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研究生: 高雅詩
Ya-shi Kao
論文名稱: 探討芝麻素對腫瘤壞死因子刺激人類主動脈內皮細胞表現細胞黏附分子的影響及相關機轉
The effects of sesame lignans on adhesion molecules expression in tumor necrosis factor treated human aorta endothelial cells and the related mechanisms
指導教授: 吳文惠
Wu, Wen-Huey
陳玉怜
Chen, Yuh-Lien
學位類別: 碩士
Master
系所名稱: 人類發展與家庭學系
Department of Human Development and Family Studies
論文出版年: 2007
畢業學年度: 95
語文別: 中文
論文頁數: 67
中文關鍵詞: 動脈硬化發炎反應黏附分子芝麻木質素
英文關鍵詞: atherosclerosis, inflammatory, adhesion molecule, sesame lignans
論文種類: 學術論文
相關次數: 點閱:258下載:26
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  • 在動脈硬化發生早期,血流中白血球的黏附和遷移入血管壁為主要因素,而內皮細胞所分泌的細胞黏附分子,則在動脈硬化過程中扮演了重要的角色。抗氧化物可以抑制細胞黏附分子的表現,並且可以降低動脈硬化發生的機會。本實驗中,主要探討以芝麻木質素sesamol與sesamin處理人類主動脈內皮細胞(HAECs)表現細胞黏附分子的影響及相關機制。以sesamol (100 μM)或sesamin (100 μM)預先處理HAEC 24小時,可以顯著的抑制U937細胞黏附於內皮細胞。以西方墨點法得知處理sesamol或是sesamin可以抑制TNF-α刺激細胞黏附分子ICAM-1的表現。觀察MAPK主要途徑ERK1/2、JNK及p38的磷酸化,發現sesamin可以抑制ERK1/2與p38的磷酸化。此外,NF-κB p65免疫染色及凝膠遲滯分析結果顯示,sesamol及sesamin均可降低NF-κB的活性。實驗結果證實,sesamin可以藉由抑制ERK1/2 與 P38的磷酸化及降低NF-κB活性,減少細胞受TNF-α刺激所引發的ICAM-1表現。由上述結果得知:芝麻木質素可藉由拮抗內皮細胞失調及發炎反應,達到保護心血管的功能。

    The adhesion and migration of circulating leukocytes into the vessel wall is an early event in atherogenesis and the expression of cell adhesion molecules by the arterial endothelium plays a major role during atherogenesis. Antioxidants have been proposed to inhibit the expression of adhesion molecules and thereby attenuate the processes leading to atherosclerosis. In this study, the effects of antioxidant of sesame lignans, sesamol and sesamin, on the expression of adhesion molecules and the associated related mechanisms in tumor necrosis factor-α (TNF-α) treated human aortic endothelial cells (HAECs) were investigated. HAECs pretreated with sesamol (100μM) or sesamin (100 μM) for 24 h significantly suppressed cellular binding between the human monocytic leukemic cell line-U937 and TNF-α treated HAECs. Western blotting analysis showed that the treatment of sesamol (100μM) or sesamin (100 μM) for 24h significantly attenuated intercellular cell adhesion molecule-1 (ICAM-1) expression in HAECs under TNF-α stimulation. Phosphorylation studies on ERK1/2, JNK, and p38, three major subgroups of mitogen activator protein kinases, demonstrated that sesamin suppressed ERK1/2 and p38 phosphorylation. In addition, sesamol and sesamin significantly decreased the expression of NF-κB p65 and the activity of NF-κB by immunostaining and gel-mobility shift assay, respectively. These results provide the evidence that sesamin attenuated ICAM-1 expression caused by TNF-α stimulation and this inhibitory effect is mediated via downregulation of ERK1/2 and P38 phosphorylation and inactivation of NF-κB. These observation support the feasibility of sesame lignans administration is as a means of protection against endothelial dysfunction and inflammation.

    第一章 Introduction 第一節 動脈硬化致病機轉 1-3 第二節 內皮細胞 (Endothelial cell) 4 第三節 動脈硬化相關之發炎反應標幟 (一) 細胞粘附分子(cellular adhesion molecules) 4-6 (二) 細胞激素(cytokines) 6-7 (A)介白素(interleukin) 8 (B)腫瘤壞死因子(tumor necrosis factor) 8 第四節 Mitogen-Activated Protein Kinase pathways與動脈硬化之相關性 9 第五節 與動脈硬化相關之transcription factors 10-11 第六節 芝麻之簡介 (一) 芝麻Lignans之效用 11-14 (二) Lignans與抗發炎 15 (三) Lignans與心血管之相關性 15 (四) Enterolactone (ENL) 16 第七節 研究目的 17 第二章 材料與方法 一、儀器設備 18 二、實驗材料與試劑 19-20 三、實驗用溶液配方 21-23 四、實驗方法 1. 細胞培養 23-24 2. 西方墨點分析法(western blotting) 25-27 3. 內皮細胞/單核球粘附試驗(endothelial cells/monocyte adhesion assay) 27 4. MAPKs活性的測定 28-29 5. NF-κB p65 細胞免疫染色 30 6.凝膠遲滯分析(Electrophoresis Mobility Shift Assay; EMSA)細胞核蛋白萃取 30-33 7. 統計分析 33 第三章 結果 34-40 第四章 討論 53-57 第五章 結論 58 參考文獻 59-67 圖片說明 圖 1 以西方墨點法觀察處理不同濃度的sesamol、sesamin及ENL之人類主動脈內皮細胞時對於TNF-α刺激細胞表現ICAM-1之情形 41 圖 2 (A)西方墨點法觀察以100μM的sesamol或sesamin處理人類主動脈內皮細胞時對於TNF-α刺激細胞表現phospho-p38 與total-p38之情形 42 圖 2 (B)西方墨點法觀察100μM 的sesamol或sesamin處理人類主動脈內皮細胞對於TNF-α刺激細胞表現phospho-JNK與total-JNK之情形 43 圖 2 (C)西方墨點法觀察經100μM的sesamol或sesamin處理人類主動脈內皮細胞對於TNF-α刺激細胞表現phospho-ERK與total-ERK之情形………………44 圖 3 西方墨點法觀察人類主動脈內皮細胞先以p38 inhibitor (SB203058)、JNK inhibitor (SP600125)及ERK inhibitor (PD98059)處理,再經TNF-α刺激,ICAM-1表現情形 45 圖4 (A)以單核球黏附試驗法觀察sesamol或sesamin影響U937細胞黏附人類主動脈內皮細胞之情形 46 圖4 (B)以單核球黏附試驗法觀察sesamol或sesamin影響U937細胞黏附人類主動脈內皮細胞之情形 47 圖 4 (C)以單核球黏附試驗法觀察sesamol或sesamin或anti-human ICAM-1抗體影響U937細胞黏附人類主動脈內皮細胞之情形 48 圖4 (D)以單核球黏附試驗法觀察sesamol或sesamin或anti-human ICAM-1抗體影響U937細胞黏附人類主動脈內皮細胞之情形 49 圖5 (A) 以凝膠遲滯分析法(Electrophoresis mobility shift assay) 觀察sesamol或 sesamin處理人類主動脈內皮細胞對於TNF-α刺激時細胞NF-κB 活化的情形 50 圖5 (B) 細胞免疫染色法分析處理sesamol或sesamin其 NF-κB subunit p65的表現 51 圖5 (C)西方墨點法觀察Pathernolide與TNF-α同時處理人類主動脈內皮細胞後,觀察ICAM-1表現之情形 52 附件 (附件一) Table 1 動脈硬化相關的細胞粘附因子與其功能 6 (附件二) Table 2 促發炎反應及抗發炎反應相關介白素的分類 8 (附件三) Table 3 MAPK家族分類 9 (附件四) Fig. 1 發炎細胞與前發炎細胞激素對於動脈粥樣瘤形成之角色 3 (附件五) Fig 2 Mitogen-activated protein kinase signal transduction 10 (附件六) Fig. 3 Model for the role of NF-kB/Rel transcription factors in the pathogenesis of atherosclerosis. 12 (附件七) Fig. 4 Chemical structure of a few bioactive compounds of sesame. 15

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