臺灣博碩士論文加值系統

行政院衛生署公佈心臟疾病與腦血管疾病分別高居國人十大死因的第二、三名，動脈粥狀硬化是導致心血管疾病的重要因子。內皮細胞受損與發炎反應是動脈粥狀硬化的初期發展過程。Intercellular adhesion molecule 1 (ICAM-1)為一發炎指標。Early growth response-1 (Egr-1)是調控動脈粥狀硬化相關基因表現的重要轉錄因子，氧化壓力刺激會誘發其表現。近年研究顯示，增加飲食中ω-3脂肪酸攝取，例如DHA，可以降低心血管疾病發生率，但目前調控機制尚未完全清楚。先前本實驗室已證實DHA透過上調HO-1表現，抑制腫瘤壞死因子α (TNFα)誘發ICMA-1表現，本研究欲進一步探討Egr-1與GPR120在DHA抑制TNFα誘發發炎反應中所扮演的角色。以TNFα活化EA.hy926內皮細胞為試驗模式下，結果顯示，TNFα增加Akt, ERK, p38磷酸化，並誘發Egr-1和ICAM-1表現。預處理PI3K抑制劑(LY294002)、ERK抑制劑(PD98059)則可抑制TNFα對Egr-1的誘發表現；而ICAM-1表現只受到PD98059的抑制。結果顯示:TNFα可能透過活化ERK增加Egr-1表現， 進而上調ICAM-1表現，促使單核球黏附至受活化的內皮細胞；利用shEgr-1技術阻斷Egr-1表現時，可減少TNFα誘發ICAM-1表現及單核球黏附作用，進一步確認Egr-1在血管發炎反應中扮演著重要角色；siGPR120阻斷GPR120表現，亦阻斷DHA抑制TNFα誘發ICAM-1表現。由此可知，DHA透過GPR120來達到抗發炎效果。結論，TNFα透過活化ERK路徑增加Egr-1表現，進而增加ICAM-1表現；而DHA則是透過GPR120達到抑制TNFα活化ERK/ Egr-1路徑，進而抑制ICAM-1表現及單核球黏附作用。根據研究結果，DHA為一抗發炎物質，具有保護心血管活性。

Background and objectives: Tumor necrosis factor-alpha (TNFα), a potent inflammatory mediator, has multiple effects on the pathogenesis of atherosclerosis. Early growth response-1 (Egr-1), a zinc finger transcription factor, participates in cell growth and differentiation. In recent years, Egr-1 has emerged as a key regulator in the development of atherosclerosis. Docosahexaenoic acid (DHA), an omega-3 fatty acid (n-3 FA), has potent anti-inflammatory effect. It has been demonstrated that G protein-coupled receptor 120 (GPR120) functions as an n-3 FA receptor. In our previous study, we have demonstrated that DHA inhibits TNFα-induced intercellular adhesion molecule 1 (ICAM-1) expression via enhancing HO-1 expression. In this study, we try to identify the role of Egr-1 and GPR120 in DHA inhibition of TNFα-induced ICAM-1 expression in EA.hy926 cells. Methods: In this study we used EA.hy926 endothelial-like cells as cell model, and used Western blotting, real-time PCR, shRNA, siRNA and adhesion assay to explore the anti-inflammatory activity of DHA and the possible mechanisms involved. Results: TNFα markedly induces the phosphorylation of ERK1/2, p38 and Akt. Treatment with ERK inhibitor, PD98059, and PI3K inhibitor, LY294002, abolishes TNFα-induced Egr-1 expression; however, ICAM-1expression is only blocked by PD98059. Transfection with shEgr-1 knocked down Egr-1 expression, and blocked TNFα-induced ICAM-1expression. Transfection with siGPR120 knocked down GPR120 expression, and reversed the DHA-mediated inhibition of TNFα-induced ICAM-1 expression. In addition, TNFα-induced adhesion of HL-60 to EA.hy926 cells was abolished by PD98059 and SB203580. Conclusions: Taken together, the anti-inflammatory effect of DHA is at least partially linked to GPR120 binding, down- regulation of ERK-1/2-dependent Egr-1 and ICAM-1 expression, and eventual suppression of HL-60 adhesion to EA.hy926 cells.

目錄…………………………………………………………………….…i
圖目錄………………………………………………………………….iv
表目錄……………………………………………………………………v
縮寫表………………………………………………………………….vi
中文摘要………………………………………………………………viii

第一部分………………………………………………………………….1
第一章 前言…………………………………………………………1
第二章 文獻回顧……………………………………………………2
一、 動脈粥狀硬化………………………………………………2
1. 動脈粥狀硬化成因…………………………………………2
2. 白血球徵募作用……………………………………………4
3. 細胞黏附分子與動脈硬化之關係…………………………5
4. 腫瘤壞死因子與動脈硬化之關係…………………………11
二、 魚油與心血管疾病之關係…………………………………12
1. 魚油概述……………………………………………………12
2. 脂肪酸概述…………………………………………………12
3. 多元不飽和脂肪酸生合成作用……………………………13
4. DHA生理作用………………………………………………15
5. n-3 脂肪酸建議攝取量………………………………….17
三、 G protein-coupled receptors (GPRs) ……………….19
1. GPRs分類…………………………… …… …………….19
2. 與脂肪酸相關之GPRs…………………………………….20
3. GPR120生理作用……………. ……………………………20
四、 早期成長反應蛋白 (Early growth response protein)24
1. 早期成長反應蛋白之分類……………………………24
2. Egr-1生理角色……………………………………………25
五、 參考文獻………………………………………………28
第三章 研究目的…………………………………………………40
實驗架構. ……………………………………………………………41

第二部分…………………………………………………………………42
Docosahexaenoic acid inhibits TNFα-induced ICAM-1 expression via binding to GRP120 and down-regulation of ERK-1/2-dependent Egr-1 expression in EA.hy926 cells
英文摘要…………………………………………………………………43
1. Introduction………………………………………………44
2. Materials and Methods…………………………………47
2.1 Chemicals…………………………………………………47
2.2 Fatty acid preparation…………………………………47
2.3 Cell cultures……………………………………………48
2.4 Western blotting analysis……………………………48
2.5 RNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR)………………………………49
2.6 Monocyte adhesion assay………………………………50
2.7 RNA interference by small hairpin RNA of Egr-1…50
2.8 RNA interference by small interfering RNA (siRNA) of GPR12…………………………………………………………………51
2.9 Plasmids, transfection and luciferase assay……51
2.10 Data analysis…………………………………………52
3.Result………………………………………………………………53
3.1 Effect of TNFα on Egr-1 gene expression…………53
3.2 ERK is involved in TNFα-induced expression of Egr-1 and ICAM-1………………………………………………………53
3.3 Silencing of Egr-1 attenuates TNFα-induced ICAM-1 gene expression and monocyte adhesion………………………54
3.4 DHA suppresses TNFα-induced ICAM-1 expression via inhibition of ERK phosphorylation and Egr-1 gene expression……………………………………………………………55
3.5 DHA inhibits TNFα-induced ICAM-1 expression and monocyte adhesion is mediated by GPR120…………………55
4. Discussion…………………………………………………57
5. Figures……………………………………………………62
6. Reference…………………………………………………82

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