口腔癌是頭頸部癌症中最常見的類型，嚴重牙周炎被認為是導致口腔癌的高危險因子。中間普雷沃氏菌 (Prevotella intermedia) 是一隻格蘭氏陰性絕對厭氧細菌，經常在牙周炎或急性齒齦炎等口腔疾病患者中發現，且可以從患者病灶處被分離出來因此被認為是會引起牙周炎的主要細菌之一。近年來隨著菌相研究興盛，有越來越多研究顯示出口腔菌叢與口腔癌之間的關係，其中，中間普雷沃氏菌被發現在口腔癌患者中的數量遠高於一般患者。然而，我們目前仍然不清楚中間普雷沃氏菌在口腔癌發展過程的作用。在本篇研究中，我們利用細胞增生實驗找出中間普雷沃氏菌對口腔細胞的影響，我們使用來自成功大學附設醫院的臨床病人口水檢體，從中以KVLB (Kanamycin Vamcomycin Laked Blood plate)分離培養基分離出中間普雷沃氏菌。有趣的是，從細胞增生實驗結果中我們發現，和中間普雷沃氏菌標準株BCRC14477經過24小時培養後，SG 細胞 (Smulow-Clickman gingival cells, 人類牙齦細胞) 具有顯著增生情形，同樣的細胞刺激增生效果也發生在與BCRC14477菌株的條件培養基 (conditioned medium) 共同培養24小時。我們將條件培養基進行加熱及利用過濾器過濾後發現培養基中細菌分泌的刺激物質具有熱不穩定特性，且其大小為30kD以上，經過文獻查詢後，得知中間普雷沃氏菌會分泌一種半胱胺酸蛋白酶- interpain A，我們假設SG 細胞增生效果是由於蛋白酶刺激細胞上的受體所造成。為了驗證這個假設，我們在細胞增生實驗中使用E64 (蛋白酶抑制劑) 及FSLLRY (PAR 2受體拮抗劑)，發現兩者皆可以抑制條件培養基刺激的細胞增生效果。接著，為了進一步確認不同菌株間的差異，我們利用毛細管電泳串聯質譜儀 (CE/LC MASS) 分析菌株的條件培養基中的成分，發現BCRC14477會分泌更多的interpain A蛋白酶，比另一株不會刺激增生的臨床菌株H31226高了13倍。這項發現為中間普雷沃氏菌的感染對口腔細胞的影響提供了一個新的研究方向，在感染過程中活化的訊號路徑可能在口腔癌發展過程中扮演重要作用。

Oral cancer, the most common type in Head and Neck Cancers, is suspected developing from severe periodontitis. Prevotella intermedia, a Gram-negative bacterium, is proposed as an important role in periodontitis due to the isolation from lesions of patients. Increasing studies have reported the strong correlation of oral microbiota with oral cancer, and higher abundance of P. intermedia is usually found in saliva of oral cancer patients. However, the role of P. intermedia during development of oral cancer is still unclear. In vitro cell proliferation assay was used to figure out the interaction between bacteria and epithelial cell line. We obtained clinical samples from NCKU hospital, and isolated P. intermedia strains by using KVLB plates. Interestingly, the standard strain BCRC14477 significantly promoted SG cell (human gingival epithelial cell) proliferation after 24 hours. Furthermore, equivalent effect was also found when treated SG cells with bacterial condition medium (CM) of BCRC14477. The results of heat-inactivated CM and CM separated by the centrifugal filters with several molecular weight cut-offs demonstrated the active component may be the secreted proteins from some P. intermedia strains. After literature search, we found P. intermedia could secret a cysteine protease interpain A and we hypothesized the active component is interpain A. To prove this hypothesis, E64 (cysteine protease inhibitor) and FSLLRY (PAR 2 antagonist) were applied to cell proliferation assay respectively and both could block the cell proliferation enhancement of condition medium. To further confirm the difference between strains, the secreted protein profiles were analyzed by CE/LC MASS. BCRC14477 strain, which enhanced more cell proliferation, secreted thirteen times more interpain A protease than another strain. This finding provides a new direction that some signal pathways were stimulated due to some P. intermedia strains infection, and it might play a role in the oral cancer development.

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