Fimbriae of Porphyromonas gingivalis induce opsonic antibodies that significantly enhance phagocytosis and killing by human polymorphonuclear leukocytes

Porphyromonas gingivalis has been strongly implicated in the pathogenesis of human periodontitis. Fimbriae mediate adherence and colonization of the oral cavity by this organism and may, therefore, have potential for use as antigen in an anti-P. gingivalis vaccine. The purpose of our study was to determine whether P. gingivalis fimbriae have opsonic target sites and whether they are accessible on the cell surfaces and cross-reactive among P. gingivalis fimbrial types and serotypes. Rabbits were immunized with a vaccine. The antiserum reacted with a 43-kDa fimbrillin monomer and a 43-kDa component in whole-cell sonicates of P. gingivalis 33277, but it showed only very weak reactivity in the 43-kDa region of Western blots of a whole-cell sonicate of strain DPG3, a mutant that does not express functional fimbriae. The antibody enhanced chemiluminescence approximately six-fold relative to preimmune serum values and significantly enhanced phagocytosis and killing of P. gingivalis 33277 by human polymorphonuclear leukocytes. Peak opsonic activity was observed at week 6 followed by a plateau that remained until week 16. The fimbria-deficient mutant DPG3 did not bind antifimbrial antibody and was not opsonized, whereas strain 381, the parent of the mutant, was opsonized. The specific antibody bound to and opsonized P. gingivalis strains 33277 and 381 (fimbria type I) but not W50, A7A-1-28, 9-14K-1 or FAY-19M-1 (fimbrial types II-V). Specific antibody bound to strain 2561 (fimbrial type I) but, as assessed by chemiluminescence, did not opsonize it. While fimbriae have opsonic target sites that are accessible on P. gingivalis cell surfaces, the relevant opsonic target sites do not appear to be shared across serotypes or fimbrial types. Thus, a vaccine containing, as antigen, fimbrial protein from a single P. gingivalis strain would likely be ineffective against infections by P. gingivalis strains expressing other fimbrial types.     

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。     

Toxicity of sonicated extracts of Bacteroides gingivalis on human pulpal cells and L929 cells in vitro.

Human pulpal fibroblasts and L929 cells were treated with sonicated extracts of two strains of Bacteroides gingivalis (W83 and ATCC 33277). The cell reaction was evaluated by monitoring cell growth and DNA synthesis. Light and scanning electron microscopic analysis were used to evaluate morphological changes of the cells. Extracts from both bacterial strains exerted a growth inhibitory effect on the cells. The pulpal cells were more sensitive than L929 cells. The ATCC 33277 strain of B. gingivalis was more cytotoxic than the W83 strain. Pulpal cells appeared to be markedly affected on the microscopic level. The diffusion of these toxic bacterial by-products, through dentin to the pulp, may account for pulpal cell damage that contributes to the initiation of pulpal pathosis.     

Characterizing the specific coaggregation between Actinobacillus actinomycetemcomitans serotype c strains and Porphyromonas gingivalis ATCC 33277

A visual coaggregation study showed specific interspecies coaggregation between an Actinobacillus actinomycetemcomitans serotype c strain and Porphyromonas gingivalis strains ATCC 33277 and 381. We mutagenized A. actinomycetemcomitans SUNYaB 67 (serotype c) with transposon IS903phikan and isolated three transposon insertion mutants that had a reduced ability to aggregate with P. gingivalis ATCC 33277. The three transposon insertions in the mutant strains mapped to the genes at ORF12, ORF13 and ORF16 of the gene cluster responsible for producing serotype c-specific polysaccharide antigen (SPA). Western blot analysis with serotype c-specific antibody showed that these strains did not produce the high-molecular-mass smear of SPA. Furthermore, two SPA-deficient mutants and an SPA-producing mutant were constructed. The two SPA-deficient mutants were deficient for ORF12 and ORF14, which are necessary for the synthesis of serotype c-SPA, and the SPA-producing mutant was deficient for ORF17, which is not related to SPA synthesis. The ORF12- and ORF14-deficient mutants showed reduced ability to aggregate with P. gingivalis ATCC 33277, while the ORF17-deficient mutant aggregated with ATCC 33277 to the same extent as wild-type SUNYaB 67. Our findings suggest that serotype c-SPA of A. actinomycetemcomitans mediates coaggregation with P. gingivalis ATCC 33277.     

Effect of host responses on the pathogenicity of strains of Porphyromonas gingivalis

Porphyromonas gingivalis is implicated in the etiology of periodontitis. Strains of P. gingivalis have been classified as invasive or noninvasive based on their ability to form abscesses in a mouse model. The purpose of this study was to investigate the ability of P. gingivalis strains to cause abscesses and periodontal bone loss in an experimental rat model and the effect of serum and salivary responses on the pathogenicity of these strains. Subcutaneous injection of animals with P. gingivalis 33277, A7A1-28, W50 or 381 resulted in abscesses in a higher percentage of mice than rats. P. gingivalis 33277 caused lesions at the site of injection, whereas strains A7A1-28 and W50 induced abscesses at distant sites in both mice and rats. Local lesions were seen in rats injected with strain 381, whereas lesions formed distant from the site of injection in mice. When periodontal bone loss was assessed in the experimental rat model, animals challenged with 33277 had the highest amount of horizontal and vertical bone loss. Rats challenged with strain A7A1-28, W50 or 381 had some or no periodontal bone loss compared with controls. Assessment of antibody responses to P. gingivalis in these animals revealed that rats challenged with 33277 had lower levels of serum immunoglobulin G (IgG) and especially salivary IgA antibody activity than A7A1-28-challenged rats. Serum IgG and in particular salivary IgA anti- P. gingivalis responses were seen in W50- and 381-challenged rats. These results indicate that the ability of P. gingivalis strains to cause abscesses does not relate directly to their periodontal pathogenicity as assessed by periodontal bone loss in the same animal model. The results further suggest the importance of salivary IgA antibody responses in protection against experimental periodontal bone loss after challenge with P. gingivalis.     

Dense fimbrial meshwork enhances Porphyromonas gingivalis adhesiveness: a scanning electron microscopic study

BACKGROUND AND OBJECTIVE: The aim of this study was to determine how the fimbriae of Porphyromonas gingivalis function in plaque formation. MATERIAL AND METHODS: We used scanning electron microscopy to examine aggregates and hemaggregates of fimbria-rich ATCC33277 (parent) and fimbra-poor OZ6301C (pgmA-knockout, mutant) strains of P. gingivalis. We also assessed the hemagglutination activity of the two strains as an indicator of P. gingivalis adhesiveness. RESULTS: Aggregates of P. gingivalis were composed of bacterial chains and clusters. Rich fimbriae projecting from cells of the parent strain tended to bunch and form a dense meshwork among bacterial cells. In contrast, cells of the mutant strain projected fewer fimbriae and the meshwork was looser. Hemaggregates including cells of the parent strain contained a detached, dense fimbrial meshwork that adhered to erythrocytes. Hemaggregates comprising cells of the mutant strain included bacterial chains and clusters that adhered to erythrocytes by shorter fimbriae than those of the parent strain. The hemagglutination titer of the parent strain was 10-fold higher than that of the mutant strain, although the number of fimbriae per cell of the parent strain was only double that of the mutant strain. CONCLUSION: The results indicate that P. gingivalis adhesiveness is prominently enhanced by the dense fimbrial meshwork. Thus, the virulence of P. gingivalis is increased by the presence of rich fimbriae.     

Interaction of Porphyromonas gingivalis with KB cells: comparison of different clinical isolates

The ability of different Porphyromonas gingivalis strains (15 clinical isolates and ATCC 33277) to attach to and invade KB cells, in relation to other properties such as release of interleukin (IL)-6 and IL-8, cytotoxicity, proteolytic activity and types of fimbriae genes present, was examined. A hierarchical cluster analysis based on adherence and internalization resulted in four groups. Eight of the 15 clinical isolates belonged to a cluster group whose adherence and internalization were about 10% those of the ATCC strain. A negative correlation between lysine-specific protease activity and adherence was found. In all cases the released concentrations of IL-6 and IL-8 were very low. Only one strain was found to be cytotoxic to KB cells. Principal components analysis demonstrated correlations between adherence, internalization and autoaggregation. Most strains had fimA type I and II, type I being associated with elastase-like activity. The ability of P. gingivalis to invade epithelial cells may be a key factor for maintaining periodontal disease.     

Immunodominant antigens of Porphyromonas gingivalis in patients with rapidly progressive periodontitis

We studied 4 isolates of Porphorymonas gingivalis , ATCC 33277, 381, A7A1-28, and W50, to identify major cell surface antigens and select the best strain from which to obtain antigen for a test vaccine. Immunoglobulin G (IgG) titers measured by enzyme-linked immunosorbent assay using whole-cell sonicates as antigen were significantly elevated for the sera of 64 rapidly progressive periodontitis patients relative to sera of 30 normal control subjects for each of the 4 strains studied. Western blots were prepared for all 4 strains and developed using sera from 22 patients and 20 control subjects to identify and determine the frequency of antibody-binding components. The intensity of binding by patient sera was greatest for the 75-kDa and 55-kDa components. The 43-kDa component was also widely recognized. Strains ATCC 33277 and 381 appeared to be antigenically similar. Because of the higher serum antibody titers, the larger proportion of seropositive patients and higher frequency of binding to specific protein components in Western blots, our efforts were focused on strain ATCC 33277. Whole-cell sonicates, proteinase K-digested sonicate, lipopolysaccharide, capsular polysaccharide, and whole-cell protein fractions were prepared and evaluated for anti-genie activity. By dot immunoblot, most of the antibody binding activity was found in the whole-cell protein fraction, with much lesser amounts in lipopolysaccharide and none in capsular polysaccharide. The antibody-binding activity was accessible on the cell surface, since 98.9% of P. gingivalis -specific antibody, including antibody binding to the 43-kDa, 55-kDa and 75-kDa components on Western blot, was removed by whole-cell adsorption. Furthermore, the 43-kDa and 55-kDa but not the 75-kDa component on intact cells were accessible for labeling with 125_I, confirming their cell surface location and accessibility.     

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。     

Serological studies of Porphyromonas (Bacteroides) gingivalis and correlation with enzyme activity

Porphyromonas gingivalis from the human oral cavity was serologically characterized using absorbed and unabsorbed rabbit antisera. The reference strains were ATCC 33277, W50, W83, 381 and hara 1. The 432 isolates were from periodontal pockets of 63 patients with adult periodontitis. Using sonicated antigens, four serotypes were identified by immunodiffusion tests and immunoelectrophoresis. Each patient harbored only one serotype of P. gingivalis , and serotypes I and IV predominated. The incidence of serotype I was four times higher than that of serotype II, and approximately seven times higher than that of serotype III. The collagenolytic and some proteolytic enzymes of representatives of each serotype were assessed. Although all strains produced these enzymes to some degree, some differences in their levels were observed. Serotype II strains were more collagenolytic than serotypes I or III, and serotype III exhibited lower activities of N-CBz-glycyl-glycyl-arginyl peptidase than other serotypes. Antibiotic sensitivity was also compared with antimicrobial disks, and serotype IV strains exhibited high sensitivity to the four antibiotics used.     

Comparative studies of the outer membranes of Bacteroides gingivalis, strains ATCC 33277, W50, W83,381

Outer membrane fractions from Bacteroides gingivalis strains ATCC 33277, 381, W50, and W83 were isolated by French pressure cell disruption and the distribution of major and minor proteins was determined by SDS-PAGE electrophoresis after treatment with 2% Sarkosyl and 2% Triton X-100. Heat-modifiability of the outer membrane proteins (OMPs) from these B. gingivalis strains was also determined after treatment at 100 degrees C and analysis by both 1- and 2-dimensional SDS-PAGE. The distribution of the OMPs on the surface of these B. gingivalis strains was determined by 125I labelling. For the most part of the OMPs of B. gingivalis presented a complex distribution, with OMPs observed between 123 kD and 13 kD. While the distribution of the OMPs was strain specific, OMPs common to all of the strains were observed. Two percent Sarkosyl treatment of the OMs at room temperature resulted in the solubilization of approximately 60% of the OMP. The Sarkosyl-insoluble MOMPs had relative molecular weights between 110 kD and 20 kD. Many of the OMPs which were separated at room temperature were heat-modified at either 65 degrees C or 100 degrees C. Heating of the OMs at 100 degrees C resulted in the heat modification of the majority of those OMPs observed at room temperature. Sarkosyl-100 degrees C OMs displayed MOMPs at apparent molecular weights between 90 kD and 15 kD. Radioiodination of the B. gingivalis strains ATCC 33277, 381, W83 and W50 revealed between 7 and 14 MOMPs at the cell surface depending upon the strain. The complexity of the OM of these B. gingivalis strains indicated the possibility of identifying and separating those OMPs involved in a variety of biological functions, including virulence, transport, and cell interaction.     

牙龈卟啉单胞菌感染MG63细胞对细胞周期的影响

目的:通过体外建立牙龈卟啉单胞菌(P. gingivalis)感染MG63细胞模型,探讨P. gingivalis ATCC 33277菌株感染MG63细胞对细胞周期的影响及发生机制。方法:P. gingivalis ATCC 33277菌株感染MG63细胞,MTT法检测细胞的增殖,流式细胞仪检测细胞周期的分布,实时定量PCR和蛋白印记方法检测Cyclin D、Cyclin E的表达。结果:MOI为100∶1,P. gingivalis ATCC 33277菌株感染MG63细胞,能够抑制细胞增殖,同时导致细胞在G1期发生停滞,Cyclin D、Cyclin E基因和蛋白在24 h、48 h均有明显下降,与对照组相比有统计学差异。结论:牙龈卟啉单胞菌感染MG63细胞Cyclin D、Cyclin E基因和蛋白表达下调,抑制细胞的增殖,导致细胞停滞在G1期。     

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。     

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.     

Strong cytotoxicity to human gingival fibroblasts by Porphyromonas gingivalis ATCC 33277

The aim of the present study was to analyze the cytotoxicity of some bacterial species associated with periodontal diseases. The specificity of cytotoxicity was estimated against cells of various origin and from different individuals. The reference bacteria were Actinobacillus actinomycetemcoinitans, Porphyromonas gingivalis and Fusobacterium nucleatum . These bacteria were cultured for 24 h in liquid media and the supernatants were used in cytotoxicity assays. The target cells used were human gingival fibroblasts (GF). dermal fibroblasts (K4), gingival epithelial cells (E) and HeLa-cells (HeLa). These cells were exposed at 4 h or 24 h, respectively, to various concentrations of culture supernatants from the selected bacteria. The influence on the viability and metabolism of the cells were estimated quantitatively as increase in neutral red uptake and lactic acid production. Growth medium supernatants of P. gingivalis 33277 were strongly cytotoxic to gingival fibroblasts after 24 h incubation, compared to supernatants of P. gingivalis 381 or W 50, A. actinomycetemcoinitans or F. nucleatum cultures. The toxic effect of P. gingivalis 33277 decreased drastically after heat inactivation, which indicates effects of proteins. By adding anti-sera the cytotoxicity of P. gingivalis 33277 could be dose dependently neutralized, which was not the case when supernatants of A. actinomycetemcoinitans was tested. Target cells of epithelial origin did not show increased cytotoxicity to P. gingivalis 33277 . The results of the present study strengthen the hypothesis that P. gingivalis remains as a suspect causative key component in periodontal diseases.     

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.     

Cytokine production induced by a 67-kDa fimbrial protein from Porphyromonas gingivalis

Fimbriae have been reported to play an important role in the adherence of Porphyromonas gingivalis to oral surfaces and possibly in triggering host responses. P. gingivalis ATCC 33277 has two distinctly different fimbriae expressed on the cell surface. The 67-kDa fimbriae differ in size and antigenicity from the earlier reported FimA, a major 41-kDa fimbrial component of P. gingivalis. Expression of the 67-kDa fimbriae on the cell surface of a fimA mutant was investigated by electron microscopy. The 67-kDa fimbrial protein was purified from the fimA mutant by sonication, precipitation, and chromatography on a DEAE Sepharose CL-6B column. The N-terminal amino acid sequence of the 67-kDa fimbrillin was distinct from that of the 41-kDa fimbrillin. Moreover, we have found that the 67-kDa fimbrial protein from P. gingivalis ATCC 33277 induced IL-1alpha, IL-beta, IL-6 and TNFalpha cytokine expression in mouse peritoneal macrophages. These results suggest that P. gingivalis 67-kDa fimbriae may play a part in the inflammatory response during the development of periodontal diseases.     

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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.     

Anti-phagocytic role of surface fibrous structure of an invasive Porphyromonas gingivalis strain

Recent studies have shown that invasive and non-invasive strains of Porphyromonas gingivalis can both be isolated from patients with periodontitis. We examined the interaction between an invasive 16-1 P. gingivalis strain and phagocytes obtained from human peripheral blood and guinea pig peritoneal cavity. Phagocytes from human peripheral blood, mainly polymorphonuclear leukocytes (PMNs) isolated by centrifugation in Ficoll Hypaque, and macrophages collected from the peritoneal cavity of guinea pigs, were exposed to P. gingivalis cells. After this exposure, greater numbers of the non-invasive P. gingivalis ATCC 33277 were observed in human PMNs and guinea pig macrophages compared with the invasive P. gingivalis 16-1. Electron microscopic observations showed that invasive 16-1 within phagosomes in human PMNs and guinea pig macrophages retained their surface fibrous structures as well as their outer membranes. Electron microscopic examination showed that destruction and damage to the cell membranes and inner structures were clear in human PMNs and guinea pig macrophages after exposure to invasive 16-1 for 6 and 24 hours; this was a clear difference from exposure to the non-invasive ATCC 33277. Release of lactate dehydrogenase (LDH) activities into the culture supernatant of PMNs after exposure to the invasive 16-1 for 4 and 6 hours was significantly greater than that after exposure to the non-invasive ATCC 33277 (p<0.05). On the other hand, the LDH activity after exposure for 21 hours to the invasive 16-1 was significantly lower than that of untreated cells and cells after exposure to the non-invasive ATCC 33277 strain (p<0.05). The PMN viabilities after exposure to cells of the invasive 16-1 for 3, 4, and 6 hours as evaluated by trypan blue staining were similar to those after exposure to cells of the non-invasive ATCC 33277, but that after exposure to the invasive 16-1 strain for 21 hours was significantly lower than that after exposure to cells of the non-invasive ATCC 33277 strain.