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

首先，转录水平的牙龈卟啉单胞菌致病基因，已被克隆先前进行了比较，通过CAT测定。两者之间无显著性差异。其次，虽然尝试通过紫外线和丝裂霉素C处理从培养物中分离牙龈卟啉单胞菌可感染的噬菌体，但尝试失败。本文尝试用平板法分离牙龈卟啉单胞菌自溶素作为新的靶点。将牙龈卟啉单胞菌涂布在含有牙龈卟啉单胞菌细胞（最终OD_ ; 2.0）的富集BHI琼脂上<550>，并在37℃下孵育18 h。牙龈卟啉单胞菌菌落周围没有形成清晰的区域。在牙龈卟啉单胞菌染色体DNA数据库中搜索与噬菌体基因同源的基因，得到编码N-乙酰胞壁酰-L-丙氨酸酰胺酶的ampD基因，通过聚合酶链反应（PCR）扩增该基因，并将其插入pBluescriptII KS+中。将得到的质粒（pPGAMP）导入大肠杆菌K12。将该培养基涂布在含有牙龈卟啉单胞菌细胞的富集BHI琼脂上。一个小的清晰的区域形成了一个殖民地周围。增加ampD基因转录水平的实验目前正在进行中。同时对60例患者的唾液和龈沟液进行PCR微生物鉴定。唾液和龈沟液是检查牙龈卟啉单胞菌的有效样本。探诊出血与牙龈卟啉单胞菌之间的关系以及牙周袋深度与牙龈卟啉单胞菌之间的关系是显著的。

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.