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系統識別號 U0026-0812200911121811
論文名稱(中文) 探討披衣菌在平滑肌細胞中造成感染時一氧化氮的影響
論文名稱(英文) Effects of Nitric Oxide on Chlamydiae Infection in Smooth Muscle Cells
校院名稱 成功大學
系所名稱(中) 醫事技術學系
系所名稱(英) Department of Medical Technology
學年度 92
學期 2
出版年 93
研究生(中文) 陳儀玲
研究生(英文) Yi-Ling Chen
學號 t3691402
學位類別 碩士
語文別 中文
論文頁數 68頁
口試委員 口試委員-邢福柳
指導教授-林尊湄
口試委員-王貞仁
口試委員-余俊強
中文關鍵字 披衣菌  平滑肌細胞  一氧化氮 
英文關鍵字 Nitric oxide  Smooth muscle cells  Chlamydiae 
學科別分類
中文摘要   披衣菌(Chlamydia)是屬於一種絕對細胞內寄生的革蘭氏陰性病原菌,此種病菌在臨床上會造成各種不同典型的慢性疾病,而潛伏性的披衣菌感染目前被認為是造成這些病變的主要致病機轉。宿主細胞受到披衣菌感染後會產生很多免疫反應,其中一氧化氮 (nitric oxide, NO),是一個已知殺菌及抗發炎的物質,其產量的改變被認為是一種可能與披衣菌潛伏性感染有關的機制。肺炎披衣菌(C. pneumoniae)主要感染呼吸道的細胞,後續會造成慢性氣喘、支氣管炎、動脈硬化生成等疾病,因此我們認為平滑肌細胞 (smooth muscle cells, SMC),可能是其感染的重要目標。在此研究中,我們想探討在披衣菌感染平滑肌細胞時,NO所扮演的角色。實驗結果證明,大白鼠冠狀動脈平滑肌細胞株 (A7r5)被C. pneumoniae感染時,與披衣菌在上皮細胞所產生的結果相似,此細胞內會發展出典型的包涵體 (inclusion body),但此時C. pneumoniae藉由抑制eNOS的表現量進而降低細胞內NO的產量。我們進一步以NO提供者 (sodium nitroprusside, SNP)處理細胞時,披衣菌包涵體形成單位 (chlamydial inclusion-forming units)則會相較於對照組減少2 log 10單位。相較之下,以iNOS抑制劑(NG-nitro-L-arginine methyl ester, L-NAME)處理後,則會增加披衣菌的生長。同時我們也證明NO所造成的影響相較之下,主要對於披衣菌的早期的生長 (<24 hr)有較高的專一性。進一步分析被感染的細胞中C. pneumoniae表現的基因發現,晚期基因omcB和早期基因hsp60表現量的比值會受到SNP的抑制,並與其劑量呈相關性,但此現象並不會在細胞受到L-NAME處理時發生。總結而言,在我們初步的結果顯示披衣菌在平滑肌細胞生長時,會抑制NO的產生,才可促使披衣菌在細胞內生長發育。然而外加NO是否可以作為一個抗披衣菌的物質?其真正的角色則須要在之後的實驗中做進一步的確認。
英文摘要   Intracellular bacteria of the genus Chlamydia cause numerous typical chronic diseases, frequently with debilitating sequelae. Persistent chlamydial infections have been proposed as a means whereby Chlamydiae evade immune responses of infection. Altered production of nitric oxide (NO), a known bactericidal and anti-inflammatory agent represents one possible mechanistic link. Smooth muscle cells (SMC) might be important targets for airway remodeling in chronic asthma, bronchitis, and atherogenesis associated with C. pneumoniae. In this study, we evaluate the NO effect on chlamydial infection in SMC. Our results demonstrated that infection of rat aortic SMC, A7r5 cell line with C. pneumoniae resulted in the development of typical inclusion bodies in the cells, similar to those seen in epithelial cells. NO production was inhibited by C. pneumoniae and mediated by down-regulation of eNOS expression. Treatment with NO donor, sodium nitroprusside (SNP) reduced chlamydial inclusion-forming units by 2 log10 units as compared with control values. In contrast, chlamydial growth was enhanced by iNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME). Furthermore, the effect of NO was demonstrated to be more specifically important in the early phase of chlamydial development (<24 hours). The analysis of bacterial messenger RNA in the cultures showed that the expression ratio of omcB/hsp60 was down-regulated by SNP and it was also dose-dependent, whereas the expression ratio was not changed by L-NAME treatment. In conclusion, our preliminary data suggests that chlamydial growth in SMC inhibit NO production, which is a necessary effecter molecule involved in the early phase of chlamydial development. However, the role of NO donor as an antichlamydial effector should be clarified by further investigation.
論文目次 中文摘要 I
英文摘要 II
誌謝 III
目錄 IV
圖目錄 VI
縮寫檢索表 VII
儀器及藥品 VIII
第一章 緒論
(一) 披衣菌概論 1
(二) 披衣菌的基本構造介紹及其與革蘭氏陰性菌的差異性 2
(三) 肺炎披衣菌感染在平滑肌細胞的重要性 3
(四) 一氧化氮之概論 6
(五) 披衣菌的潛伏感染 7
(六) 本研究的主要目的 9
第二章、材料與方法
(一)細胞培養 10
(二)披衣菌大量培養 13
(三)披衣菌效價測定 14
(四)反轉錄反應與聚合酶連鎖反應 17
(五)西方墨點法 20
(六)細胞免疫螢光染色 25
(六)亞硝酸的定量 29
(七)一氧化氮存在與否對C. pneumoniae生長之影響 30
(八)SNP或L-NAME的處理對披衣菌hsp60或omcB gene表現的影響 33
第三章結果
(一)肺炎披衣菌在平滑肌細胞中的生長情形 35
(二)披衣菌感染改變細胞中eNOS及iNOS mRNA及蛋白質的表現量 35
(三)披衣菌的生長抑制細胞亞硝酸的產生 36
(四)不同SNP或L-NAME濃度對披衣菌生長的影響 36
(五)不同時間加SNP或L-NAME對披衣菌生長的影響 37
(六)SNP或L-NAME的處理對披衣菌hsp60或omcB gene表現的影響 38
第四章、討論 39
第五章、參考文獻 46
圖 51
附錄 64
表格 67
自述 68
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