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系統識別號 U0026-1801201813192100
論文名稱(中文) 動脈層流刺激後靜脈內皮細胞之發炎與預防
論文名稱(英文) Venous endothelial cell inflammation and prevention after arterial flow transition
校院名稱 成功大學
系所名稱(中) 細胞生物與解剖學研究所
系所名稱(英) Institute of Cell Biology and Anatomy
學年度 106
學期 1
出版年 107
研究生(中文) 方少禹
研究生(英文) Shao-Yu Fang
學號 T96044049
學位類別 碩士
語文別 英文
論文頁數 62頁
口試委員 指導教授-吳佳慶
口試委員-吳華林
口試委員-裘正健
口試委員-江美治
中文關鍵字 靜脈嫁接疾病  靜脈內皮細胞  微型核醣核酸  動脈層流剪切力 
英文關鍵字 vein graft  venous endothelial cells  microRNA  arterial shear stress 
學科別分類
中文摘要 靜脈嫁接疾病(vein graft disease)中,不正常的血流會引發血管內皮細胞損傷以及伴隨著發炎反應的產生,而在病徵發展的過程當中少有研究針對兩者之間的連結進行探討。近期,我們發現在動靜脈系統轉換(venous-to-arterial transition)下靜脈內皮細胞(endothelial cells, ECs)會透過引發過量的自噬反應以及發炎而導致細胞的損傷。先前文獻指出微型核醣核酸(microRNA, miRs)在調控血管內皮細胞功能扮演重要角色。本實驗的研究目的是要去探討靜脈嫁接疾病的病程中詳細的發炎機制和找出潛在的預防方法。為了模擬因mechanical overload所造成的靜脈嫁接疾病產生,我們利用動脈層流剪切力(arterial shear stress)去引發靜脈血管內皮細胞的發炎,並從研究中發現活性氧物質(ROS)和COX-2/NFκB訊號路徑應皆參與於ALS所誘發之靜脈發炎。而靜脈內皮細胞之miR-4488表現在以動脈層流刺激後則會有下降的趨勢。為了建立有效的治療方式,我們試著使用ROS與NFκB抑制劑藉此抑制由動脈層流所引發的靜脈血管內皮細胞之發炎反應,但是兩個抑制劑的使用皆沒辦法達到很好效果。由於我們在體外以及細胞實驗中皆觀察到在靜脈內皮細胞中Cyclooxygenase-2 (COX-2)會被動脈層流所誘導而產生,因此我們合併處理COX-2專一性抑制劑SC-236與L-arginine (L-Arg)去抑制ALS所誘發之發炎反應。在靜脈內皮細胞接受動脈層流刺激之前先以SC-236和L-Arg合併處理為一個成功的治療策略,能有效預防靜脈內皮細胞的發炎以及損傷。同時我們也發現miR-4488會涉及這樣的一個保護作用。本研究為動靜脈層流轉換而引起的靜脈內皮細胞發炎之詳細機制提供了新的認識,而合併處理SC-236和L-Arg的方式也可做為日後靜脈嫁接手術後新的治療策略。
英文摘要 Little is known about the interrelations among the pathological progression of abnormal flow-induced endothelial damage and inflammatory response in vein graft disease. Recently, we discovered that venous-arterial transition induced the damage of venous endothelial cells (ECs) via excessive autophagy and inflammation. Previous studies show that microRNAs (miRs) play an important role in modulating ECs functions. The aim of our study is to investigate the detailed mechanisms of inflammatory response and potential prevention for pathological progression of vein graft disease. To mimic the vein graft disease induced by mechanical overload, we use the arterial laminar shear stress (ALS) to elicit inflammatory responses in venous ECs. We discovered that ROS and COX-2/ NFκB both involved in the ALS-induced venous inflammation. We found decrease of miR-4488 in venous ECs after ALS stimulation. To establish therapeutic treatment, the ROS and NFκB inhibitors were used to suppress the ALS-induced inflammation in venous EC. However, both ROS and NFκB inhibition could not rescue the inflammatory response. Since the COX-2 induction was observed in both ex vivo and in vitro experiments, the specific COX-2 inhibitor (SC-236) combined with L-arginine (L-Arg) was used to block the ALS-induced inflammatory response. The pretreatment of venous ECs with combined treatment prior to the ALS stimulation provides a successful therapeutic strategy to inhibit the inflammation and venous ECs damage. The miR-4488 was found to involve in the protective effect of combined treatment. This study provides novel knowledge for the detailed mechanism of inflammation in venous endothelial damage resulting from flow transition, and the combination of SC-236 and L-Arg may provide protective effect on vein graft from pathological changes.
論文目次 Table of Content
中文摘要 I
Abstract II
Introduction 1
1.1 Vein graft disease 1
1.2 Anatomical structural differences between artery and vein 2
1.3 The function of endothelial cells 4
1.4 Shear stress in endothelial physiological and pathological function 4
1.5 Inflammatory response in endothelial cells 7
1.6 Therapeutic effect of anti-inflammation drugs on preventing endothelial dysfunction 8
1.7 MicroRNA in endothelial function and inflammation 10
Specific Aims 11
Materials and Methods 12
2.1 Cell culture 12
2.2 Apply fluid shear stress to cells or vessels by using the flow chamber system 14
2.3 RNA isolation 16
2.4 Western Blot 21
2.5 Immunofluorescence staining 25
2.6 Luciferase reporter assay 28
2.7 Reactive Oxygen Species Detection Assay 30
Results 31
3.1 Venous-to-arterial flow transition induced venous ECs inflammation 31
3.2 The inflammation mechanism in ECs 35
3.3 Potential miRs in regulating arterial flow induced inflammation in venous ECs 38
3.4 Involvement of ROS and NFκB pathway in arterial flow induced inflammatory responses in venous ECs 40
3.5 Protective effect of COX inhibitors on preventing ALS-induced EC damage 43
3.6 To investigate the protective effect of combined treatment on vein graft 47
Discussion 50
Conclusion 56
Reference 57

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