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研究生:許芳瑜
研究生(外文):Fung-Yu Hsu
論文名稱:新型蛋白C129對人類主動脈平滑肌細胞的抑制增生作用
論文名稱(外文):Anti-proliferative activity of a novel protein C129 in human vascular smooth muscle cells
指導教授:陳俊翰陳俊翰引用關係
指導教授(外文):Jiun-Han Chen
學位類別:碩士
校院名稱:元培科技大學
系所名稱:醫學檢驗生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
畢業學年度:102
語文別:中文
論文頁數:70
中文關鍵詞:平滑肌細胞肺炎鏈球菌動脈粥狀硬化C129
外文關鍵詞:vascular smooth muscle cellStreptococcus pneumoniaatherosclerosisC129
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  • 收藏至我的研究室書目清單書目收藏:0
動脈粥狀硬化症(atherosclerosis)是一個臨床上重要的課題,此病症會導致其他嚴重的致死疾病,像是心肌梗塞或主動脈瘤等。動脈粥狀硬化症是一個緩慢的發炎過程,形成的原因複雜,包含了白血球聚集、內皮細胞的損傷、平滑肌細胞增生和移行等等。其中,平滑肌細胞的增生和移行在動脈粥狀硬化症中扮演了重要的角色,而抑制平滑肌增生是治療動脈硬化的一個方向。肺炎鏈球菌(Streptococcus pneumoniae)是一個上呼吸道常見的感染菌株,利用其不同的毒力因子間的相互作用來入侵宿主並造成傷害。本研究自肺炎鏈球菌的基因庫中隨機選取了數個蛋白片段,探討其對人類平滑肌細胞增生的影響和細胞內機轉,藉此評估此蛋白用於治療動脈粥狀硬化的可能性。本研究中我們利用細胞存活率分析(MTS assay)發現其中一個自肺炎鏈球菌基因庫選取的蛋白片段C129具有抑制生長因子誘發血管平滑肌細胞增生的能力,但C129蛋白對血管平滑肌細胞具有高毒性。另外一個截短的蛋白C129-2具有相似的抗增生能力,但相對的毒性較低。該片段有用於治療動脈粥狀硬化或血管再阻塞的可能性。我們利用西方墨點法(western blot)觀察此抑制平滑肌細胞生長作用的細胞內機轉,發現在細胞內訊息傳遞路徑中沒有觀察到明顯的抑制作用,因此推測C129-2蛋白可能藉由影響細胞週期或其他機制來抑制平滑肌細胞的生長。C129蛋白可能具有臨床治療的潛力,然而,詳細機制則需要更進一步的研究。
Atherosclerosis is important issue in clinical practice. This disease may lead to severe outcome such as myocardial infarction or aortic aneurysm. The process of atherogenesis is a chronic inflammatory response, included of leukocytes activation, vascular endothelium damage and smooth muscle proliferation. Vascular smooth muscle proliferation and migration play an important role in atherogenesis, inhibition of VSMC proliferation might be a direction in treatment of atherosclerosis.
Streptococcus pneumoniae is a common encountered microbe in upper respiratory tract. Several virulent factors have been reported. In this study, we obtained several fragments from the gene library of Streptococcus pneumoniae by random cloning and study the clones in vascular smooth muscle proliferation. Results of MTS demonstrated that one of the clones (C129) have the anti-proliferation activity in vascular smooth muscle cells. However, C129 is with high toxicity to VSMCs. Truncated protein C129-2 has similar anti-proliferative activity but lower toxicity. This fragment may have the potential in treatment of atherosclerosis or restenosis. We also investigated the intracellular signals by western blotting, but no significant change was found included of MAPK and Akt pathways. The anti-proliferative potential might mediated by other factor in cell cycle. We assumed that C129-2 may be the candidate in treatment of atherosclerosis. However, the detail mechanisms need more studies.

誌謝 I
中文摘要 II
英文摘要 IV
目錄 VI
圖目錄 IX
中英文對照表 X
第一章 緒論 1
1.1 研究動機 1
1.2 血管的架構 2
1.3 動脈粥狀硬化 3
1.4 平滑肌細胞 5
1.5 肺炎鏈球菌 6
1.6 肺炎鏈球菌的致病因子 7
1.7 肺炎鏈球菌與平滑肌的關係 9
1.8 細胞訊息傳遞 10
1.9 細胞週期 12
第二章 研究材料與方法 14
2.1 研究材料: 14
2.2 以JM109(DE3) 勝任細胞表現C129蛋白質體DNA 15
2.3 大量表現與純化C129蛋白: 15
2.4 內毒素濃度測定 16
2.5 蛋白質濃度測定法 16
2.6 蛋白質電泳 18
2.7人類主動脈平滑肌細胞培養: 19
2.8 C129蛋白對人類主動脈平滑肌細胞的毒性試驗: 19
2.9 C129蛋白對人類主動脈平滑肌細胞的生長抑制試驗: 20
2.10 細胞存活率分析 20
2.11 細胞計數: 22
2.12 西方墨點法: 23
2.13 細胞時間點試驗: 24
2.14 資料處理與統計分析: 25
第三章 結果 26
3.1 C129蛋白對人類主動脈平滑肌細胞的毒性 26
3.2 C129蛋白對人類主動脈平滑肌細胞生長的抑制情形 26
3.3 C129-1蛋白對人類主動脈平滑肌細胞的毒性 26
3.4 C129-1蛋白對人類主動脈平滑肌細胞生長的抑制情形 27
3.5 C129-2蛋白對人類主動脈平滑肌細胞的毒性 27
3.6 C129-2蛋白對人類主動脈平滑肌細胞生長的抑制情形 27
3.7 C129-3 C70A蛋白對人類主動脈平滑肌細胞的毒性 27
3.8 C129-3 C70A蛋白對人類主動脈平滑肌細胞的抑制生長情形 28
3.9 C129-2蛋白對FBS誘發MAPK-ERK1/2 pathways活化作用影響 28
3.10 C129-2蛋白對FBS誘發p38 MAPK pathways活化作用影響 28
3.11 C129-2蛋白對FBS誘發MAPK-JNK pathways活化作用影響 29
3.12 C129-2蛋白對FBS誘發Akt/mTOR pathways活化作用影響 29
第四章 討論 30
第五章 圖與圖表說明 36
參考文獻 49


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