臺灣博碩士論文加值系統

金黃色葡萄球菌 (S. aureus)是常見的革蘭氏陽性菌，它會造成廣泛的人類免疫疾病，像是氣喘和慢性阻塞性呼吸性等疾病。而許多免疫反應是藉由血管內皮與循環性多形核白血球之間複雜的交互作用所調節。許多的研究指出表皮細胞膜表現的黏附因子在發炎反應中扮演重要的角色。目前已知ICAM-1 (細胞間黏附分子)和VCAM-1 (血管黏附分子)在許多不同的細胞中都會有表現，但是在人類肺泡上皮細胞 (HPAEpiCs)中，金黃色葡萄球菌誘發VCAM-1的表現其機制尚未明瞭。利用西方點墨法、RT-PCR、Real-time PCR和單核球黏附分析可以證明金黃色葡萄球菌會誘發人類肺泡上皮細胞產生VCAM-1的mRNA和蛋白質的表現；且給予細胞不同訊號分子 (如PDGFR、MyD88、c-Src、PI3K、ERK、p38、JNK、NF-kB、AP-1或p300)的抑制劑或干擾性RNA(siRNA)分別都會減弱VCAM-1的表現。此外也觀察到這些訊號分子在金黃色葡萄球菌都有活化的現象；若前處理不同的抑制劑之後，分別會有不同程度的抑制或無影響，藉以得知其互相的上下游關係。總結來說，在此篇研究當中我們觀察到VCAM-1的表現是經過轉錄和轉譯的層面。金黃色葡萄球菌的感染刺激之下，TLR2會與c-Src、MyD88形成複合體並且活化兩條路徑，分別為PI3K/Akt/ERK1/2/JNK1/2以及PDGFR/p38/JNK1/2。這兩條路徑都會活化轉錄因子NF-kB、AP-1和p300並使人類肺泡上皮細胞產生出具有功能性的VCAM-1。本篇論文對於金黃色葡萄球菌作用在人類肺泡上皮細胞產生呼吸系統發炎反應的訊號傳遞機制提供了新的觀點，希望可以對於肺部發炎相關致病機制以及未來臨床治療或研究提供參考方向。

Staphlococcus aureus (S. aureus), the common Gram-positive bacteria, causes a wide range of human inflammatory diseases, such as asthma and chronic obstructive pulmonary disease (COPD). These inflammatory responses are mediated by complex interactions between both circulating polymorphonuclear cells (PMNs) and the vascular endothelium. Several studies have indicated that expression of adhesive molecules on the cell surface of epithelial cells plays a critical role in these inflammatory responses. Although S. aureus has been shown to induce ICAM-1 or VCAM-1 expression in various cell types, the mechanisms underlying S. aureus-induced VCAM-1 expression in human pulmonary alveolar epithelial cells (HPAEpiCs) remain unknown. Western blot, Real time-PCR, and monocytes adhesion analyses showed that in HPAEpiCs, S. aureus induced VCAM-1 mRNA and protein expression in a time-dependent manner, which were attenuated by the inhibitors of c-Src (PP1), PDGF receptor (AG1296), PI3K (LY294002), JNK (SP600125), p38 MAPK (SB202190), MEK1/2 (U0126), AP-1 (Tanshinone IIA), NF-kB (Bay11-7082) and p300 (GR343), or transfection with siRNA of TLR2, MyD88, p42, p38, JNK1, c-Src, PDGFR, Akt, p65, c-Jun, ATF2, p300. These results suggest that PDGFR transactivation participates in VCAM-1 expression induced by S. aureus. Accordingly, S. aureus-stimulated phosphorylation of Akt, p38 MAPK, JNK and ERK1/2 was inhibited by pretreatment with PP1, AG1296, LY294002, SP600125, SB202190 or U0126. Taken together, in this study we investigated the effect of S. aureus induced VCAM-1 expression at transcriptional and translational levels. S. aureus-stimulated TLR2 induced association of MyD88 and c-Src, which mediated through two pathways. First, S. aureus activated c-Src, PI3K/Akt, ERK1/2 and JNK1/2. Second, S. aureus activated dependent PDGFR transactivation, p38 and JNK1/2. The two pathways both linked to AP-1, NF-B and p300, and induced VCAM-1 expression which led to monocytes adhesion. These results provide new insight into the mechanisms of S. aureus action, supporting that S. aureus may contribute to promote inflammatory responses involved in the development of respiratory diseases. Increased understanding of signal transduction mechanisms underlying VCAM-1 gene regulation creates opportunities for the development of anti-inflammation therapeutic strategie.

目 錄 (Contents)
論文指導教授推薦書
論文口試委員審定書
長庚大學碩士紙本論文著作授權書. . . . . . . . . . . . . . . . . . . . . . . . . .. . iii
誌謝(Acknowledgements) . . . . . . . . . . . . . . . . . iv
縮寫表 (Abbreviations).. . . . . . . . . . . . . . . . . . v
抑制劑表 (Inhibitors) .. . . . . . . . . . . . . . . . . .vii中文摘要 (Abstract in Chinese.) . . . . . . . . . . . . .viii
英文摘要 (Abstract in English).. . . . . . . . . . . . . ..xi
目 錄 (Contents) . . . . . . . . . . . . . . . . . . . . . x
緒論 (Introduction) . . . . . . . . . . . . . . . . . . . .1
Specific aims . . . . . . . . . . . . . . . . . . . . . . 29材料與方法 (Materials and Methods) . .. . . . . . . . . . .31實驗結果 (Results) . . . . . . . . . . . . . . . . . . . .41
圖表 (Figures and Legends) . . . . . . . . . . . . . . . .58
討論 (Discussion). . . . . . . . . . . . . . . . . . . .100
參考文獻 (References) . . . . . . . . . . . . . . . . . .105

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