Extracrevicular Invasion by Periodontal Pathogens

牙周病原体的沟外侵袭

• 批准号: 6765101
• 负责人: Joel D. Rudney
• 金额: $ 26.08万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-08-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6765101
• 关键词: Actinobacillus actinomycetemcomitans; Bacteroides gingivalis; atherosclerotic plaque; bacteria infection mechanism; chlorhexidine; clinical research; confocal scanning microscopy; cytokine; denaturing gradient gel electrophoresis; disease /disorder proneness /risk; fluorescent in situ hybridization; gene expression; green fluorescent proteins; human subject; messenger RNA; oral mucosa; periodontitis; polymerase chain reaction; ribosomal RNA; saliva; stroke; tissue /cell culture

Gingival infection with periodontal pathogens often persists after treatment, and mucosa are thought to be a reservoir for recolonization. Invaded mucosal cells may provide a protected environment for these fastidious anaerobes. Preliminary studies used fluorescent in situ hybridization (FISH) with universal and specific rRNA probes and confocal microscopy (LSCM) to detect Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, and unidentified bacteria inside buccal cells from 23 of 24 subjects. This suggests that intracellular mucosal bacteria exist in a multi-species community which may maintain itself by modulating or overcoming host cell defenses, with exfoliated cells providing a protected route for bacterial transmission. Those postulates will be tested by these specific aims: 1.) Determine the composition of the mucosal intracellular community by probing for additional periodontal pathogens and other mucosal species. 16S rRNA temperature gradient electrophoresis will be used to design probes for uncultured species. Multicolor FISH will determine whether more than one species can occupy a cell. 2.) Determine whether intracellular bacteria modulate or overcome cell defenses in vivo by using multicolor FISH/LCSM to compare invaded and uninvaded cells for expression of cytokine and antimicrobial peptide mRNA. 3.) Determine whether exfoliated invaded cells may be a vector for transmission by using a tissue culture model. Washed mucosal cells will be suspended in autologous or heterologous clarified saliva with or without antibiotics, and then incubated with green fluorescent protein (GFP)-labeled KB cells. Transmission will be evaluated by multicolor FISH/LSCM. 4.) Determine whether the intracellular mucosal community can establish and maintain itself in the absence of the gingival crevice by using FISH/LSCM to look for such communities in predentate infants, and to verify whether they occur in edentulous adults. 5.) Determine whether mucosal invasion protects pathogens from elimination in periodontal patients who require aggressive treatment, by using FISH/LSCM and multiplex PCR to compare mucosal and gingival colonization before and after scaling and root planing, topical chlorhexidine, and systemic antibiotics.

牙周病原体感染通常在治疗后持续存在，粘膜被认为是再分化的储存库。入侵的粘膜细胞可能为这些挑剔的厌氧菌提供了一个受保护的环境。初步研究使用通用和特定rRNA探针的荧光原位杂交(FISH)和共聚焦显微镜(LSCM)检测24名受试者中23名口腔细胞中的伴生放线杆菌、牙龈卟啉单胞菌和不明细菌。这表明细胞内的粘膜细菌存在于一个多物种的群落中，可以通过调节或克服宿主细胞的防御来维持自身，脱落的细胞为细菌的传播提供了一条受保护的途径。这些假设将受到以下具体目标的检验：1.)通过探查其他牙周病原体和其他粘膜物种来确定粘膜细胞内群落的组成。16S rRNA温度梯度电泳法将用于设计未培养物种的探针。五颜六色的鱼将决定是否一个以上的物种可以占据一个细胞。2.)通过使用多色FISH/LCSM比较侵入和未侵入的细胞中细胞因子和抗菌肽mRNA的表达，确定细胞内细菌是否调节或克服体内的细胞防御。3.)使用组织培养模型确定脱落的侵袭细胞是否可能是传播的媒介。将洗涤的粘膜细胞悬浮在含有或不含抗生素的自体或异种澄清的唾液中，然后与绿色荧光蛋白(GFP)标记的KB细胞孵育。传播将通过多色FISH/LSCM进行评估。4.)通过使用FISH/LSCM在有牙齿的婴儿中寻找这样的群落，并验证它们是否发生在无牙成人中，来确定细胞内粘膜群落是否能够在没有牙缝的情况下建立和维持自己。5.)通过使用FISH/LSCM和多重聚合酶链式反应来比较洁治和根面平整、外用洗必泰和全身抗生素前后的粘膜和牙龈定植情况，确定黏膜侵袭是否保护了需要积极治疗的牙周患者的病原体不被清除。