory factors.     

Porphyromonas gingivalis stimulates IL-18 secretion in human monocytic THP-1 cells

Porphyromonas gingivalis is a major pathogen implicated in chronic periodontitis. We examined whether P. gingivalis affected the secretion of the pro-inflammatory cytokine interleukin-18 (IL-18) in macrophage-like THP-1 cells and in monocytic THP-1 cells in suspension. Live P. gingivalis-induced significant IL-18 secretion. Heat-inactivation of P. gingivalis greatly reduced the IL-18 stimulation; the IL-18 levels were similar to that observed with P. gingivalis LPS alone. Live P. gingivalis caused a cytotoxic effect that was reduced greatly by heat-inactivation. Our observations indicate that P. gingivalis specifically stimulates the production and release of the active form of IL-18, which may contribute to the progression of periodontitis.     

Development of a noninvasive reporter system for gene expression in Porphyromonas gingivalis

Several reports have supported the association of Porphyromonas gingivalis with periodontal disease. Genetic studies are vital for understanding the relative importance of virulence factors in this organism. Thus, gene reporters may prove useful for the study of gene expression in this organism. We have investigated the use of the green fluorescent protein (GFP), bacterial luciferase, and bifunctional xylosidase/arabinosidase enzyme (XA) as reporters of gene expression in P. gingivalis. Fusion cassettes containing the promoterless tetracycline resistant gene [tetA(A)Q2] and the promoterless gfp, luxAB, or xa gene were placed under the control of the rgpA promoter in P. gingivalis W83 using recombinational allelic exchange. The rgpA gene encodes for an arginine-specific protease in P. gingivalis. No GFP activity was detected in P. gingivalis isogenic mutants carrying the rgpA::gfp-tetA(Q)2 fusion construct. Luciferase activity in P. gingivalis mutants carrying the rgpA::luxAB-tetA(Q)2 fusion was only detected in the presence of exogenous FMNH(2). xa gene expression in P. gingivalis with the rgpA::xa-tetA(Q)2 fusion construct was detected in crude extracts using rho-nitrophenol derivatives as substrate and on agar plates with methylumbelliferyl derivatives under long-wave ultraviolet light. This indicates that both luxAB and xa genes can be used as reporters of gene expression in P. gingivalis. However, only the xa gene can be used as a noninvasive reporter gene.     

Por secretion system-dependent secretion and glycosylation of Porphyromonas gingivalis hemin-binding protein 35

The anaerobic Gram-negative bacterium Porphyromonas gingivalis is a major pathogen in severe forms of periodontal disease and refractory periapical perodontitis. We have recently found that P. gingivalis has a novel secretion system named the Por secretion system (PorSS), which is responsible for secretion of major extracellular proteinases, Arg-gingipains (Rgps) and Lys-gingipain. These proteinases contain conserved C-terminal domains (CTDs) in their C-termini. Hemin-binding protein 35 (HBP35), which is one of the outer membrane proteins of P. gingivalis and contributes to its haem utilization, also contains a CTD, suggesting that HBP35 is translocated to the cell surface via the PorSS. In this study, immunoblot analysis of P. gingivalis mutants deficient in the PorSS or in the biosynthesis of anionic polysaccharide-lipopolysaccharide (A-LPS) revealed that HBP35 is translocated to the cell surface via the PorSS and is glycosylated with A-LPS. From deletion analysis with a GFP-CTD[HBP35] green fluorescent protein fusion, the C-terminal 22 amino acid residues of CTD[HBP35] were found to be required for cell surface translocation and glycosylation. The GFP-CTD fusion study also revealed that the CTDs of CPG70, peptidylarginine deiminase, P27 and RgpB play roles in PorSS-dependent translocation and glycosylation. However, CTD-region peptides were not found in samples of glycosylated HBP35 protein by peptide map fingerprinting analysis, and antibodies against CTD-regions peptides did not react with glycosylated HBP35 protein. These results suggest both that the CTD region functions as a recognition signal for the PorSS and that glycosylation of CTD proteins occurs after removal of the CTD region. Rabbits were used for making antisera against bacterial proteins in this study.