细菌膜泡（ECV）,外膜蛋白复合物（OMPC）的氨基酸组成分析

采用酸水解法比较分析了ＰｇＷ５０、Ｐｇ３８１的ＥＣＶ、ＯＭＰＣ中１７种氨基酸的不同含量，结果显示：ＥＣＶ的蛋白结构基本上与同菌的ＯＭＰＣ相近，而在某些氨基酸浓度（ｎｍｏｌ／Ｌ）存在差异，说明在ＥＣＶ形成过程中其外膜可能发生某些生化改变；不同菌的ＯＭＰＣ之间、ＥＣＶ之间在氨基酸含量上也存在着差异，表明不同种的细菌，其ＯＭＰＣ在结构上各有差异，相应形成的ＥＣＶ都会发生某些特殊的变化；另外，天冬氨酸、甘氨酸可能也是组成Ｐｇ、Ｐｅ细菌细胞壁的重要的氨基酸成分。     

细菌膜泡、外膜蛋白复合物的十二烷基磺酸钠－聚丙烯凝胶电泳分析

本实验用连续测定法观察了细菌的生长率的变化，结果表明：三株卟啉菌在３ｄ的生长率基本相似，均以指数方式生长，其中ＰｇＷ５０在以后的几天中仍基本保持平衡，Ｐｇ３８１在５～６ｄ后开始呈下降趋势，而Ｐｅ在３～４ｄ到达顶点后，即开始呈下降趋势。ＳＤＳ－ＰＡＧＥ显示：ＥＣＶ的泳带要比同菌ＯＭＰＣ的泳带多，且大多集中在２０ＫＤ到１００ＫＤ之间．各带的含量（峰高）亦不相同。     

细菌胞外膜泡的电镜观察

本实验对分离的５株细菌的ＥＣＶ作了电镜观察，证实４株菌都能形成ＥＣＶ，但在量上有差异。以ＰｇＷ５０最为典型，菌体及其ＥＣＶ的结构层次清晰可见；ＰｅＥＣＶ的荚膜层较薄且直径相对小而均一；Ｐｍ和Ｐｉ的ＥＣＶ大小不均一，标本中破裂现象和不规碎片出现较早。     

牙龈卟啉菌（Pg）的胰酶样蛋白酶（TLP）检测

对Ｐｇ的ＥＣＶ和ＯＭＰＣ的ＴＬＰ检测结果显示：ＥＣＶ中的酶（ＴＬＰ）和蛋白含量均高于ＯＭＰＣ，ＥＣＶ中的ＴＬＰ活性在细菌生长早期随细菌的快速增长而升高，与细菌的生长基本同步，说明：（１）ＴＬＰ的酶合成是发生在细菌生长早期；（２）ＥＣＶ的形成是生长期细菌的一个特征，而不是老化的细菌或者非生长期细菌的代谢产物。     

8—甲氧基补骨脂素对粘液表皮样癌细胞的抑制作用

目的：研究８－甲氧基补骨脂素（８－ＭＯＰ）对粘液表皮样癌细胞（ＭＥＣ－１）的抑制作用。方法：应用常规ＭＴＴ法、细胞计数法和流式细胞术研究８－ＭＯＰ作用于粘液表皮样癌细胞的剂量－效应关系和时间－效应关系以及３０％抑制浓度的药物对细胞群体倍增时间和细胞周期的影响。结果：１）小剂量８－ＭＯＰ在短时间内对ＭＥＣ－１细胞没有抑制作用，当剂量大于２５ｍｇ／Ｌ时，药物的抑制作用呈剂量依赖性；作用时间在２４ｈ～７２ｈ之间时表现出时间依赖性关系；药物的抑制作用有延迟性。２）１２０ｈ作用组３０％抑制浓度ＩＣ３０＝３３．８６ｍｇ／Ｌ，５０％抑制浓度ＩＣ５０＝４５．３ｍｇ／Ｌ，ＲＡＡ值＝２．９。３）它阻止细胞进入Ｓ期完成细胞分裂。结论：ＭＥＣ－１细胞对８－ＭＯＰ较敏感，８－ＭＯＰ在人粘液表皮样癌的防治方面可能有一定意义。     

60例腭部小涎腺癌临床分析

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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.     

Inhibitory effect of cranberry polyphenol on biofilm formation and cysteine proteases of Porphyromonas gingivalis

BACKGROUND AND OBJECTIVE: The purpose of this study was to investigate the effects of cranberry polyphenol fraction on biofilm formation and activities of Arg-gingipain and Lys-gingipain in Porphyromonas gingivalis. MATERIAL AND METHODS: The polyphenol fraction was prepared by using a glass column packed with Amberlite XAD 7HP and 70% aqueous ethanol as an elution solvent. RESULTS: Synergistic biofilm formation by P. gingivalis and Fusobacterium nucleatum was significantly inhibited by the polyphenol fraction at a concentration of 250 microg/mL compared with untreated controls (p < 0.01). Arg-gingipain and Lys-gingipain activities in P. gingivalis ATCC 33277 and FDC 381 were inhibited significantly at a polyphenol fraction concentration of > or = 1 microg/mL (p < 0.05). CONCLUSION: These findings indicate that the polyphenol fraction inhibits biofilm formation and the Arg-gingipain and Lys-gingipain activities of P. gingivalis.     

Effects of chlorhexidine digluconate and hydrogen peroxide on Porphyromonas gingivalis hemin binding and coaggregation with oral streptococci

Porphyromonas gingivalis, a gram-negative anaerobe, is one of the major causative agents of periodontal disease. In this study, the effects of chlorhexidine digluconate and hydrogen peroxide on the hemin binding of P. gingivalis and coaggregation of this bacterium with oral streptococci were examined. The pretreatment of P. gingivalis W50 and 381 with chlorhexidine digluconate and hydrogen peroxide increased the hemin binding of these bacteria. The hemin binding of P. gingivalis was increased by the subminimal inhibitory concentration (MIC) of chlorhexidine digluconate. However, concentrations of hydrogen peroxide below the MIC had no effect on the hemin binding of P. gingivalis W50 and 381. Coaggregation of P. gingivalis 381 with Streptococcus oralis ATCC 9811 and Streptococcus gordonii DL1 was diminished by chlorhexidine digluconate. The coaggregation-inhibitory effect was concentration-dependent. Hydrogen peroxide also showed inhibitory effects on the coaggregation of P. gingivalis 381 with S. oralis 9811 and S. gordonii DL1 at concentrations below that used clinically. Concentrations of chlorhexidine digluconate below the MIC inhibited coaggregation. However, concentrations of hydrogen peroxide below the MIC were not effective in reducing the coaggregation of P. gingivalis with oral streptococci. These observations show that chlorhexidine digluconate and hydrogen peroxide could confer variable effects on P. gingivalis hemin binding and coaggregation of this bacterium with oral streptococci.     

Porphyromonas gingivalis invades oral epithelial cells in vitro

The aim of the present study was to analyze the adhesive and invasive potential of a number of P. gingivalis strains, in an in vitro system utilizing cultures of human oral epithelial cells (KB cell line, ATCC CCL 17). P. gingivalis strains W50 and FDC 381 (laboratory strains) and OMGS 1738, 1743 and 1439 (clinical isolates) as well as E. coli strain HB 101 (non-adhering, non-invasive control) were used. Adherence was assessed by means of scintillation counting and light microscopy, after incubation of radiolabelled bacteria with epithelial cells. In the invasion assay, monolayers were infected with the P. gingivalis and E. coli strains and further incubated with an antibiotic mixture (metronidazole 0.1 mg/ml and gentamicin 0.5 mg/ml). Invasion was evaluated by (i) assessing presence of bacteria surviving the antibiotic treatment, and (ii) electron microscopy. All P. gingivalis strains adhered to and entered into the oral epithelial cells. After 3 hours of incubation, bacteria were frequently identified intracellularly by means of electron microscopy. The cellular membranes, encapsulating the microorganisms in early stages of the invasive process, appeared later to disintegrate. The presence of coated pits on the epithelial cell surfaces suggested that internalization of P. gingivalis was associated with receptor-mediated endocytosis (RME). Formation of outer membrane vesicles (blebs) by intracellular bacteria indicated that internalized P. gingivalis was able to retain its viability. E. coli strain HB 101 neither adhered to nor invaded epithelial cells.     

应用表面增强激光解吸电离飞行时间质谱技术筛选牙龈卟啉单胞菌共同外膜蛋白

目的 应用表面增强激光解吸电离飞行时间质谱(surface enhanced laser desorption/ionization time-of-flight mass spectrometry,SELDI-TOF-MS)技术筛选多种牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)共同外膜蛋白(outer membrane protein,OMP),为针对多种Pg设计具有交叉保护作用的疫苗提供候选靶抗原.方法 应用超速离心法提取PgATCC33277、Pgw83、Pg301和Pg381菌株外膜蛋白,SELDI疏水性蛋白芯片H50检测OMP,Biomarker Wizard软件分析4种菌株OMP质谱图.结果 OMP质谱图显示,PgATCC33277、PgW83、Pg301和Pg381分别有71、74、76和72个蛋白质峰,并存在13种共同OMP;在美国国立生物信息中心蛋白库中鉴定到一种已知蛋白gil116636495,其功能尚不清楚.结论 发现13种共同OMP可作为牙周病疫苗候选抗原,证实基于SELDI-TOF-MS技术适合检测小相对分子质量(＜20000)、低丰度、疏水性蛋白,且操作简单、重复性好,可用于大规模寻找Pg的共同OMP.     

Strain-dependent activation of the mouse immune response is correlated with Porphyromonas gingivalis-induced experimental periodontitis

Aims: To evaluate the effect of oral infection with three Porphyromonas gingivalis strains on alveolar bone loss (ABL) and its correlation with the mouse immune response.
Materials and Methods: Mice were orally infected with P. gingivalis strains 381, 33277 and 53977. After 42 days, maxillae were analysed for ABL using micro-computed tomography and the serum for anti- P.gingivalis IgG1 and IgG2a levels. The cytokine response to P. gingivalis was tested using the subcutaneous chamber model.
Results: The P. gingivalis 53977-infected group showed the highest ABL, which was significantly different from all other groups ( p <0.001). In addition, the humoral response to P. gingivalis 53977 was significantly lower than the response to P. gingivalis 381 and 33277 ( p 0.01). The IgG2a/IgG1 ratio was higher in the P. gingivalis 33277-infected group (1.6) compared with the P. gingivalis 381-infected group (0.51). Four days post-infection, interleukin (IL)-1 β levels remained significantly higher in the P. gingivalis 53977-infected group only (1198.2±260.0, p <0.05), while IL-4 levels remained significantly higher in the P. gingivalis 381-infected group (265.8±131.6, p <0.05).
Conclusions: The high levels of ABL induced by P. gingivalis 53977 were inversely correlated with the humoral response to this bacterium. In addition, ABL was correlated with an elevated pro-inflammatory response.     

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.     

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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.     

Clonal analysis of Porphyromonas gingivalis by the arbitrarily primed polymerase chain reaction

Genetic analysis of Porphyromonas gingivalis strains may distinguish between virulent and nonvirulent strains and also may be used to trace individual strains in epidemiological studies. The present study examined the utility of the arbitrarily primed polymerase chain reaction for genotypic fingerprinting of P. gingivalis. DNA was extracted according to conventional methods. Ten-base oligonucleotide primers with arbitrary sequences were used with the polymerase chain reaction to amplify P. gingivalis genomic DNA. The amplification products were analyzed by agarose gel electrophoresis. The primer GACCGCTTGT grouped 73 P. gingivalis strains into 23 genotypes, including 16 genotypes containing a single strain each. The primer AGGGGTCTTG identified 45 different genotypes, 33 of which contained a single strain. P. gingivalis strains ATCC 332771^T and 381 belonged to the same genotype. Likewise, strains W50 and W83 were of the same genetic clone. The present study indicates that the arbitarily primed polymerase chain reaction represents a valuable and easy method for clonal analysis of P. gingivalis.