牙龈卟啉单胞菌诱导的耐受对小鼠牙槽骨吸收的影响

目的:研究牙龈卟啉单胞菌(P.gingivalis)诱导的耐受对小鼠骨吸收相关因子白细胞介素-1β(IL-1β)、骨保护素(OPG)/NF-κB受体激活蛋白配体(RANKL)表达水平,以及牙槽骨吸收的影响。方法:采用1×10~9CFU P.gingivalis口内涂布给菌5 d,磷酸盐缓冲液(PBS)洗脱后,再次口内涂布P.gingivalis给菌2 d,构建Balb/c小鼠牙周组织耐受模型。10 d后收集新鲜牙龈组织,采用western bolt检测牙龈组织中IL-1β、OPG和RANKL表达水平的变化。6周后,收集上颌骨,采用亚甲基蓝染色法检测牙槽骨吸收水平的改变。结果:耐受组小鼠牙龈组织中IL-1β表达水平较非耐受组降低,OPG/RANKL比值较非耐受组升高。亚甲基蓝染色结果显示,耐受组小鼠釉牙骨质界至牙槽嵴顶(ABC-CEJ)的距离小于非耐受组(P<0.01),说明耐受组小鼠牙槽骨吸收较非耐受组降低。结论:P.gingivalis诱导小鼠牙周组织耐受后,小鼠牙龈组织中骨吸收相关因子IL-1β分泌降低,OPG/RANKL比值升高,抑制了牙槽骨吸收。     

牙龈卟啉单胞菌菌毛蛋白FimA基因在大肠杆菌中的融合表达和纯化

目的克隆牙龈卟啉单胞菌菌毛蛋白FimA基因,构建原核表达载体,诱导其在大肠杆菌中融合表达,并鉴定、纯化其表达产物。方法克隆牙龈卟啉单胞菌菌毛蛋白FimA基因,构建表达载体pET15b-FimA,转化大肠杆菌BL21（DE3）pLyS感受态细胞;异丙基-β-D-硫代半乳糖苷（IPTG）诱导表达融合蛋白,以抗6×His Tag单克隆抗体为一抗,Western blot鉴定、Co2+柱亲和层析纯化融合蛋白。结果克隆的FimA基因序列及插入表达载体中的FimA序列均与GenBank数据库中的序列呈现100%同源性;IPTG诱导后Western blot鉴定4.1×104处有目的蛋白表达;Co2+柱亲和层析法获得纯化的高浓度FimA蛋白。结论本实验成功构建了牙龈卟啉单胞菌菌毛蛋白FimA基因的原核表达载体pET15b-FimA,并在大肠杆菌中获得成功表达和纯化,为进一步制备牙龈卟啉单胞菌菌毛蛋白单克隆抗体和研制开发预防牙周炎的亚单位蛋白疫苗奠定了实验基础。     

牙龈卟啉单胞菌与动脉粥样硬化

大量流行病学证据已经证实牙周病和动脉粥样硬化存在一定的相关性,目前的研究主要偏向于牙周病在动脉粥样硬化病因中的作用机制,牙周病的病原体如牙龈卟啉单胞菌是否为动脉粥样硬化的病原体尚不清楚.本文对牙龈卟啉单胞菌在动脉粥样硬化发生、发展过程中所起的作用作一综述.     

牙龈卟啉单胞菌与冠心病

牙周病是引起冠心病的危险因素之一,牙龈卟啉单胞菌是引起牙周病的主要致病菌,它可以侵入血管内膜导致血管壁增厚及释放内毒素,释放细胞因子促进血小板的聚集,血栓形成,从而导致心血管疾病的发生。     

牙龈卟啉单胞菌菌毛fimA基因的克隆及表达纯化

目的克隆牙龈卟啉单胞菌菌毛fimA基因并使其在大肠杆菌中正确表达。方法利用PCR方法克隆牙龈卟啉单胞菌fimA基因,构建其原核表达质粒pT-BAD/fimA,获得其在大肠杆菌中的表达,基质辅助激光解吸电离-飞行时间质谱（MALDI-TOF-MS）鉴定重组蛋白。以不同质量浓度咪唑缓冲液（250、200、150、100、50 μmol·L-1）洗脱结合在His-tag纯化柱的目的蛋白,选择最佳洗脱质量浓度。结果克隆基因测序结果与GeneBank数据库中的序列呈现99.9%同源性;诱导表达后可观察到相对分子量3.8×104的融合蛋白,经MALDI-TOF-MS检测进一步证实为FimA蛋白。100 μmol·L-1咪唑缓冲液洗脱得到的蛋白质纯度最高,其纯度为95%。结论本实验成功克隆了fimA基因,并获得在大肠杆菌中的正确表达,同时得到纯度较高的FimA蛋白,为后续研制增强免疫应答的高效牙周炎真核表达质粒奠定了基础。     

牙龈卟啉单胞菌与免疫细胞凋亡

牙周病是口腔的常见病和多发病。大量研究证实，牙周病是由口腔微生物所引起，微生物与宿主之间的相互作用同时会影响牙周病的发展与严重程度。近年来，口腔疾病与细胞凋亡的关系的研究日益受到普遍重视，牙周病与免疫细胞凋亡的关系已成为研究热点。本文就成人牙周炎主要致病菌——牙龈卟啉单胞菌与免疫细胞凋亡的关系作一综述，意在探讨其在牙周病病因中的作用。     

牙龈卟啉单胞菌与免疫细胞凋亡

牙周病是口腔的常见病和多发病。大量研究证实,牙周病是由口腔微生物所引起,微生物与宿主之间的相互作用同时会影响牙周病的发展与严重程度。近年来,口腔疾病与细胞凋亡的关系的研究日益受到普遍重视,牙周病与免疫细胞凋亡的关系已成为研究热点。本文就成人牙周炎主要致病菌———牙龈卟啉单胞菌与免疫细胞凋亡的关系作一综述,意在探讨其在牙周病病因中的作用。     

牙龈卟啉单胞菌菌毛fimA基因在大肠杆菌的融合表达

目的：克隆牙龈卟啉单胞菌菌毛蛋白fimA基因,并使其在大肠杆菌中融合表达。方法：利用PCR方法克隆fimA,并用基因重组融合表达技术获得在大肠杆菌中的表达。结果：克隆基因测序结果与GenBank数据库中的序列呈现99．9％同源性;经诱导表达后可观察到Mr38kDa的融合蛋白。结论：成功克隆了fimA基因,并在大肠杆菌中表达了FimA蛋白,为后续研究奠定了基础。     

多聚酶链反应检测牙龈卟啉单胞菌

近年来，随着Ｐｇ菌分子生物学研究工作的深入，已有Ｐｇ菌纤毛基因得以分离并测序。本文根据Ｐｇ菌特异的纤毛亚单位旦白结构基因设计寡苷酸引物，采用ＰＣＲ技术增了其中１３１ｂｐ的片段，并对其进行特异性鉴定。     

牙龈卟啉单胞菌的分型及其致病作用

牙龈卟啉单胞菌被认为是牙周疾病最重要的致病菌之一，与多种牙周疾病特别是成年人牙周炎关系密切，本文对其生物学特性，血清分型，遗传分型的临床流行病学特点及其致病性等进行了综述。     

Heterogeneity of Porphyromonas gingivalis strains in the induction of alveolar bone loss in mice

These experiments examine alveolar bone loss in a model in which specific pathogen-free mice are exposed orally with Porphyromonas gingivalis. Alveolar bone loss was induced as a result of a specific infection with P. gingivalis, rather than other environmental antigens. Infection with live P. gingivalis was required, as significant bone loss did not follow gavage with formalin-killed P. gingivalis. The virulence of different strains of P. gingivalis was compared. Two laboratory strains of the bacteria (ATCC 53977 and W50) and a mutant strain lacking the 43-kDa fimbrillin (strain DPG3) induced bone loss. P. gingivalis 381, however, did not induce bone loss. There was a strong immunoglobulin G (IgG) antibody response to infection with each strain but a significant serum IgA response only to strain 381. These studies show that in mice with a background oral microflora bone loss is induced by a specific infection with P. gingivalis and that bacterial strain variation is important in determining whether alveolar bone loss will ensue.     

Porphyromonas gingivalis heat shock protein vaccine reduces the alveolar bone loss induced by multiple periodontopathogenic bacteria

OBJECTIVES: Heat shock protein (HSP) can be utilized as a vaccine to cross-protect against multiple pathogenic species. The present study was performed to evaluate Porphyromonas gingivalis heat shock protein 60 (HSP60) as a vaccine candidate to inhibit multiple bacteria-induced alveolar bone loss. MATERIAL AND METHODS: Recombinant P. gingivalis HSP60 was produced and purified from P. gingivalis GroEL gene. Rats were immunized with P. gingivalis HSP60, and experimental alveolar bone loss was induced by infection with multiple periodontopathogenic bacteria. RESULTS: There was a very strong inverse relationship between postimmune anti-P. gingivalis HSP immunoglobulin G (IgG) levels and the amount of alveolar bone loss induced by either P. gingivalis or multiple bacterial infection (p=0.007). Polymerase chain reaction data indicated that the vaccine successfully eradicated the multiple pathogenic species. CONCLUSIONS: We concluded that P. gingivalis HSP60 could potentially be developed as a vaccine to inhibit periodontal disease induced by multiple pathogenic bacteria.     

A tumor necrosis factor-alpha antagonist inhibits inflammatory bone resorption induced by Porphyromonas gingivalis infection in mice

BACKGROUND: A tumor-necrosis factor-alpha (TNF-alpha) antagonist, the WP9QY peptide, was designed based on the crystal structure of the TNF-beta/TNF-receptor complex in order to overcome the disadvantages of macromolecules such as antibodies or soluble receptors by reducing the molecular size of TNF-alpha antagonists. It efficiently antagonizes the effect of TNF-alpha binding to the TNF receptor (I). OBJECTIVES: The aim of the present study was to assess the effects of the WP9QY peptide on inflammatory bone resorption and osteoclast formation in the periodontal pathogen-infection model. MATERIAL AND METHODS: Live Porphyromonas gingivalis ATCC 33277 was injected once daily for 6 days into the subcutaneous tissue overlying the calvariae in mice. At the same time, the WP9QY peptide (1 mg/kg, 2 mg/kg or 4 mg/kg per day) was administrated via osmotic minipumps for 7 days. Histological observations and the radiological assessments of the calvariae as well as bone mineral density measurements were performed. RESULTS: The WP9QY peptide significantly prevented the P. gingivalis-induced reduction in the bone mineral density at the calvariae. The histomorphometric assessments revealed the inhibitiory effects of the WP9QY peptide on the P. gingivalis-induced increase in the number of the inflammatory cells and in the area of sagittal suture at the calvariae. Furthermore, there was also an inhibitory effect on the P. gingivalis-induced increase in the number of osteoclasts per unit bone surface at the calvariae. CONCLUSION: These results suggest that the strategy for the design to reduce the molecular size of the TNF-alpha antagonists would be beneficial for the treatment of local inflammatory bone loss induced by periodontal-pathogen infection.     

Porphyromonas gingivalis mutY is involved in the repair of oxidative stress-induced DNA mispairing

The ability for DNA mismatch repair, after oxidative stress‐induced DNA damage, is critical for the persistence of Porphyromonas gingivalis in the inflammatory environment of the periodontal pocket. Our previous report demonstrated that, in contrast to other organisms, the repair of oxidative stress‐induced DNA damage involving 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) may occur by a yet‐to‐be described mechanism in P. gingivalis. 8‐oxoG does not block DNA replication; rather, it mispairs with adenine, which can be repaired by the MutY glycosylase. To determine the function of the P. gingivalis MutY homologue in DNA repair, it was insertionally inactivated using the ermF‐ermAM antibiotic cassette and used to create a mutY‐deficient mutant (FLL147) by allelic exchange mutagenesis. FLL147 had an increased rate of spontaneous mutation and was more sensitive to hydrogen peroxide compared with the wild‐type W83 strain. DNA oligomers containing a site‐specific 8‐oxoG:A mispair was repaired similarly in both the P. gingivalis mutY‐defective mutant and wild‐type strains. The P. gingivalis mutY homologue was shown to complement the mutY mutation in Escherichia coli. In a gel mobility shift assay, the purified recombinant MutY is able to bind an oligo containing an 8‐oxoG:A mispair. Taken together, MutY may play the expected role in oxidative stress resistance in P. gingivalis. However, there may exist other redundant mechanism(s) for the removal of 8‐oxoG:A mismatch in this organism.     

Impact of Porphyromonas gingivalis inoculation on ligature-induced alveolar bone loss. A pilot study in rats

Meulman T, Peruzzo DC, Stipp RN, Gonçalves PF, Sallum EA, Casati MZ, Goncalves RB, Nociti FH Jr. Impact of Porphyromonas gingivalis inoculation on ligature‐induced alveolar bone loss. A pilot study in rats. J Periodont Res 2011; 46: 629–636.©2011 John Wiley & Sons A/S Background and Objective: Periodontitis is a polymicrobial infection characterized by the loss of connective tissue attachment, periodontal ligament and alveolar bone. The aim of this study was to evaluate the impact of Porphyromonas gingivalis inoculation on the ligature‐induced alveolar bone loss (ABL) model in rats. Material and Methods: Forty male Wistar rats were randomly assigned to the following groups: G1, control (n = 10); G2, ligature‐induced ABL (n = 15); and G3, ligature‐induced ABL + P. gingivalis inoculation (n = 15). Rats in G2 and G3 were killed 15, 21 and 30 d after ligature placement, and the following parameters were assessed: microbiological load; ABL; and interleukin (IL)‐1β (Il1beta)/Il1ra, Il6/Il10 and Rankl/osteoprotegerin (Opg) mRNA ratios in the gingival tissues, as determined by quantitative PCR. Results: Microbiological analyses demonstrated that rats in G1, G2 and G3 were positive for the presence of bacteria (determined using PCR amplification of the 16S gene), but that only the treatment sites of rats in G3 were positive for P. gingivalis at all time‐points investigated. Histometrically, significant bone loss (p < 0.001) was observed for both ligated groups (G2 and G3) compared with the nonligated group (G1), with higher ABL observed for G2 at all the experimental time‐points. Furthermore, gene‐expression analysis demonstrated that the presence of P. gingivalis in the dentogingival area significantly decreased the Il1β/Il1ra, Il6/Il10 and Rankl/Opg mRNA ratios compared with ligature alone. Conclusion: Within the limits of this pilot study, it was concluded that inoculation of P. gingivalis affected the ligature‐induced ABL model by the induction of an anti‐inflammatory and antiresorptive host response.     

T-cell contributions to alveolar bone loss in response to oral infection with Porphyromonas gingivalis

We have previously shown that mice lacking CD4⁺, but not CD8⁺, T cells lose less alveolar bone loss in response to oral infection with Porphyromonas gingivalis than do immunocompetent mice of the same genetic background, indicating that CD4⁺ T cells contribute to bone resorption. The CD4⁺ and CD8⁺ T-cell knockouts were produced by targeted deletions of, respectively, major histocompatibility complex II (MHCII) or β₂-microglobulin (producing non-expression of MHCI). Because MHC deletions can have other effects in addition to those on T-cell selection, we wanted to confirm that the lessened bone loss was truly an effect of the lack of T cells. Consequently, we repeated our experiments with C57B1/6J-Tcra mice that have a targeted deletion of the alpha chain of the T-cell receptor (Tcra). Six weeks after oral infection with P. gingivalis ATCC 53977 the total bone loss at buccal maxillary sites was 0.28 mm in infected immunocompetent mice (P = 0.002 compared with sham-infected mice), whereas in Tcra knockouts the bone loss was only 0.08 mm (P = 0.04 compared with shams). The T-cell-deficient mice thus lost 70% less bone after infection than did genetically matched immunocompetent mice (P = 0.003). These experiments confirm that T cells, and their responses to oral infection with P. gingivalis, help to push bone remodeling in the direction of net loss of bone.     

T-cell contributions to alveolar bone loss in response to oral infection with Porphyromonas gingivalis

We have previously shown that mice lacking CD4+, but not CD8+, T cells lose less alveolar bone loss in response to oral infection with Porphyromonas gingivalis than do immunocompetent mice of the same genetic background, indicating that CD4+ T cells contribute to bone resorption. The CD4+ and CD8+ T-cell knockouts were produced by targeted deletions of, respectively, major histocompatibility complex II (MHCII) or beta2-microglobulin (producing non-expression of MHCI). Because MHC deletions can have other effects in addition to those on T-cell selection, we wanted to confirm that the lessened bone loss was truly an effect of the lack of T cells. Consequently, we repeated our experiments with C57B1 /6J-Tcra mice that have a targeted deletion of the alpha chain of the T-cell receptor (Tcra). Six weeks after oral infection with P. gingivalis ATCC 53977 the total bone loss at buccal maxillary sites was 0.28 mm in infected immunocompetent mice (P=0.002 compared with sham-infected mice), whereas in Tcra knockouts the bone loss was only 0.08 mm (P=0.04 compared with shams). The T-cell-deficient mice thus lost 70% less bone after infection than did genetically matched immunocompetent mice (P =0.003). These experiments confirm that T cells, and their responses to oral infection with P. gingivalis, help to push bone remodeling in the direction of net loss of bone.