牙龈卟啉单胞菌菌毛FimA基因型在牙周病患者中的分布情况

目的：探讨牙龈卟啉单胞菌FimA基因型在牙周患者的分布情况。方法：采用PCR技术对40例牙周患者龈下菌斑中牙龈卟啉单胞菌及其基因型进行检测。结果：牙龈卟啉单胞菌在牙周患者的牙周袋内检出率为87．5％,牙龈卟啉单胞菌FimA各基因型在牙龈卟啉单胞菌携带者的检出率分别为：I型22．9％,Ⅱ型60．0％,Ⅲ型17．1％,Ⅳ37．1％．V型未检出;基因I型在牙周袋内未单独检出,与基因Ⅱ型混合感染。结论：牙龈卟啉单胞菌FimA基因Ⅱ型和Ⅵ型是牙龈卟啉单胞菌在牙周病损部位的主要定植菌,两基因型可能与牙周病的发生发展有关。     

牙龈卟啉单胞菌分子生物学研究进展

牙龈卟啉单胞菌已公认为是牙周炎的致病菌,它的一些表面结构诸如细胞外囊泡,菌毛,外膜蛋白,凝集素介导该菌对牙周组织粘附、定植,或作为毒性因子破坏牙周组织,随着分子生物学技术的发展,已对这些结构进行了分子克隆,本文拟就牙龈卟啉单胞菌(Porphyromonasgingivalis)简称Pg)分子生物学进展作一简要综述。1　菌毛基因的分子克隆牙龈卟啉单胞菌菌毛作为该菌表面结构之一介导了该菌对牙周组织的粘附和定植。已纯化了41kda菌毛亚单位蛋白,并克隆了菌毛蛋白基因[1]。使用寡核苷酸M1和M2作为引物,采用PCR从9株Pg菌株中扩增了1.3kb的DNA片段,E…     

牙龈卟啉单胞菌蛋白酶及疫苗的研究进展

牙龈卟啉单胞菌 (P g) ,革兰阴性厌氧菌 ,是人类牙周炎的主要致病菌[1] 。动物实验表明它在小鼠、大鼠和灵长类动物的龈下定植与牙周炎的发生和进展相关。P g可以调整真核细胞信号转导途径 ,为了满足新陈代谢的需要 ,P g基因表达的调节可以控制在转录水平。证据表明 ,P g的感染会导致严重的全身系统疾病如心血管疾病和分娩早产儿。P g含有大量毒性因子[2 ] ,如菌毛 ,血凝素[3 ] ,脂多糖等 ,其中Arg、Lys样半胱氨酸蛋白酶在牙周致病作用中占据了一个重要地位 ,他们通过激活宿主前体酶 ,例如 :血纤维蛋白酶原 ,或通过暴露细胞隐位及改变血…     

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.     

牙龈卟啉单胞菌血凝素2氯化血红素结合位点多肽对牙龈卟啉单胞菌生长抑制作用

目的探讨牙龈卟啉单胞菌血凝素2（Porphyromonas gingivalis hemagglutinin-2,PgHA-2）的氯化血红素结合位点多肽对牙龈卟啉单胞菌（Porphyromonasgingivalis,Pg）摄取氯化血红素生长的影响。方法合成多肽DHYAVMISK（肽1）,DEYAVMISK（肽2,肽1中第2位氨基酸突变为谷氨酸）,ALHPDHYLI（肽3,HA-2结合位点不相关多肽）。将肽l、肽2、肽3分别与氯化血红素琼脂糖珠预孵育,加入Pg重组血凝素2（Porphyromonas gingivalis recombinant HA-2,PgrHA-2）,收集与氯化血红素结合的PgrHA-2,SDS—PAGE电泳,分析多肽对PgrHA-2与氯化血红素结合的抑制作用。肽1、肽2、肽3与氯化血红素预孵育后,加入到CDC液体培养基中培养Pg,测定菌液A600值,分析多肽对Pg生长的抑制作用。结果肽1浓度依赖性抑制PgrHA-2与氯化血红素结合,而肽2和肽3对PgrHA-2与氯化血红素的结合无抑制作用。在24、48和72h时间点,肽1组的A600值较肽2、肽3和PBS组明显降低（P〈0．05）。结论本研究表明PgHA-2氯化血红素结合位点多肽DHYAVMISK与Pg竞争结合氯化血红素,抑制Pg的生长,为开发新的牙周病防治方法奠定基础。     

抗牙龈卟啉单胞菌血凝素2单克隆抗体的制备

目的制备抗牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)血凝素2(hemagglutinin-2,HA-2)的单克隆抗体(monoclonal antibody,mAb)。方法用重组HA-2(recombinant HA-2,rHA-2)免疫BALB/C小鼠,取其脾细胞与小鼠骨髓瘤细胞SP2/0融合,间接ELISA方法筛选杂交瘤细胞.用ELISA方法测效价。结果获得1株能够高效识别rHA-2的mAb,命名为4F11。此单克隆抗体的免疫球蛋白亚类为IgG1,效价达1?106。结论成功制备了重组牙龈卟啉单胞菌血凝素2的单克隆抗体mAb.将进一步用于牙龈卟啉单胞菌的诊断,并为牙周疾病的治疗研究奠定基础。     

牙龈卟啉单胞菌HA2基因和白细胞介素IL-15基因重组质粒的构建

构建抗牙龈卟啉单胞菌的牙周炎基因疫苗p VAX1-HA2、pVAX1-HA2/IL-15,体外检测其在293T细胞的表达。以HA2基因(牙龈卟啉单胞菌牙龈素—血凝素基因编码区的核心功能区)为目的基因与IL-15基因为免疫佐剂构建真核表达质粒,用Lip2000介导瞬时转染293T细胞,RT-PCR检测目的基因mRNA水平及酶联免疫吸附试验检测IL-15蛋白表达水平。重组质粒p VAX1-HA2、pVAX1-HA2/IL-15经酶切及DNA测序鉴定构建正确,转染的293T细胞能够检测到目的基因的表达,也可以检测到IL-15蛋白的表达。说明我们成功构建了真核共表达质粒pVAX1-HA2和p VAX1-HA2/IL-15,为下一步研制抗牙龈卟啉单胞菌DNA疫苗奠定了基础。     

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...     

牙龈卟啉单胞菌血凝素2与氯化血红素结合位点分析

目的 通过竞争性ELISA方法明确牙龈卟啉单胞菌血凝素2(Porphyromonas gingivalis hemagglutinin-2,Pg HA-2)与氯化血红素结合的多肽位点,为牙周病保护性抗体的制备奠定基础.方法 人工合成疑为Pg HA-2氯化血红素结合位点的多肽片段,制备抗Pg HA-2单克隆抗体(MAb QB),通过间接竞争性ELISA,进一步分析血红素特异性结合位点.结果 Pg HA-2氯化血红素结合位点的氨基酸序列为DGFPGDHYAVMISK.MAb QB可以抑制Pg HA-2与氯化血红素结合.结论 明确了Pg HA-2氯化血红素结合位点的氨基酸序列,确定了Pg HA-2氯化血红素结合位点的位置.为今后Pg HA-2氯化血红素结合位点的鉴定、功能结构区分析和多肽疫苗的制备奠定基础.     

牙龈卟啉单胞菌与阿尔茨海默病相关性致病机制

牙龈卟啉单胞菌(Porphyromonas gingivalis)作为牙周炎的关键致病细菌之一,与阿尔茨海默病(Alzheimer’s disease, AD)的发生发展密切相关。P.gingivalis通过分泌多种毒力因子,如脂多糖、牙龈蛋白酶及外膜囊泡,促进了AD的发展。这些毒力因子能够引发机体的炎症反应、β-淀粉样蛋白的沉积、Tau蛋白磷酸化、神经毒性作用及血脑屏障(blood brain barrier, BBB)的损害,在AD的发病机制中有重要作用。因此本文综述了P.gingivalis与AD之间的潜在致病机制,以便为AD的预防和治疗提供新的靶点。     

Development of a simple chemically defined medium for Porphyromonas gingivalis: requirement for α-ketoglutarate

Abstract The aim of this study was the development of a simple, defined medium for the growth of laboratory and clinical isolates of Porphyromonas gingivalis . A medium was designed in which the carbon and nitrogen requirements were provided by a single protein source — bovine serum albumin. High cell yields were achieved in this medium but growth was accompanied by a heavy blackening of the cells due to the deposition of metal sulfide(s), most probably iron(II) sulfide, at the cell surface. Good growth in the absence of blackening was achieved when the iron salt in the medium was substituted with α-ketoglutarate. The resultant α-ketoglutarate/BSA medium was able to support the growth of all laboratory and clinical P. gingivalis strains examined and should prove useful in the investigation of the physiology and nutritional regulation of virulence of this organism.     

Occurrence of Porphyromonas gingivalis with Prevotella intermedia in periodontal samples

Abstract To further examine the previously suggested inverse relationship between Porphyromonas gingivalis and Prevotella intermedia in periodontal disease, 1016 samples taken from single or multiple (pooled) subgingival sites were cultured anaerobically and examined for the simultaneous occurrence of the microorganisms. P. gingivalis was isolated from 297 (29%) and Pr. intermedia from 501 (49%) samples. P. gingivalis was found as frequently with (14%) as without (15%) Pr. intermedia . The type of sampling had no effect on the occurrence of P. gingivalis with Pr. intermedia . However, female subjects harboured them in combination more frequently than male subjects. The mean proportions of P. gingivalis in the cultivable flora appeared to be lower when found with than without Pr. intermedia . Whether the detection of the combination, or P. gingivalis alone, has clinical relevance needs further clarification.     

Immunochemical characterisation and epitope mapping of a novel fimbrial protein (Pg-II fimbria) of Porphyromonas gingivalis

Abstract Mouse monoclonal antibody (mAb) Pgf-II specific for a 72-kDa major cell-surface protein (72K-CSP) derived from Porphyromonas gingivalis OMZ 409 was prepared. Immunoblotting analysis revealed that mAb Pgf-II reacted with 72K-CSP but not with 41-kDa fimbrial subunit protein (41K-fimbrilin) derived from P. gingivalis 381. Electron microscopic observation revealed that P. gingivalis OMZ 409 possessed peritrichous, thin fimbriae on their surface. Immunogold electron microscopy also demonstrated that mAb Pgf-II bound to the 72K-CSP examined with the gold particles arranged along the fibril array originating from the cell surface of the bacteria. These findings suggested that P. gingivalis 72K-CSP was identifiable as another fimbriae (termed Pg-II fimbriae) different from the fimbriae (termed Pg-I fimbriae) composed of a 41K-fimbrilin. Using multipin peptide synthesis technology, 102 sequential overlapping peptides covering the entire 514 amino-acid stretch of Pg-II fimbriae were synthesised. Seven immunodominant regions within Pg-II fimbrial protein molecule, which definitely reacted with the serum of patients with periodontal diseases, were detected.     

Heat shock protein 60 (GroEL) from Porphyromonas gingivalis: Molecular cloning and sequence analysis of its gene and purification of the recombinant protein

Abstract Porphyromonas gingivalis is associated with human periodontal disease. We cloned and sequenced the gene for heat shock protein 60 (GroEL, HSP60) from P. gingivalis FDC381. The identified clone carried a 2.6 kb DNA fragment which contained two open reading frames (ORFs) encoding a 9.6- and a 58.4-kDa protein. The translated amino acid sequence of these ORFs showed a high degree of homology with known sequences for GroES and GroEL from several bacterial species and humans. Escherichia coli carrying this clone expressed a 65-kDa protein which was recognized by anti- Mycobacterium leprae HSP60 monoclonal antibody. We purified the 65-kDa protein by DEAE-sepharose chromatography and hydroxyapatite chromatography. This protein was immunogenic and was recognized by sera from a number of patients with periodontal disease. This immunological reactivity and the existence of molecular mimicry between the P. gingivalis GroEL and other HSP homologs may indicate an important role for this molecule in periodontal lesion.     

T cell knockout mice have diminished alveolar bone loss after oral infection with Porphyromonas gingivalis

Periodontal diseases are chronic inflammatory diseases that can result in resorption of the alveolar bone of the jaw. We have developed a murine model in which alveolar bone loss is induced by oral infection with Porphyromonas gingivalis, an oral anaerobic bacterium associated with periodontal disease in humans. Here we compared a strain of immunocompetent mice (C57BL/6J) to the same strain of mice made T cell deficient by genetic deletion of the alpha chain of their T cell receptors (C57BL/6J-Tcra). T cell deficiency did not affect the ability of P. gingivalis to implant in the oral cavity. The two strains of mice had equal percentages of P. gingivalis among their cultivable anaerobes 7 weeks after infection. The same bacterial load led to much less bone resorption in the T cell deficient mice than in the immune normal mice, measured as either the number of sites with significant loss, or as the total amount of bone resorbed. T cell deficient mice lost bone at only three out of 14 measurement sites, compared with eight out of 14 sites in the wild-type mice. The total amount of bone lost was 70% less in the T cell deficient mice. T cell deficient mice had lower titers of P. gingivalis-specific IgG than the wild-type mice after oral infection did, but the same titers of specific IgA. Lower titers did not correlate with greater bone loss. Antigen-activated T lymphocytes are known to induce osteoclastogenesis; here we demonstrate that T cell deletion decreases the amount of alveolar bone loss induced by infection of the murine oral cavity.     

Selective growth inhibition of Porphyromonas gingivalis by bestatin

Abstract Recent work in our laboratory indicates that selected protease/peptidase inhibitors interfere with the growth of Porphyromonas gingivalis . The aim of the present study was to further investigate the inhibitory effect of bestatin on the growth of P. gingivalis . Complete growth inhibition of P. gingivalis (11 strains) was observed when bestatin was incorporated at 2.5 μg ml⁻¹ in a complex broth medium. Fifty percent inhibition was still obtained with bestatin at a final concentration of 0.5 μg ml⁻¹. The inhibitory effect of bestatin was highly specific as the growth of 20 different oral bacterial species, including Gram-positive and Gram-negative as well as saccharolytic and asacharolytic bacteria, was not affected even at bestatin concentrations up to 50 μg ml⁻¹. Bestatin did not significantly affect the viability of P. gingivalis indicating that it has a bacteriostatic rather than a bactericidal effect. Growth assays using other specific inhibitors suggested that the effect of bestatin on the growth of P. gingivalis was unlikely to be related to its aminopeptidase inhibitor activity. Cultivation of P. gingivalis with a subinhibitory concentration of bestatin did not modify the cell envelope protein profile, as determined by SDS-PAGE analysis, but significantly decreased the number of extracellular vesicles produced. The present study indicated that bestasin is a highly effective inhibitor of cell growth of P. gingivalis . Additional studies will indicate whether bestatin should be considered as a potential drug in the control of P. gingivalis , a suspected pathogen in adult chronic periodontitis.     

Porphyromonas gingivalis fimbriae and their synthetic peptides induce proinflammatory cytokines in human peripheral blood monocyte cultures

Abstract Porphyromonas gingivalis fimbriae as well as synthetic peptides that mimic the fimbrial subunit protein, which includes the amino acid sequence XLTXXLTXXNXX, induced high production of proinflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-8, tumor necrosis factor-α in human peripheral blood monocyte/macrophage cultures. Responses induced by some peptide segments were comparable to those induced by Escherichia coli lipopolysaccharides. A chemically modified peptide analogous to an active peptide segment was found to be antagonistic with regard to interleukin-6 production induced by the native fimbriae. It may be suggested that P. gingivalis fimbriae and their degraded peptides function as proinflammatory agents in vivo, while certain analog peptides inhibited the process.     

Immunological characterization and localization of a Porphyromonas gingivalis BApNA-hydrolyzing protease possessing hemagglutinating activity

Abstract A monoclonal antibody (mAb-PC) was produced against a BA p NA-hydrolyzing protease possessing hemagglutinating activity (Pase-C) from Porphyromonas gingivalis . Other P. gingivalis BA p NA-hydrolyzing enzymes (Pase-B and Pase-S) did not react with this antibody. By ELISA or SDS-PAGE and Western immunoblotting analysis, mAb-PC recognized all P. gingivalis and P. endodontalis strains tested but did not recognize other members of the Porphyromonas genus nor other putative periodontopathogenic organisms. Pase-C, extracellular vesicles (ECV) and human strains of P. gingivalis showed two major immunoreactive bands (44 kDa and 40 kDa), whereas a different pattern was obtained with animal strains of P. gingivalis . Biotinylarginyl chloromethane, an irreversible inhibitor of trypsin-like proteases, did not affect the reactivity of Pase-C with mAb-PC on immunoblot. By reversed-phase electronmicroscopy following immunogold labeling, the antibody was shown to bind to the cell surface of P. gingivalis . mAb-PC inhibited the hemagglutinating activity of both P. gingivalis cells and ECV whereas a monoclonal antibody against LPS of P. gingivalis did not. These results suggest that Pase-C is located on the cell surface of P. gingivalis and may participate in erythrocyte binding.     

Flowcell culture of Porphyromonas gingivalis biofilms under anaerobic conditions

We have developed an anaerobic biofilm culture system. The system is inexpensive, simple to use and, unlike an anaerobic glovebox, requires no dedicated space. As a test of the system, Porphyromonas gingivalis was cultured under low oxygen (1–2 ppm) and under anaerobic conditions (≤0.1 ppm O₂). In the presence of small amounts of oxygen, the organism attached and formed an initial biofilm over the course of 4 h, but the biofilm was unable to maintain its growth and had lost biomass after 18 h. Also, ambiguous results were obtained when the biofilm was stained with a viability stain. Under anaerobic conditions, the biofilm was able to continue growth — biomass was greater after 18 h than after 4 h, and the anaerobic biofilm had a less ambiguous staining pattern than did the low-O₂-grown biofilm.     

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。