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PG0717基因与慢性牙周炎的相关性研究

目的检测慢性牙周炎患者和牙周健康者龈下菌斑中牙龈卟啉单胞菌（P.gingivalis）PG0717基因,探讨PG0717基因与牙周临床指数之间的关系。方法选取慢性牙周炎（CP）患者90例和牙周健康者90例,共采集龈下菌斑标本540个;记录临床牙周指数（牙周探诊深度、临床附着丧失和探诊出血）;设计特异性引物检测P.gingivalis阳性龈下菌斑标本的PG0717基因。结果在P.gingivalis阳性龈下菌斑中,CP组PG0717基因检出率显著高于对照组,分别为56.22%和41.27%（掊2=4.50,P＜0.05）;随着牙周探诊深度、临床附着丧失加重和探诊出血趋势的增加,CP组该基因检出率呈现增高趋势。结论PG0717基因与P.gingivalis的致病性有关。     

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.     

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。     

牙龈卟啉单胞菌与牙周病相关性的聚合酶链反应研究

目的利用PCR检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,P. g)的16S rDNA水平,通过检测该基因水平来探讨牙龈卟啉单胞菌的水平与牙周病的相关性.方法采集慢性牙周炎患者12例共36个龈下菌斑标本,记录临床指标(探诊深度、临床附着水平、牙龈指数、菌斑指数、龈沟出血指数),PCR检测龈下菌斑标本中的P. g 16S rDNA基因,扩增产物经琼脂糖电泳、拍照后,应用计算机软件GeneTools扫描并计量其中的相对核酸含量.结果P. g16S rDNA水平与探诊深度(P＜0.001)及牙龈指数(P＜0.01)之间存在正相关关系.结论P.g16S rDNA水平与牙周状态密切相关,对于P.g16S rDNA水平的监测有望成为牙周病诊断和治疗方案制定的辅助检查手段.     

龈下菌斑中牙龈卟啉单胞菌牙龈素基因片段的检测

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,P.gingivalis）的2个基因片段kgp-cd和rgpB-cd,探讨2个基因片段的存在和缺失与牙周临床指标之间的关系.方法：选择慢性牙周炎患者龈下菌斑84个,对P.gingivalis阳性的龈下菌斑样本进行kgp-cd和rgpB-cd基因片段检测;根据2个基因片段的有无,将P.gingivalis分为A型和B型,采用SPSS11.5统计软件包,用t检验和x2检验分析不同基因型P.gingivalis与牙周临床指标的关系.结果：A型P.gingivalis在慢性牙周炎中的检出率高于B型（P＜0.05）,分别为85.29%和14.71%.不同基因型P.gingivalis引起的牙周袋探诊深度和牙龈出血倾向存在显著性差异（PD：t=2.85,P＜0.05;BOP：P＜0.05）.结论：kgp-cd和rgpB-cd基因与P.gingivalis的致病性有关.     

应用实时荧光定量PCR定量检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌

目的 检测慢性牙周交患者和牙周健康人龈下菌斑中牙龈卟啉单胞菌、总菌量和牙龈卟啉单胞菌所占比例,探讨牙龈卟啉单胞菌与牙周炎发生发展的关系.方法 采集经常规PCR方法检测牙龈卟啉单胞菌为阳性的76例慢性牙周炎患者和25例牙周健康者的龈下菌斑,应用TaqMan实时荧光定量PCR方法定量检测样本中牙龈卟啉单胞菌、总菌量;构建含有牙龈卟啉单胞菌和真细菌扩增片断的重组质粒,建立定量标准.结果 本研究设计的引物和探针具有良好的特异性及敏感性.病变位点龈下菌斑中牙龈卟啉单胞菌数量和总菌量均比健康位点高(P<0.001),两组位点牙龈卟啉单胞菌在菌斑中的比例没有差异(P>0.05);细菌数量与探诊深度间存在显著正相关关系(P<0.001),不同的探诊深度牙龈卟啉单胞菌所占比例无统计学差异(P>0.05).结论 龈下菌斑中牙龈卟啉单胞菌的数量水平及细菌总量与牙周状况、牙周炎发展有密切关系,实时荧光定量PCR对牙周病学研究具有广泛的应用前景.     

龈沟液中有机酸与龈下牙龈卟啉单胞菌间的关系

目的分析慢性牙周炎患者相同龈沟液样本中挥发性有机酸与牙龈卟啉单胞菌（Porphyromonas gingivalis,P.g）间的关系。方法应用高效毛细管电泳技术分析慢性牙周炎患者治疗前龈沟液中有机酸的浓度;应用多聚酶链反应（polymerase chain reaction,PCR）技术检测相同样本中牙龈卟啉单胞菌。结果慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌检出率显著高于牙周健康对照者;慢性牙周炎患者P.g阳性组龈沟液中丁酸与异戊酸浓度显著高于P.g阴性组。结论结果表明相同样本龈沟液中丁酸与异戊酸的表达与P.g的检出相关联,P.g是重要牙周致病菌     

牙龈卟啉单胞菌PG0839基因突变株生物学特性和毒力的初步研究

目的:通过对牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)PG0839基因突变菌株的生物学特性和毒力进行研究,为进一步明确毒力岛基因PG0839的功能提供实验依据。方法:培养P.gingivalis W83和PG0839基因突变菌株,观察2个菌株菌落形态特征和革兰染色镜下特征,绘制菌株的生长曲线。制备1%绵羊红细胞,取2种细菌细胞,进行绵羊红细胞凝集活性检测。分别接种浓度为2×1010、1×1010、5×109 CFU/ml的P.gingivalis W83和PG0839基因突变株菌液于小鼠背部。采用SPSS 13.0软件包中的皮尔森卡方检验和对数秩检验对不同组别小鼠生存情况进行分析。结果:与P.gingivalis W83比较,PG0839突变菌株产生黑色素和红细胞凝集活性显著降低。2个菌株生长曲线未见显著差异(U=25.50,P=0.19)。小鼠皮下感染模型表明,PG0839基因突变菌株毒性显著低于W83菌株(P<0.05)。结论:PG0839基因的缺失改变了细菌的一些生物学特征,提示PG0839基因可能调控P.gingivalis某些基因或者基因产物的表达。同时,PG0839基因在P.gingivalis致病过程中发挥一定作用。     

牙龈卟啉单胞菌HA2基因与牙周病关系的研究

目的 利用PCR及RT-PCR检测慢性牙周炎患者龈下菌斑中牙龈卟啉单胞菌(Porphyromonas gingivalis,P.g)HA2的DNA和RNA水平,探讨其分布规律及与牙周病的关系.方法 12例慢性牙周炎患者共采集36个龈下菌斑标本,记录临床指标(探诊深度、临床附着水平、牙龈指数、菌斑指数、龈沟出血指数),PCR及RTPCR扩增龈下菌斑标本中的P.g HA2基因,扩增产物经琼脂糖电泳、拍照,应用计算机软件GeneTools扫描并计量其中的相对核酸含量.结果 P.g HA2在DNA水平上的检出率为92%,在RNA水平上的检出率为86%.P.g HA2的DNA水平与探诊深度及牙龈指数之间存在正相关关系(P＜0.01);P.g HA2的RNA水平与牙龈指数之间存在正相关关系(P＜0.05).结论 P.g HA2基因广泛存在于慢性牙周炎患者的龈下菌斑中,且P.g HA2在DNA和RNA水平上与牙周状态关系密切,提示P.g HA2可能是P.g致病因子之一.     

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。     

不同fimA基因型牙龈卟啉单胞菌在青春期龈炎患者中的分布

目的:分析不同fimA基因型牙龈卟啉单胞菌(Porphyromonas gingivalis,P.gingivalis)在青春期龈炎患者和青春期牙周健康者中的分布。方法:收集51例青春期龈炎患者和46例青春期牙周健康者的龈下菌斑,采用16SrRNA PCR检测P.gingivalis,并根据各fimA基因型的特异引物,用PCR检测不同fimA基因型菌株的分布。结果:龈炎组各fimA基因型P.gingivalis的总检出率:Ⅰ型34.2%,Ⅱ型55.3%,Ⅳ型18.4%,Ⅲ型和Ⅴ型未检出;另有7例(占18.4%)未分出型别。健康组各fimA基因型P.gingivalis的总检出率:Ⅰ型14.3%,Ⅱ型85.7%,Ⅲ型14.3%,Ⅳ型28.6%,Ⅴ型未检出;另有2例(占14.3%)未分出型别。结论:青春期龈炎和青春期牙周健康者龈下菌斑中的P.gingivalis存在fimA基因多态性。fimA基因Ⅱ型是其主要存在的基因型,其次是Ⅰ型和Ⅳ型,Ⅲ型和Ⅴ型较少或不能检出。     

Association of Eubacterium nodatum and Treponema denticola with human periodontitis lesions

BACKGROUND: The purpose of the present investigation was to compare the levels, proportions and percentage of sites colonized by 40 bacterial species in subgingival plaque samples from periodontally healthy subjects and patients with chronic periodontitis to seek possible pathogens other than the consensus pathogens Porphyromonas gingivalis and Tannerella forsythia. METHOD: Subgingival plaque samples were taken from the mesial aspect of each tooth in 635 subjects with chronic periodontitis and 189 periodontally healthy subjects. The samples were individually analyzed for their content of 40 bacterial species using checkerboard DNA-DNA hybridization (total samples = 21,832). Mean counts, % DNA probe counts and percentage of sites colonized at >10(5) were determined for each species in each subject and then averaged in each clinical group. Significance of difference between groups was determined using the Mann-Whitney test. Association between combinations of species and periodontal status was examined by stepwise logistic regression analysis. Analyses were repeated using a subset of subjects from both clinical groups who had proportions of P. gingivalis plus T. forsythia less than the median (4.42%) found in periodontally healthy subjects. All analyses were adjusted for multiple comparisons. RESULTS: For the 824 subjects the consensus pathogens P. gingivalis and T. forsythia as well as Eubacterium nodatum and Treponema denticola had significantly higher mean counts, proportions and percentage of sites colonized in samples from subjects with periodontitis than from periodontally healthy subjects. There were significantly more Capnocytophaga gingivalis, Streptococcus gordonii and Veillonella parvula in periodontally healthy subjects. E. nodatum, T. denticola, Streptococcus oralis, Streptococcus intermedius, Fusobacterium nucleatum ssp. vincentii all had higher counts and proportions in diseased than healthy subjects who had low proportions of P. gingivalis and T. forsythia. Logistic regression analysis indicated that the same species groups were associated with disease status after adjusting for the proportions of the other species. CONCLUSIONS: This investigation confirmed the strong association of P. gingivalis and T. forsythia with chronic periodontitis and emphasized a strong association of E. nodatum and T. denticola with periodontitis whether in the presence or absence of high levels of the consensus pathogens. Other species, including S. oralis, Eikenella corrodens, S. intermedius and F. nucleatum ssp. vincentii, were associated with disease when P. gingivalis and T. forsythia were present in low proportions.     

不同fimA基因型牙龈卟啉单胞菌在牙周健康人群中的分布

目的调查不同fimA基因型牙龈卟啉单胞菌 (P.gingivalis)在牙周健康人群中的分布情况。方法收集136例牙周健康者的龈下菌斑样本,采用16S rRNAPCR法检测P.gingivalis;并根据各fimA基因型特异性引物,用PCR法检测不同fimA基因型P.gingivalis的分布。 结果136例牙周健康者的龈下菌斑样本中携带P.gingivalis的阳性率为22.1%。大多数受检者龈下菌斑中只检测到1种fimA基因型 P.gingivalis菌株(80.0%);5例样本检出了2种fimA型P.gingivalis(16.7%),且均为ⅠfimA型与其它fimA 型P.gingivalis的联合检出。各fimA型P.gingivalis的检出情况:Ⅰ型(66.7%)、Ⅰb型(6.7%)、Ⅱ型(6.7%)、 Ⅲ型(10%)、Ⅳ型(6.7%)、Ⅴ型(16.7%)。Ⅰ型的检出率明显高于其它各型,差异有统计学意义(P<0.05);其它各fimA型 P.gingi-valis间的检出差异均无统计学意义。结论本研究条件下ⅠfimA型P.gingivalis在牙周健康人群中检出例数最高,提示:我 国牙周状况不同人群携带的P.gingivalis菌株fimA基因型可能存在差异。?     

Prevalence of fimA genotypes of Porphyromonas gingivalis and other periodontal bacteria in a Spanish population with chronic periodontitis

Objectives: The aim of this study was to determine the prevalence of the different fimA genotypes of Porphyromonas gingivalis in adult Spanish patients with chronic periodontitis, patients with gingivitis and periodontally healthy subjects, and the relationship between these genotypes and other periodontopathogenic bacteria. Study design: Samples of subgingival plaque were taken from 86 patients (33 with chronic periodontitis, 16 with gingivitis, and 37 periodontally healthy) in the course of a full periodontal examination. PCR was employed to determine the presence of the 6 fimA genotypes of Porphyromonas gingivalis (I-V and Ib) and of Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola. Results: Porphyromonas gingivalis fimA genotypes II and Ib were present in significantly higher percentages in periodontal patients (39.4% and 12.1% respectively) than in healthy or gingivitis subjects. The prevalence of Tannerella forsythia, Treponema denticola, and Porphyromonas gingivalis fimA genotype IV was significantly higher in the group that presented bleeding greater than 30%. A positive correlation was found between Porphyromonas gingivalis fimA genotype IV and Treponema denticola. Conclusions: A strong association between Porphyromonas gingivalis fimA genotypes II and Ib and chronic periodontitis exists in the Spanish population. The most prevalent genotype in periodontal patients is II. Key words:Periodontitis, Porphyromonas gingivalis, fimA genotype, periodontal bacteria, polymerase chain reaction.     

Real-time polymerase chain reaction for detection and quantification of bacteria in periodontal patients

BACKGROUND: Accurate laboratory tests for the detection and quantification of periodontopathogens in subgingival plaque samples of periodontal disease patients are becoming essential to study the pathogenesis of this polymicrobial condition. We used a real-time polymerase chain reaction (PCR) assay for the quantification of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Dialister pneumosintes, Campylobacter rectus, and Micromonas micros as well as total eubacteria in subgingival plaque samples from individuals with periodontitis. METHODS: Eighty-three subgingival samples from periodontally diseased patients and 43 samples from periodontally healthy subjects were tested and the results of bacterial quantification were correlated to clinical parameters. Quantification was performed with specific 16S rRNA target sequences with double fluorescence labeled probes and serial dilutions of plasmid standards by real-time PCR. RESULTS: Results showed that patients as well as healthy subjects were positive for the presence of target periodontopathogens; however, median values were higher in samples from periodontitis subjects. In addition, a positive association was observed between colonization at high levels by P. gingivalis and M. micros and the presence of deep periodontal pockets. CONCLUSION: Real-time PCR provides a reliable high-throughput method for quantification of periodontopathogens and may be useful for understanding the complex etiology observed in periodontal diseases.     

牙龈卟啉单胞菌对正畸治疗病人牙周状况影响的研究

目的:观察牙龈卟啉单胞菌对正畸治疗病人牙周状况的影响,以便有效地控制正畸过程中牙龈炎性反应的发生发展,避免不可逆性的牙周损害。方法:收集41例正畸治疗中发生牙龈炎性反应的病人、20例正畸治疗前牙周健康者和35例慢性牙周炎病人的龈沟液标本,应用16SrDNA PCR技术检测各样本中的牙龈卟啉单胞菌,并分析其与临床指标(GI)之间的关系。结果:牙龈卟啉单胞菌的检出率分别为:正畸治疗组70.73%、正常对照组35.00%、牙周炎组85.71%;正畸治疗组的检出率明显高于正常对照组;牙周炎组显著高于正畸治疗组和正常对照组,三者之间有显著性差异(P<0.05);牙龈卟啉单胞菌的检出率随着牙周病变程度的增加而升高,与牙龈指数呈正相关关系(P<0.05)。结论:牙龈卟啉单胞菌与正畸治疗中的牙龈炎性反应和发展密切相关。