博碩士論文 100331011 詳細資訊




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姓名 陳睿翔( Reui-siang Chen)  查詢紙本館藏   畢業系所 生物醫學工程研究所
論文名稱 以血流動力系統探討血管內皮細胞在尼古丁刺激下對層流剪應力之型態異常與自體凋亡之表現變化
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摘要(中) 尼古丁對於血管內皮細胞之影響已被廣泛研究,但由於缺少了血流所造成之層流剪應力,使得許多研究結果無法說明部分臨床統計上的發現。我們以血流動力系統模擬層流剪應力(Laminar Shear Stress;LSS)的流動,對人類臍帶靜脈內皮細胞進行12小時的不同強度刺激(6、8、12 dynes/cm2)並將刺激前後的細胞型態進行量化分析。我們發現,隨著層流剪應力強度的增加,血管內皮細胞的長短軸比變化量(Variation of Aspect Ratio; ?AR)與流軸夾角變化量(Variation of Angle Difference;?AD)兩者都隨之提高。另一方面,我們將同時給予10-4 M尼古丁與12 dynes/cm2 LSS刺激的處理組與單獨給予12 dynes/cm2 LSS刺激的處理組相比,細胞的?AR提升32.5 % (P < 0.05)、?AD提升23.5 % (P < 0.05)。為了瞭解上述的型態重組與自體凋亡之關聯性,我們將沖刷刺激後之細胞進行細胞核染色並量化自體凋亡程度。我們發現細胞在同時接受尼古丁與12 dynes/cm2 LSS刺激下整體凋亡情形比單純給予12 dynes/cm2 LSS刺激時相比,增加了2.5倍 (P < 0.05)。最後,為了證實尼古丁所產生的活性氧自由基會干擾細胞對層流剪應力的型態重組,我們將血管內皮細胞在10-4 M尼古丁、10-4 M抗壞血酸和12 dynes/cm2 LSS刺激後的處理組與10-4 M尼古丁、12 dynes/cm2LSS刺激後的處理組相比,?AR降低24 % (P < 0.05)、?AD降低40 % (P < 0.05);在自體凋亡方面,同時添加抗壞血酸和尼古丁再以12 dynes/cm2 LSS刺激後的處理組,其自體凋亡情形也比僅添加10-4 M尼古丁、12 dynes/cm2 LSS刺激後的處理組降低了24 % (P < 0.05)。此研究說明了層流剪應力在尼古丁對心血管內皮層的傷害中扮演了相當關鍵的角色,同時也提供一項心血管疾病與吸菸之間的關聯性並說明吸菸者較常在肱動脈等處罹患心血管疾病的可能原因。
摘要(英) Nicotine is a major risk factor for various cardiovascular diseases such as atherosclerosis. Although the cytotoxicity of nicotine has been widely investigated, the precise pathogenesis of nicotine-induced endothelial dysfunction remain unclear due to lack of hemodynamic factors in most of prior in vitro studies that play a crucial role in endothelial physiology. To understand how nicotine affects endothelium in vasculature, human umbilical vein endothelial cells were exposed to different laminar shear stress (LSS; 6, 8, and 12 dynes/cm2) with and without 10-4 M nicotine simultaneously in a parallel plate flow system for 12 h, following detections of 1) morphological response to LSS and 2) synergistic effect of nicotine and LSS on cellular apoptosis. Our results showed that cells sheared by 12 dynes/cm2 LSS with nicotine for 12 h excessively elongated and aligned with the flow direction, and exhibited significant apoptosis as compared to the groups with either nicotine or LSS alone (P < 0.05 for both). Furthermore, the irregular morphological reorganization and elevated apoptosis can be mitigated by using ascorbic acid (10-4 M) to scavenge nicotine-induced reactive oxygen species. In summary, our findings demonstrated an essential role of LSS in nicotine-mediated endothelial impairment occurring in the physiological environment.
關鍵字(中) ★ 層流剪應力
★ 尼古丁
★ 人類臍帶靜脈內皮細胞
★ 自體凋亡
★ 動脈粥樣硬化
★ 長短軸比
★ 流軸夾角
★ 型態重組
關鍵字(英) ★ Laminar shear stress
★ Atherosclerosis
★ Nicotine
★ Endothelial cells
★ Apoptosis
★ Morphological reorganization
論文目次 ABSTRACT........vi
誌謝........vii
目錄........viii
圖目錄........x
第一章 緒論........1
1-1 前言........1
1-2 文獻回顧........2
1-2-1 吸菸與心血管疾病之相關聯性........2
1-2-2 吸菸與活性氧自由基之相關聯性........3
1-2-3 層流剪應力與內皮細胞之相關研究........3
1-2-4 尼古丁與自體凋亡之相關研究........4
1-3 研究目的........6
第二章 實驗藥品、儀器與研究方法........7
2-1 實驗儀器與材料........7
2-1-1 血流動力系統 ........7
2-1-2 實驗儀器........15
2-1-3 實驗藥品........15
2-2 實驗方法........17
2-2-1 內皮細胞自臍帶分離之方法........17
2-2-2 內皮細胞繼代培養........18
2-2-3 尼古丁毒性測試........19
2-2-4 流體沖刷實驗 ........20
2-2-5 血管內皮細胞刺激後之型態重組量化分析........21
2-2-6 血管內皮細胞刺激後之自體凋亡定性分析........23
2-2-7 血管內皮細胞刺激後之自體凋亡量化分析........24
2-2-8 數據統計分析 ........25
第三章 實驗結果........26
3-1 血管內皮細胞在尼古丁刺激下之存活率........26
3-2 血管內皮細胞在尼古丁與層流剪應力共同刺激下之型態重組分析.......28
3-3 血管內皮細胞在尼古丁與層流剪應力共同刺激下之自體凋亡分析.......32
3-4 血管內皮細胞以含抗壞血酸流體沖刷刺激後之型態重組分析 ........35
3-5 血管內皮細胞以含抗壞血酸流體沖刷刺激後之自體凋亡分析 ........38
第四章 討論........40
參考文獻........43
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指導教授 李宇翔(Yu Hsiang Lee) 審核日期 2014-1-27
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