牙齦紫質單孢菌臨床分離株16S rRNA及毒性因子 Arg-gingipain A 之演化分析

Phylogenetic analyses of the 16S rRNA gene and Arg-gingipain A of Porphyromonas gingivalis clinical isolates

陳宜欣(Yi-Hsin Chan)

指導教授 ： 林克亮 ； 陳志豪

中山醫學大學/醫學科技學院/醫學檢驗暨生物技術學系碩士班/碩士(2015年)

摘要

背景與目的: 牙周病在台灣的流行率很高，尤以牙齦紫質單孢菌 (Porphyromonas gingivalis, P. gingivalis) 為人類破壞型牙周病的主要致病菌。牙齦紫質單孢菌會產生一種特別的半胱胺酸蛋白酶，被稱為牙齦素 (Arg-gingipain, RgpA)。其中，牙齦素是導致慢性牙周炎的重要致病因。我們的研究目的是去分析不同品系間的臨床P. gingivalis 在16S RNA與 RgpA基因上在演化時序上的差異。
方法: 從牙周患者的牙齦囊袋採取口腔檢體，將臨床檢體以DNA萃取，經PCR擴增其16S RNA以及RgpA基因後以定序結果作為分析的依據，並使用MEGA 6.01進行演化分析。此外，將分析得到的16S RNA基因提交給國家生物訊息中心的基因資料庫做為保存，並申請Accession numbers。
結果: 本實驗共收集了54筆臨床檢體。得到未經培養的檢體之陽性率38.9%，經培養後再分離之陽性率18.5%。其中，有10株臨床株的16S RNA 與RgpA基因被標定擴增，以定序結果結果作為討論。P. gingivalis 在16S RNA與其他參考株分成兩個群，而臨床分離株彼此間也分成兩個群落。在RgpA的基因演化可以發現P.g_CSMU24、P.g_CSMU33、P.g_CSMU38、P.g_CSMU41 和 P.g_CSMU51與其它分離株相比，是有較顯著差異的。
結論: P. gingivalis 在16S RNA 以及 RgpA基因上呈現穩定而緩慢的演化。在這些基因變異與人類疾病嚴重程度的變化關係是有待進一步的研究。

關鍵字

牙齦紫質單孢菌 ； 牙周病 ； 牙齦素 ； 16S RNA 基因 ； 演化分析

並列摘要

Background and aim: Periodontitis has a high prevalent rate in Taiwan and Porphyromonas gingivalis is a major pathogen in destructive periodontal disease in humans. Porphyromonas gingivalis produces a unique class of cysteine proteinases termed gingipains that comprises Arg-gingipain A
(RgpA). Gingipains have been implicated in the pathogenicity of Porphyromonas gingivalis, a major etiologic agent of chronic periodontitis. The purpose of our studies is to determine the phylogenies of 16S RNA gene and Arg-gingipain (RgpA) gene of clinical P. gingivalis strains among different years.
Methods: Samples were collected from gingival pockets of periodontitis patients. All samples were treated with genomic DNA extraction, and PCR was used to amplify 16S RNA and RgpA genes for DNA sequencing. DNA sequences of all amplicons were subjected to phylogenetic analysis by using MEGA 6.01. In addition, the DNA sequences of 16S RNA genes were also deposited and submitted to National Center for Biotechnology Information Genbank database to acquire accession numbers.
Results: Fifty four samples were collected from gingival pockets of periodontitis patients. The positive rates of P. gingivalis were 38.9% and 18.5% by PCR and by culture, respectively. 16S RNA and RgpA gene fragments from ten samples were amplified and sequencing. Phylogenetic analysis of 16S RNA genes indicated that P. gingivalis strains were divided into two clusters and our isolates from each other were similar to two clusters. Similarly, phylogenetic analysis of RgpA genes showed that compared with the other strains, the P.g_CSMU24、P.g_CSMU33、P.g_CSMU38、P.g_CSMU41 and P.g_CSMU51 of our clinical isolates were significantly different.
Conclusion: P. gingivalis was steadily evolved on both 16S RNA gene and RgpA gene. The correlation between gene variations, fitness on human being and disease severity is requires further studies.

並列關鍵字

Porphyromonas gingivalis ； periodontitis ； Arg-gingipain ； 16S RNA gene ； phylogenetic analysis

參考文獻

曾春祺、袁國、廖連宏、溫敏杰、張智仁。1998。台南市民眾的口腔狀態及治療需求研究。中華牙周醫誌。3:205-212。

曾春祺、袁國、黃振勳、汪忠川、溫敏杰。1998。阿蓮地區志願受檢民眾的口腔狀態及治療需求研究。中華牙周醫誌。3:153-158。

American Academy of Periodontology. 2000. Parameter on chronic periodontitis with slight to moderate loss of periodontal support. Journal of Periodontology. 71:853–855.

American Academy of Periodontology. 2003. Diagnosis of periodontal diseases (position paper). Journal of Periodontology. 74:1237-1247.

Armitage, G.C. 1999. Development of a classification system for periodontal diseases and conditions. Ann Periodontol. 4:1-6.