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The detection of bacteria as the pathogen of chronic immflamatory granulomatosis

慢性炎症性肉芽肿病原菌的检测

基本信息

批准号：
07670512
负责人：
MUKAI Masaya
金额：
$ 1.47万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07670512/
关键词：
rheumatoid arthritis in situ PCR bacterial infection 16s rRNA intracellular bacterial survival chronic inflammatory granuloma 16srRNA

项目摘要

[Purpose] Because some noncultivable bacteria such as chlamydia cause reactive arthritis, the pathogenesis of rheumatoid arthritis (RA), or chronic inflammatory granuloma may be such bacteria that stimulate continuously immune system in synovium, or in granuloma. This hypothesis was evaluated with the very sensitive method, in situ polymerase chain reaction (ISPCR) and with 16s rRNA primers for almost all bacteria. [Subject and method] Nineteen synovial fluids were sterilely punctured from sixteen patients with RA.Synovial cells were fixed, and were proteolised. Digoxigenin labeled dUTP was directly incorporated to amplified bacterial DNA in cells in suspension with ISPCR.Positive cells were detected with fluorescence conjugated antidigoxigenin antibody using fluorescence-activated cell sorter (FACS). Products of PCR in supernatants were also investigated with agarose gel electrophoresis. [Results] Cells from one RA synovial fluid was obviously positive for bacterial DNA.Others were negative for bacterial DNA.[Conclusion] This system with direct incorporation in ISPCR could be useful for cell suspension materials to detect DNA.Because the specificity and sensitivity of this method are unknown, it was suggested that noncultivable bacteria could be some pathogenesis of RA.Also, because it had taken long time to establish reverse transcriptase ISPCR in tissue with the bacterial primers, there was not enough time to investigate many clinical materials. Many samples have been collected and prepared for this method.

[目的]由于某些不可培养的细菌如衣原体引起反应性关节炎，类风湿关节炎（RA）或慢性炎性肉芽肿的发病机制可能是这些细菌在滑膜或肉芽肿中持续刺激免疫系统。这一假设进行了评估与非常敏感的方法，原位聚合酶链反应（ISPCR）和16 S rRNA引物几乎所有的细菌。[对象与方法] 16例RA患者的19份关节液经无菌穿刺后，取滑膜细胞进行固定，蛋白水解。用地高辛标记的dUTP直接掺入悬浮细胞中的细菌DNA，用荧光激活细胞分选仪（fluorescence-activated cell sorter，FACS）检测阳性细胞。琼脂糖凝胶电泳检测上清中的PCR产物。[结果] 1例RA关节液细胞细菌DNA明显阳性，其余关节液细胞细菌DNA均阴性。[结论]直接掺入ISPCR的方法可用于细胞悬液材料的DNA检测，但由于该方法的特异性和敏感性尚不清楚，提示不可培养的细菌可能是RA的致病菌，同时由于用细菌引物在组织中建立逆转录酶ISPCR的时间较长，许多临床材料没有足够的时间进行研究。已经为该方法收集和制备了许多样品。