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Title: | 探討棕櫚酸鈉加上高醣處理對人類臍靜脈內皮細胞與小鼠主動脈表現細胞間黏附因子的作用機制 The Mechanisms of Sodium Palmitate plus High Glucose on ICAM-1 Expression in Endothelial Cells and Aortas |
Authors: | Zhe-Yu Shi 石哲宇 |
Advisor: | 陳玉怜(Yuh-Lien Chen) |
Keyword: | 棕櫚酸鈉,動脈硬化,MAPKs,NF-kB,ICAM-1,OGT, Sodium palmitate,atherosclerosis,MAPKs,NF-kB,ICAM-1,OGT, |
Publication Year : | 2015 |
Degree: | 碩士 |
Abstract: | 根據衛生福利部102年度公佈的國人十大死因,心臟疾病、腦血管疾病、糖尿病分別位居第二、三、四名,而高糖高脂飲食常造成這些疾病的主要成因之一。 棕櫚酸為十六碳飽和脂肪酸,是我們飲食攝入飽和脂肪酸的大宗,已有許多研究證實棕櫚酸會造成多種細胞產生發炎反應、細胞凋亡及動脈硬化。在心血管疾病剛形成時,常見血管內皮細胞功能異常,進而導致血管動脈硬化與急性心肌梗塞。內皮細胞功能異常包括發炎、自由基在細胞內堆積,嚴重時常會導致細胞凋亡。而動脈硬化已被證實屬於一種慢性的發炎反應,發炎反應促使內皮細胞黏附因子大量表現,驅使單核球黏附、進入血管管壁以及吞噬囤積在血管管腔的脂質,進而促進動脈硬化斑塊的產生。高糖高脂如何造成內皮細胞發炎及其相關機轉,到目前為止仍相當不清楚,有待研究。因此我們研究的主軸是去探討高糖高脂肪酸對於血管內皮細胞的影響與其機制。我們使用人類臍靜脈內皮細胞做為in vitro的實驗材料,經實驗證實,高糖與高棕櫚酸鈉會誘發內皮細胞顯著表現黏附因子ICAM-1。而針對ICAM-1表現的相關機轉探討時,發現高糖高棕櫚酸鈉會促使內皮細胞p-p38、p-JNK、p-p65的表現量上升。進一步地加入p-38、ERK、JNK、p-65的抑制劑,再加入高糖高棕櫚酸鈉刺激會降低ICAM-1的表現,因此推測高糖高棕櫚酸鈉是經由JNK、p38、ERK與NF-B這三個路徑去誘發ICAM-1的表現。 另外,我們在in vitro實驗中初步發現,在高糖環境下,越高濃度之棕櫚酸鈉,對於人類臍靜脈內皮細胞內之OGT (O-GlcNAc transferase)會有抑制作用;而在高糖環境下,以0.5 mM 棕櫚酸鈉刺激人類臍靜脈內皮細胞後,隨著時間拉長,OGT表現也會明顯地被抑制。OGT為一可進行O-linked glycosylation之酵素,可將特定結構UDP-GlcNAc修飾在細胞核與細胞質蛋白上,這樣的修飾可能造成蛋白的表現改變以及泛素化 (Ubiquitination)。 我們也針對餵食western diet的ApoE-/-小鼠 (實驗組),犧牲取其胸主動脈進行觀察,發現與C57BL/6 (控制組)相比,實驗組老鼠之動脈硬化斑塊非常明顯,並且我們以ICAM-1、VCAM-1、E-selectin、IL-6抗體對發病組與控制組小鼠胸主動脈分別進行免疫組織化學染色。結果發現,與控制組相比,發病組小鼠在動脈硬化斑塊及斑塊周圍之ICAM-1、VCAM-1、E-selectin、IL-6表現相當明顯。在高倍視野下,發現了血管管腔周圍染有CD31 (內皮細胞marker)之動脈內皮細胞也同時有ICAM-1表現,代表形成動脈硬化的過程中,內皮細胞會有慢性發炎的反應產生。在細胞實驗和動物實驗中,我們證實在高糖高脂肪酸環境下會促使黏附因子表現。 Population ageing is a foregone conclusion and becomes an important issue in Taiwan. People with higher levels of glucose and fat in diet resulted in the greater risk of cardiovascular diseases. According to the statistics of Ministry of Health and Welfare in 2013, cardiovascular disease, cerebrovascular disease, and diabetes mellitus have remained in the top ten leading causes of death. The previous studies proved that sodium palmitate, a 16-carbon saturated fatty acid, can induce inflammation and apoptosis in many cell types. Either hyperglycemia or hyperlipidemia often bring about vascular dysfunction, including inflammation and accumulation of reactive oxygen spices which lead to up-regulation of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), E-selectin expression. Furthermore, monocytes adhere to endothelial cells and migrate into subendothelial space to engulf ox-LDL and free fatty acid, then the monocytes were transformed into foam cells. Finally, smooth muscle cells, collagen and foam cells were accumulated in subendothelial space and formed the plaque. Moreover, the atherosclerotic plaques became unstable and they would detach from vascular wall and enter blood circulation, which can seriously lead to acute myocardial infarction and stroke. However, the detailed mechanisms of high glucose and high fatty acid which are involved in the cardiovascular diseases are not clarified. Therefore, we focus on their effects on the expression of ICAM-1 in sodium palmitate plus high glucose-treated human umbilical vein endothelial cells (HUVECs) and their related mechanisms. Palmitate plus high glucose significantly induced ICAM-1 expression as well as the phosphorylation of extracellular-regulated kinase (ERK), p38 MAPK, c-jun N-terminal kinase (JNK), and nuclear factor kappa B (NF-B) in HUVECs. Pretreatment with PD98059 (an ERK1/2 inhibitor), SP600125 (a JNK inhibitor), SB203580 (a p38 inhibitor) and Bay11-7082 (a NF-B inhibitor) significantly reduced ICAM-1 expression under the stimulation of sodium palmitate plus high glucose. Furthermore, we found that cells treated with 0.5 mM sodium palmitate for 24 hours significantly decreased high glucose-induced O-GlcNAc transferase (OGT) expression. In animal studies, we used apoE-/- mice and C57BL/6 mice fed with high -sucrose high-palmitate diet (HSHP diet). The thoracic aorta of apoE-/- mice in HSHP group significantly expressed high level of adhesion molecule (ICAM-1, VCAM-1, E-selectin) and pro-inflammatory cytokine IL-6 by immunohistichemistry. We also find that the co-expression of CD31 (endothelial marker) and ICAM-1 in the endothelial cell, indicating that high-glucose high-palmitate diet will cause the dysfunction of endothelial cells. Based on in vitro and in vivo studies, high palmitate plus high glucose can cause endothelial dysfunction and then lead to the progression of cardiovascular diseases. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19828 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 解剖學暨細胞生物學科所 |
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