Gene profiling of periodontal pathogens in periodontal lesion

1.伴放线放线杆菌的宿主诱导基因具有多种毒力因子。然而，毒力因子的潜在作用还没有得到很好的描述。此外，许多未知的毒力因子被认为参与了发病机制。放线菌伴生菌在初代菌株(R型)上呈粗糙菌落生长。通过反复继代培养，菌落转化为光滑的表型(S型)。由于表型变化反映了从体内到体外对环境变化的不同基因表达，因此在R型菌株中诱导基因的分离实际上是宿主诱导基因的分离，包括毒力因子。在本研究中，我们利用cDNA消减杂交技术鉴定了R型伴生放线菌中诱导表达的基因，其中mip、prx和OmpA三个基因被鉴定为R型特异性基因。将注意力集中在MIP上，并创建了一个重组的MIP样蛋白和缺陷突变体。重组蛋白与患者血清发生反应，提示牙周病变中有MIP样蛋白的产生。用该突变体进行了侵袭试验，结果表明该突变体对宿主细胞的侵袭能力减弱。MIP、Prx和OmpA在宿主体内的表达可能增强，MIP可能在伴生放线菌侵袭过程中起重要作用。2.新的微生物检测系统。因此，在活体内分析牙周病原体和毒力因子需要进行微生物学检查。在目前的研究中，我们建立了一个新的考试制度。该体系采用环介导的等温扩增(LAMP)方法。根据16S核糖体RNA基因的核苷酸序列设计了引物，LAMP方法检测牙周病原菌具有较高的敏感性、特异性和快速。

1.Host-induced genes of A.actinomycetemcomitansActinobacillus actinomycetemcomitans possess many virulence factors. However, the potential roles of the virulence factors are not well characterized. In addition, many unknown virulence factors are considered to be involved in the pathogenesis. A.actinomycetemcomitans grows as a rough colony on primary isolates (R-type). The colony converts to a smooth phenotype (S-type) through repeated subculture. Since the phenotypic alteration reflects different gene expressions in response to environmental changes from the in vivo to the in vitro, isolation of genes induced in the R-type strain turns out to be an isolation of host-induced genes, including the virulence factors. In the current study, we identified genes induced in the R-type of A.actinomycetemcomitans using the cDNA subtractive hybridization technique.Three genes, mip,prx and ompA were identified as R-type specific genes. Attention was focused on the mip, and a recombinant Mip-like protein and the deficient mutant were created. The recombinant protein reacted with the patients sera, suggesting the production of the Mip-like protein in periodontal lesions. The mutant was used for an invasion assay and demonstrated impaired ability for the invasion of the host cells. The expression of the mip, prx and ompA may be enhanced in the host, and the Mip may play an important role for invasion of A.actinomycetemcomitans.2.New system for microbiological examination.Microflora of subgingival plaque is a very complicated community. Therefore, microbiological examination is required for the analysis of periodontal pathogens and virulence factors in vivo. In the current study, we established a new system for the examination. Loop-mediated isothermal amplification (LAMP) method was employed for the system. Primers were designed from the nucleotide sequence of 16S ribosomal RNA gene, and the LAMP method detected periodontal pathogens with high sensitivity, specificity and rapidity.