Development of a new diagnostic strategy of periodontal disease based on the gene profile associated with host defense mechanisms.

本研究的目的是建立一种新的基于基因谱的诊断策略，以确定牙周病病变中发生的宿主-寄生虫相互作用。我们已经成功开发了2种应用敏感PCR技术的方法；1)利用16sRNA基因RT-PCR检测少量菌斑样本中25种牙周病细菌；2)采用实时荧光定量PCR技术定量牙周病患者牙龈组织活检中多种细胞因子mRNA水平。为了进行微生物分析，从龈下和龈上菌斑样本中提取基因组DNA，并通过PCR检测16S rRNA基因进行细菌检测。牙周炎(P)组龈下菌斑中牙龈卟啉单胞菌、牙密螺旋体、中间普雷沃菌等11种细菌的检出率显著高于健康(H)组。仅在P组检测到牙龈卟啉单胞菌，提示该细菌可作为牙周病鉴定的候选者。龈上菌斑的菌群也反映龈下菌斑的菌群。实时荧光定量PCR检测细胞因子mRNA显示与半定量常规RT-PCR相当的水平。接下来，我们应用实时技术定量多个细胞因子基因水平，并将mRNA水平与临床参数进行比较。在本研究的靶细胞因子中，IL-6和IL-1α与PPD和GI呈强正相关。牙周炎患者牙龈标本IL-1ra mRNA水平持续升高，提示其在牙周病变中具有免疫抑制活性。我们的数据表明，这些方案可以为细胞因子网络和宿主-寄生虫相互作用的复杂性提供强有力的见解。该网络的阐明可能会在个体水平上阐明疾病敏感性和在位点水平上阐明疾病活性的机制。

The purpose of this study is to develop a new diagnostic strategy based on the gene profile that determine the host-parasite interaction occurring in periodontal disease lesion. We have successfully developed 2 methods applying the sensitive PCR technique ; 1)RT-PCR of 16sRNA genes to detect 25 periodontopathic bacteria from small amount of plaque samples, 2)real-time PCR protocol for quantification of multiple cytokine mRNA levels in the gingival tissue biopsies from periodontal disease patients. For microbial analyis, Genomic DNA was extracted from subgingival and supragingival plaque samples, and bacterial detection was performed by PCR of the 16S rRNA genes. the detection frequencies of 11 bacteria including Porphyromonas gingivalis, Treponema denticola and Prevotella intermedia in subgingival plaque were significantly higher in Periodontitis (P) group than in Healthy (H) group. P. gingivalis was detected only in P group, suggesting this bacterium as the candidate for identifying periodontal diseases. The bacteria flora of supragingival plaque was also found to reflect that of subginginval plaque. Real-time PCR detection of cytokine mRNA showed a comparable levels of semi-quantitative conventional RT-PCR. We next applied the real-time technique for the quantification of multiple cytokine gene levels, and compared the mRNA levels with clinical parameters. Among the target cytokines in this study, IL-6 and IL-1α showed a positive and strong association with PPD and GI. IL-1ra mRNA levels of gingival specimens from periodontitis patients were consistently high, suggesting the evoke of immunosuppressive activity in periodontal lesion. Our data suggest that these protocols could provide powerful insights into the complexities of the cytokine network and host-parasite interactiuon. The elucidation of the network may clarify the mechanisms of disease sensitivity at individual level and disease activity at site level.