牙龈卟啉单胞菌毒力岛基因检出与慢性牙周炎的相关性研究

目的:检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis)毒力岛中PG0836、PG0838和PG0839基因,探讨3个基因与临床牙周指数之间的关系.方法:选取慢性牙周炎(chronic periodoiltitis,CP)患者90例,共采集龈下菌斑标本270个.记录受试位点的牙周探诊深度、临床附着丧失和探诊出血情况.设计特异性引物,检测P.gingivalis阳性龈下菌斑标本的PG0836、PG0838和PG0839基因.采用SPSS 11.0软件包进行统计学分析,采用X2检验对不同牙周临床指数基因的检出率进行比较.结果:在CP患者P.gingivalis阳性龈下菌斑中,PG0836、PG0838和PG0839基因检出率分别为66.17%、24.88%和27.86%.探诊出血阳性位点的PG0839基因检出率(28.35%)显著高于探诊出血阴性位N(14.29%,P<0.05).在牙周探诊深度为4～6mm和>6mm的受试位点,PG0839基因检出率均显著高于牙周探诊深度<4mm的位点(P<0.05).PG0836和PG0838基因在不同牙周指数组检出率无显著差异.结论:PG0839基因的检出与CP病损部位的临床指标呈正相关关系,提示该基因可能与P.gingivalis的致病性有关.     

牙龈卟啉单胞菌PG1055基因在临床菌株中的表达

目的应用基因芯片技术检测PG1055基因在不同人群的牙龈卟啉单胞菌(P.gingivalis)中分布,探讨这些基因与牙周临床指数之间的关系。方法取龈下菌斑进行细菌分离培养,以临床采集样本提取的DNA为探针,以抑制消减杂交技术获得P.gingivalisW83的特异基因片段PG1055为目标序列,采用Cy5荧光标记目标序列。应用基因芯片技术检测PG1055基因在牙周病患者及健康人群的牙龈卟啉单胞菌中的分布。结果PG1055基因在牙周病患者及健康人群中的检出率有统计学差异,并且与牙周临床指数相关。结论PG1055基因与P.gingivalis的致病性有关。     

牙龈卟啉单胞菌对慢性牙周炎致病作用研究进展

牙龈卟啉单胞菌是公认的牙周主要致病菌之一,是与慢性牙周炎密切相关的红色复合体的重要成员,在引发牙周组织破坏的过程中发挥重要作用。其中牙龈卟啉单胞菌的毒力因子及牙龈卟啉单胞菌与牙龈上皮细胞的相互作用在这一过程中发挥关键作用。本文对牙龈卟啉单胞菌在慢性牙周炎中发挥的作用及其自身在此过程中的改变做一综述。     

慢性牙周炎状态下牙龈卟啉单胞菌rag-1基因的表达变化

目的:分析不同牙周炎症状态下牙龈卟啉单胞菌(Porphyromonal gingivalis,P.gingivalis)rag-1基因mRNA的表达规律.方法:收集150例慢性牙周炎患者的龈下菌斑,PCR法检测P.gingivalis和rag-1基因.对P.gingivalis rag-1基因阳性的菌斑样本,采用RT-PCR法检测rag-1 mRNA的表达水平,并比较不同炎症状态下的表达差异.应用SPSS11.0软件包对数据进行配对t检验和秩相关检验.结果:轻度组、中度组和重度组的rag-1基因mRNA的相对表达水平分别为1.19±0.06、0.46±0.05和2.22±0.10(P<0.05).结论:rag-1基因的mRNA表达水平随着牙周组织破坏程度及炎症状态的不同而改变.     

慢性牙周炎合并慢性阻塞性肺疾病患者牙龈卟啉单胞菌rag位点基因分析

目的:分析不同rag基因型牙龈卟啉单胞菌在慢性牙周炎合并慢性阻塞性肺疾病(chronic obstructive pulmonary disease,COPD)患者中的分布状况.方法:选择慢性牙周炎及合并COPD的慢性牙周炎患者各30例,收集唾液样本,采用16S rDNA聚合酶链反应(PCR)法检测牙龈卟啉单胞菌的检出...     

牙龈卟啉单胞菌与动脉粥样硬化发病的关系

慢性牙周炎是心血管疾病特别是冠心病的危险因素之一,然而其相关的生物学基础目前尚不清楚.笔者下面就与慢性牙周炎关系最为密切的牙龈卟啉单胞菌与动脉粥样硬化的相关性和牙龈卟啉单胞菌致动脉粥样硬化的可能机制以及Toll样受体在牙龈卟啉单胞菌与动脉粥样硬化相关性中的作用作一综述.     

牙龈卟啉单胞菌牙龈素在牙周炎症过程中的作用及其机制

牙周炎是口腔临床实践中最为常见的疾病之一。牙龈卟啉单胞菌与牙周炎密切相关,牙龈卟啉单胞菌表面的牙龈素(gingipains)、菌毛和脂多糖被认为是其最重要的毒力因子,是近年来牙周炎机制研究的热点,引起研究者的广泛关注。本文结合近年来的研究进展,就牙龈卟啉单胞菌gingipains的结构、引起牙周炎症的机制和所涉及的信号通路作一综述。     

慢性牙周炎唾液牙龈卟啉单胞菌的定量PCR检测

目的:定量检测慢性牙周炎患者、牙周健康人群唾液中牙龈卟啉单胞菌的含量,比较其在各组人群分布的差异。方法:应用SYBR Green模式的实时荧光定量PCR技术,针对Pg特异基因Arg-gingipain设计引物,检测20例慢性牙周炎和20例牙周健康者唾液内Pg的含量,t检验分析比较在各组人群中Pg定植的差异。结果:慢性牙周炎患者唾液中,Pg的检出数目为(1.78×10³~1.99×10⁵),检出率为85%;牙周健康者唾液中,Pg的检出数目为(2.19×10³~2.30×10³),检出率为10%。Pg在慢性牙周炎和牙周健康者唾液中检出数目和检出率的差异具有统计学意义(P<0.05)。结论:慢性牙周炎唾液中,牙龈卟啉单胞菌较正常人群明显升高,在今后的研究及防治中,不仅要观察龈沟液中的牙龈卟啉单胞菌,也应注意其在唾液中含量的变化。     

牙龈卟啉单胞菌脂多糖引起牙周炎症的机制和特点

革兰阴性厌氧菌牙龈卟啉单胞菌和牙周炎密切相关，脂多糖是革兰阴性菌外膜中的主要结构成分，具有广泛的生物学活性，被认为是革兰阴性菌的主要毒力因子之一。本文就牙龈卟啉单胞菌脂多糖的化学组成和结构，所启动炎症信号通路及其起始受体，引起牙周炎症的机制和特点作一综述。     

慢性牙周炎患者龈下菌斑中不同基因型牙龈卟啉单胞菌的检测

摘要 目的:建立临床标本中牙龈卟啉单胞菌(P.g)的PCR检测方法,探讨慢性牙周炎患者不同牙位的龈下菌斑中P.g基因型的差异。方法:采用培养法分离鉴定慢性牙周炎患者不同牙位龈下菌斑中P.g,同时采用PCR检测 P.g16SrDNA、prtC和fimA基因。部分扩增产物测定了核苷酸序列。结果:在66例患者的127个龈下菌斑标本中, P.g16SrDNA、prtC和fimA多重引物扩增的阳性率为9814%;PCR阳性率显著高于培养法P.g的检出率(P< 0101)。3010%的患者(18/60)同时感染了不同基因型的P.g菌株。P.g16SrDNA、prtC和fimA扩增片段的核苷酸序列同源性在98162%~100%之间。结论:本文所建立的P.g的PCR检测方法具有较高的敏感性和特异性,适用于P.g的快速临床诊断。同一患者可被不同感染来源的多株P.g同时感染。     

牙龈卟啉单胞菌脂蛋白基因在慢性牙周炎及牙周健康者中的检测

目的 应用基因芯片技术检测3种脂蛋白基因PG0717、PG0183、PG2135在慢性牙周炎患者和牙周健康者牙龈卟啉单胞菌(Porphyromonas gingivalis,Pg)中的分布,探讨这些基因与牙周临床指数之间的关系,为研究脂蛋白在Pg致病过程中的作用提供依据.方法 选取41例慢性牙周炎患者(牙周炎组)及76例牙周健康者(健康对照组),记录探诊深度、附着丧失、探诊出血及牙齿松动度,取龈下菌斑进行细菌分离培养,以临床采集的样本提取的DNA为探针,以抑制消减杂交技术获得的PgW83特异基因片段PG0717、PG0183、PG2135为目标序列,采用Cy5荧光标记目标序列.应用基因芯片技术检测PG0717、PG0183、PG2135基因在牙周炎组病变部位、非病变部位和健康对照组Pg中的分布.结果在牙周炎组病变部位PG0717、PG0183、PG2135基因的检出率分别为90%(18/20)、70%(14/20)、70%(14/20),非病变部位的检出率分别为60%(12/20)、45%(9/20)、40%(8/20),而在健康对照组PG0717、PG0183、PG2135的检出率分别为55%(11/20)、25%(5/20)、30%(6/20).3种脂蛋白基因在牙周炎组病变部位和健康对照组中的检出率差异均有统计学意义(P＜0.05),并且与牙周探诊深度、临床附着丧失、探诊出血及牙齿松动度相关.结论 带有PG0717、PG0183、PG2135基因的Pg菌株致病力强.     

Antibody reactive with Porphyromonas gingivalis hemagglutinin in chronic and generalized aggressive periodontitis

BACKGROUND: Porphyromonas gingivalis, a black-pigmented, gram-negative anaerobe, is found in periodontitis lesions and its presence in subgingival plaque significantly increases the risk for periodontitis. We have previously shown that patients with aggressive forms of periodontitis that are seropositive for P. gingivalis have less attachment loss than those that are seronegative. This suggests that antibody reactive with antigens of P. gingivalis may be protective and decrease disease severity and extent. Recent studies in the murine abscess model and in the host antibody response in chronic periodontitis patients suggest that antibody reactive with P. gingivalis hemagglutinin may be an important protective antibody response. OBJECTIVES: In this study, we tested the hypothesis that there was a significant relationship between antibody reactive with P. gingivalis hemagglutinin and measures of periodontal attachment loss. METHODS: We determined the immunoglobulin G (IgG) antibody concentration reactive with recombinant P. gingivalis hemagglutinin in 117 chronic periodontitis and 90 generalized aggressive periodontitis patients. We also determined the IgG subclass distribution for antibody reactive with P. gingivalis hemagglutinin. RESULTS AND CONCLUSIONS: We found IgG reactive with P. gingivalis hemagglutinin in both chronic periodontitis and generalized aggressive periodontitis patients. Most of this IgG antibody was of the IgG1 and IgG3 subclasses. Antibody reactive with P. gingivalis hemagglutinin, however, did not have a significant relationship with measures of periodontal attachment loss.     

不同rag基因型牙龈卟啉单胞菌在慢性牙周炎患者中的分布

目的分析不同rag基因型牙龈卟啉单胞菌在慢性牙周炎患者中的分布状况。方法收集50例慢性牙周炎患者的150个龈下菌斑样本,采用16S rDNA 聚合酶链反应（PCR）法检测牙龈卟啉单胞菌在牙周炎病变位点的检出率,并根据各rag基因型的特异性引物检测牙龈卟啉单胞菌不同rag基因型在慢性牙周炎病变位点的分布。结果经16S rDNA PCR法检测,病变位点牙龈卟啉单胞菌阳性检出率为70.7%。各rag基因型在牙龈卟啉单胞菌阳性位点的总检出率：rag-1为60.4%,rag-2为23.6%,rag-3为44.3%,rag-4为15.1%;经统计学检验,rag-1和rag-3型检出率较高,高于rag-2和rag-4型（P<0.05）。结论慢性牙周炎患者龈下菌斑中的牙龈卟啉单胞菌存在rag基因多态性,rag-1和rag-3基因型牙龈卟啉单胞菌与中国辽宁地区人群慢性牙周炎的发生发展关系密切。     

EB病毒和牙龈卟啉单胞菌协同感染与妊娠期慢性牙周炎的相关性研究

目的：研究妊娠期女性EB病毒（Epstein?Barr virus,EBV）和牙龈卟啉单胞菌（Porphyromonas gingivalis, Pg）的协同感染情况和牙周炎严重程度的关系。方法收集36例患慢性牙周炎（妊娠期慢性牙周炎组）的妊娠期女性和36例牙周健康（妊娠期牙周健康组）的妊娠期女性唾液样本,应用巢式PCR技术检测EBV感染率,应用16S rRNA为基础的PCR技术检测Pg感染率,结合口腔临床检查收集的牙周临床指标,对EBV和Pg的协同感染与牙周炎严重程度的相关性加以分析。结果妊娠期慢性牙周炎组和妊娠期牙周健康组EBV、Pg感染率的检出率差异无统计学意义（P＞0.05）,2组EBV和Pg协同感染的检出率差异具有统计学意义（χ2=4.800,P=0.028）。EBV和Pg协同感染与探诊出血指数相关（t=3.058,P=0.003）,与牙周袋深度和附着丧失无关（P＞0.05）。结论 EBV和Pg协同感染与妊娠期慢性牙周炎的相关性仍需进一步研究。     

Intracellular localization of Porphyromonas gingivalis thiol proteinase in periodontal tissues of chronic periodontitis patients

OBJECTIVES: Porphyromonas gingivalis is a significant periodontal pathogen that has been shown in vitro to be able to invade gingival epithelial cells and grow intracellularly. The aim of the present study was to detect P. gingivalis in gingival tissues from chronic periodontitis (CP) patients. MATERIALS AND METHODS: Monoclonal antibodies specific to a cell membrane-bound thiol proteinase of P. gingivalis were used to detect the microbe in gingival tissues of CP patients (n = 13) by immunohistochemistry. The presence of P. gingivalis was also analysed by polymerase chain reaction (PCR). RESULTS: Immunohistochemical analysis of the periodontal tissues revealed positive staining for P. gingivalis thiol proteinase in 11 of the 13 patients. Positive staining was mainly located intracellularly in the perinuclear region of the cytoplasm in the periodontal epithelial cells and it could be detected throughout the whole depth of both pocket and oral epithelium. The sensitivity of immunohistochemistry was found to be comparable with that of PCR. CONCLUSIONS: Our results provide in vivo evidence of the ability of P. gingivalis to enter human gingival epithelial cells. Intracellular localization of P. gingivalis contributes to its evasion of the host immune surveillance and eventually increases its resistance to conventional treatments of periodontal diseases.     

牙龈卟啉单胞菌PG0839基因突变株的构建

目的构建牙龈卟啉单胞菌毒力岛基因PG0839突变菌株,为研究PG0839基因功能提供实验基础。方法扩增1 584 bp PG0839基因片段,对聚合酶链反应（PCR）产物和pUC19载体进行BamH Ⅰ和EcoRⅠ双酶切,连接酶切产物得到质粒pPG0839-1。将2 101 bp erm基因产物插入到pPG0839-1中PG0839基因的EcoRⅤ位点,构建质粒pPG0839-2,作为电穿孔的供体质粒。电穿孔转化于受体菌牙龈卟啉单胞菌W83菌株,红霉素抗性培养基筛选阳性克隆,命名为PG0839基因突变菌株。结果运用插入失活方法构建PG0839基因突变菌株,进而通过酶切、测序、PR和反转录PCR对PG0839基因突变菌株进行验证,证实PG0839基因突变菌株构建成功。结论本实验成功构建PG0839基因突变菌株。     

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.     

Analysis of cultivable Porphyromonas gingivalis with trypsin-like protease enzyme activity and serum antibodies in chronic adult periodontitis

OBJECTIVE: Trypsin-like protease (TLPase) enzyme produced by Porphyromonas gingivalis has been implicated as a virulence factor in the pathogenesis of periodontal disease. The aims of this study were to investigate the relationship between cultivable P. gingivalis , TLPase enzyme activity (BANA hydrolysis) and serum antibody levels against cell sonicate and a purified TLPase antigen from P. gingivalis W50.
MATERIALS AND METHODS: Sub-gingival plaque samples were cultured for levels of P. gingivalis together with a chairside analysis of TLPase enzyme activity (Perioscan) from periodontitis and gingivitis sites of adult periodontitis patients. A TLPase from P. gingivalis was purified by gel filtration and ion exchange chromatogra-phy from the vesicle fraction for use as a test antigen. RESULTS: Elevated levels of P. gingivalis were found at periodontitis sites, however, there was no correlation with sub-gingival plaque TLPase enzyme activity. Adult periodontitis patients had higher levels of IgG and IgA against cell sonicate and TLPase antigens than did controls. Those patients who were P. gingivalis culture-positive demonstrated an elevated immune response against both cell sonicate and TLPase when compared to P. gingivalis culture-negative patients. Treatment resulted in an improvement of clinical indices and no cultivable P. gingivalis could be recovered from the treated sites and there was a concomitant decrease in IgG levels against the TLPase. There was no significant difference in BANA hydrolysis at gingivitis sites or periodontitis sites after treatment.
CONCLUSIONS Further longitudinal studies are suggested to investigate the role of the TLPase in the response to treatment of chronic adult periodontitis patients.