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研究生:何梓豪
研究生(外文):Chi-Hou Ho
論文名稱:研究茶成份調控尼古丁受體作用於單核球引起動脈硬化及其未來臨床應用
論文名稱(外文):Investigate the roles of Tea extract through modulating neuro-Nicotine Acetylcholine receptor on monocytes involved in atherosclerosis and it''s potential clinical application
指導教授:何元順
指導教授(外文):Yuan-Soon Ho
口試委員:潘敏雄陳志榮楊順發
口試委員(外文):Min-Hsiung PanChih-Jung ChenShun-Fa Yang
口試日期:2013-06-07
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:醫學檢驗暨生物技術學系
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
論文出版年:2013
畢業學年度:101
語文別:中文
論文頁數:115
中文關鍵詞:尼古丁受體
外文關鍵詞:Nicotine Receptors
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近年來腦血管疾病、糖尿病、高血壓疾病與抽煙被證實會增加冠狀動脈疾病的風險,而在罹患動脈粥狀硬化病人之血管斑塊中,發現大量泡沫細胞(單核球為其前驅物)之累積是動脈粥狀硬化發生的誘因之一。然而,煙害與泡沫細胞之形成機制目前尚未明朗。因此本研究中我們希望釐清尼古丁(香煙之主要成分)與泡沫細胞形成之交互關係,而天然物之添加是否可抑制泡沫細胞之形成進而達到預防動脈硬化之作用。
我們發現人類單核球能高表現尼古丁受體─α9-nAchR(相較於其他種類之白血球),而尼古丁環境之曝露能增加其表現量及泡沫細胞之形成。為了進一步釐清尼古丁受體在煙害與泡沫細胞之形成所扮演之角色,以及尋找有效減緩煙害之天然物,根據先前之研究成果發現兒茶素(茶中成份)能有效抑制α9-nAchR之表現以及癌細胞之生長作為基礎,啟發我們進行人體飲用普洱茶試驗。我們發現飲用普洱茶能有效降低各受試者體內單核球α9-nAchR之表現量以及減少煙害誘發之泡沫細胞。此外,抽煙者試驗前期單核球α9-nAchR之表現量以及曝露於煙害環境而誘發之泡沫細胞皆高於非吸煙者。從統計分析結果發現,服用普洱茶膠囊後能有效改善人體多項生化指標之表現,進而改善受試者身體之健康狀況。為了印證人體試驗之研究成果,我們利用紅茶、綠茶之粗萃取物進行細胞實驗,我們發現兩種茶萃取物皆能抑制單核球上α9-nAchR表現以及減少泡沫細胞形成。說明茶中皆具有某些活性成份(如:EGCG)能減緩煙害對於單核球之影響,進而減少泡沫細胞形成。
本研究發現,尼古丁環境之曝露能刺激尼古丁受體表現及誘導泡沫細胞之形成,而此反應能受到茶中活性成份所抑制。然而,尼古丁受體與泡沫細胞形成之機轉還需繼續釐清。在未來應用層面上,我們希望進一步證實茶中活性成份能有效減緩煙害以及泡沫細胞之形成,進而推廣喝茶之好處以及臨床上之應用。
Recently, stroke, diabetes, high blood pressure and smoking were proved to be the major risk factors of cardiovascular diseases. There were a large number of foam cells which were formed by monocytes, found in the atheroma plaques of atherosclerosis patients. Though, the tobacco and foam cell formation mechanism is not yet apparent, which anticipate us to make clear of nicotine (cigarettes is the major component) and foam cell formation interactive relationship, and whether the addition of natural products would inhibit the formation of foam cells and as a result to achieve the prevention of atherosclerosis. We investigate that human monocytes could be high performance in nicotine receptor ─ α9-nAchR (in comparison to other types of white blood cells), and nicotine exposure to environment can increase the performance of its volume and foam cell formation.
Previous research found that catechins (tea ingredients) could effectively reduce the performance of α9-nAchR and the growth of cancer cells, hence, we are inspired to clarify that tobacco and nicotine receptors in the formation of foam cells and the function played, as well as to discover the effective mitigation of natural products of tobacco; therefore, human trials drinking tea was preformed.
We found that drinking tea can effectively reduce the α9-nAchR performance of each individual monocyte and the amount of tobacco-induced foam cell. In addition, theα9-nAchR monocytes expression and exposure to tobacco environment induced foam cell in the pre-trail smokers, which were higher than non-smokers. From the statistical results, taking tea capsules can effectively improve number of biochemical parameters, thereby improving the health status of the human body. In order to prove human trials and study results, we make use of black tea, green tea extract for cell crude experiments, and discover that two kinds of tea extracts inhibitα9-nAchR monocyte performance and reduce foam cell formation. All tea have certain active ingredients (eg: EGCG), which can slow down the monocytes, thereby reducing foam cell formation.
The study investigate that nicotine exposure to environment can stimulate nicotinic receptor expression, hence, inducing the formation of foam cells. This reaction can be inhibited by the active ingredient in tea. However, the nicotine receptors and foam cell formation mechanisms should to be further clarified. Application level in the future, we wish to prove tea active ingredients attenuate the effect of smoking and reduce foam cell formation. Finally, we promote the advantages of tea drinking and it’s clinical applications.
誌謝 II
中文摘要 IV
英文摘要 VI
目錄 VII
第一章 緒論(Introductions) 1
背景 2-8
第二章 研究動機 (Study Aims) 9
研究動機 10
第三章 材料與實驗方法 (Materials and Methods) 11
實驗材料 (Materials) 12
藥品試劑 13-17
常用儀器 17-18
常用溶液 19-20
實驗方法 (Methods) 21
普洱清茶膠囊之製備 21
人體飲用普洱茶試驗 21-22
人體周邊血液白血球之分離 22
人體周邊血液單核球細胞之純化 22-23
細胞培養 23-24
反轉錄-聚合酶連鎖反應 24-25
即時聚合酶連鎖反應 25-26
流式細胞儀偵測法 27
西方墨點法 28-30
免疫組織化學染色法 31-32
單核球細胞在尼古丁環境下之培養 32
單核球細胞在氧化型低密度脂蛋白膽固醇環境下之培養 33
Oil Red O染色法 33
Coumarin VI染色法 34-35
利用ELISA對血清、尿液中之Cotinine進行訂量 35-36
第三章 實驗結果 (Results) 37
觀察人體血液細胞中尼古丁受體之表現種類及位置 38
觀察人體血液細胞中尼古丁受體表現之細胞族群 38-39
人類周邊血液單核球之分離純化 40-41
尼古丁可刺激單核球並增加其尼古丁受體及氧化型低密度脂蛋白膽固醇受體之表現 41-43
茶萃取物活性成分抑制尼古丁所誘發之受體表現 43-44
尼古丁刺激人類單核球之活化及泡沫細胞之形成 44-45
茶萃取物有效阻礙尼古丁刺激人類單核球之活化及減緩泡沫細胞之形成 45-46
藉由Coumarin VI觀察泡沫細胞形成之方法建立 47-48
利用顆粒性對Coumarin VI訂量系統,探討尼古丁及紅、綠茶萃取物對泡沫細胞形成之影響 49-51
人體飲用普洱茶試驗之收案進度及受試者健康狀況分佈 51
人體飲用普洱茶能降低人類周邊血液單核球尼古丁受體之表現 51-54
人體飲用普洱茶試驗有效改善受試者之健康狀況 54-56
人體飲用普洱茶試驗能降低人類周邊血液單核球對尼古丁之敏感度、減緩泡沫細胞之形成 57-60
從人類血管斑塊及脂肪壞死之組織切片中發現尼古丁受體α9-nAchR之表現 60-62
第四章 討論 (Discussions) 63
第五章 圖表 (Tables and Figures) 79
第六章 參考資料 (References) 110
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