Counteractive therapy against periodontal bacteria by introduction of antisense-RNA of virulent genes

首先，用CAT法比较了先前克隆的牙龈卟啉单胞菌致病基因的转录水平。各组间无显著性差异。接下来，虽然尝试用紫外线和丝裂霉素C处理从培养物中分离出可感染牙龈假单胞菌的噬菌体，但未能成功。采用平板法对作为新靶点的牙龈假单胞菌的自溶素进行了分离。将牙龈假单胞菌撒于含有牙龈假单胞菌细胞（最终OD_<550>; 2.0）的强化BHI琼脂上，37℃孵育18 h。牙龈假单胞菌菌落周围未形成清晰区。在牙龈卟啉卟啉染色体DNA数据库中寻找噬菌体基因同源基因，找到编码n -乙酰muramyl - l-丙氨酸氨基酶的ampD基因。该基因经聚合酶链反应（PCR）扩增后插入pBluescriptII KS+中。将合成的质粒（pPGAMP）导入大肠杆菌K12。这种转化被扩散到含有牙龈假单胞菌细胞的富集的BHI琼脂上。在一个变形蚁群周围形成了一小块清晰的区域。提高ampD基因转录水平的实验目前正在进行中。采用PCR法对60例患者的唾液和龈沟液进行微生物鉴定试验。发现唾液和龈沟液是检查牙龈假单胞菌发生的有效样本。探诊出血与牙龈卟啉卟啉有显著关系，牙袋深度与牙龈卟啉卟啉有显著关系。

First, transcriptional levels of the Porphyromonas gingivalis pathogenic genes that had been cloned previously were compared by CAT assay. There was no significant difference among them. Next, though the bacteriophage infectable to P. gingivalis was tried to be isolated from the culture by ultraviolet and mitomicin C treatments, the attempt was failed. Isolation of autolysins of P. gingivalis as a new target was tried by the plate assay method. P. gingivalis was spread on enriched BHI agar containing P. gingivalis cells (final OD_<550>; 2.0) and incubated at 37℃ for 18 h. No clear zone was formed around a P. gingivalis colony. The gene homologous to the bacteriophage gene was searched for the P. gingivalis chromosomasl DNA database and the ampD gene that encodes N-acetylmuramoyl-L-alanine amidase was found. This gene was amplified by polymerase chain reaction (PCR) and inserted into pBluescriptII KS+. The resultant plasmid (pPGAMP) was introduced into Escherichia coli K12. This transformant was spread on enriched BHI agar containing P. gingivalis cells. A small clear zone was formed around a transformant colony. Experiments to increase the transcriptional level of the ampD gene are currently in progress. Also, the microbial identification test was performed on saliva and gingival crevicular fluid of 60 patients by using PCR method. Saliva was found to be an effective sample for examination of occurrence of P. gingivalis as well as gingival crevicular fluid. The relationship between bleeding on probing and P. gingivalis was significant as well as the relationship between pocket depth and P. gingivalis.