動脈粥狀硬化 (atherosclerosis) 為誘發心血管疾病的主因，好發於血管彎曲和分歧處附近，此處流體剪應力 (fluid shear stress) 主要為振盪型剪應力 (oscillatory shear stress)，容易引起內皮細胞產生發炎、增生、氧化等反應；另一種流體剪應力為脈衝型剪應力 (pulsatile shear stress)，作用於不易發生動脈硬化的區域，能抑制內皮細胞產生病變。因此血液流動所產生的流體剪應力被認為是調控血管壁發炎反應及動脈粥狀硬化生成的重要因子。最近研究指出，miRNA-10a具有抗發炎效用，在好發動脈粥狀硬化區域表現量較低，但不同流體剪應力調控miRNA-10a的相關訊息傳導機制尚未被闡明。本研究透過體外流體實驗證實振盪型剪應力 (0.5 ± 4 dynes/cm2) 與脈衝型剪應力 (12 ± 4 dynes/cm2) 分別具有抑制和提升內皮細胞miRNA-10a表現的能力，受流體剪應力調控的miRNA-10a將進一步影響下游分子第六型GATA (GATA-6) 及血管細胞黏著分子-1 (VCAM-1) 的表現，調控細胞內部的發炎反應。轉染視黃酸受體-α (RARα) 與視黃醇X受體-α (RXRα) 的專一性siRNA至內皮細胞，可以分別完全抑制與部份抑制脈衝型剪應力所誘導的miRNA-10a表現。在動物實驗中，ApoE缺損 (ApoE-/-) 的小鼠餵食高膽固醇飼料的血管會展現動脈硬化損傷的型態。給予小鼠PreR-10a將能增加血管內皮細胞miRNA-10a的表現，並避免動脈硬化損傷的形成。這些結果顯示視黃酸受體-α扮演主要調控miRNA-10a的角色，可與視黃醇X受體-α建構複合體，調控不同流體剪應力對miRNA-10a表現的影響，進而調控內皮細胞發炎反應及血管動脈硬化生成。

The major cause of cardiovascular disease is atherosclerosis, which develops preferentially at arterial branches and curvatures. The fluid shear stress (FSS) in these regions is oscillatory shear stress (OSS). OSS has been shown to play important roles in modulating various responses involved in endothelial cell (EC) dysfunction. The other kind of fluid shear stress is pulsatile shear stress (PSS), which generally occurs in areas spared from atherosclerotic lesions. Therefore, FSS is considered as an important factor to modulate inflammation and atherosclerosis in vascular wall. Recently, miRNA-10a has been identified as an anti-inflammatory molecule in athero-susceptible regions in vivo. However, whether miRNA-10a can be regulated by different FSS patterns and the detailed mechanisms involved in this mechanotransduction remain unclear. In the present study, we investigated the role of miRNA-10a in regulating inflammatory responses of ECs to different FSS patterns. Application of OSS (0.5 ± 4 dynes/cm2) and PSS (12 ± 4 dynes/cm2) to HUVECs can sustainably down-regulate and up-regulate miRNA-10a expression, respectively. This flow-regulated miRNA-10a expression could consequently regulate the expression of its directed target GATA-6 and downstream vascular cell adhesion molecule-1 (VCAM-1). Transfecting ECs with RARα-specific siRNA could totally inhibit PSS-induced miRNA-10a expression, but RXRα-specific siRNA could only partially inhibit this response. The vascular of ApoE-/- mice fed with high-cholesterol diet express atherosclerotic lesions formation. Administration of PreR-10a can enhance miRNA-10a expression in the vascular endothelial cell and prevent atherosclerotic lesions formation. These results suggest that RARα/RXRα could constitute regulatory complex to switch miRNA-10a expression in response to different FSS patterns in regulating inflammatory response and atherosclerosis formation.

目錄
中文摘要 I
Abstract II
致謝 III
目錄 IV
圖表目錄 VI
壹、緒論 1
1.1 研究動機 1
1.2 心血管疾病對國人的影響 1
1.3 動脈粥狀硬化為慢性發炎疾病 2
1.4 血液流體型態與動脈粥狀硬化發炎反應的關係 3
1.5 血液流體型態與剪應力對內皮細胞基因的調控 5
1.6 微型核醣核酸與動脈粥狀硬化的相關研究 6
1.7 視黃酸受體和視黃醇X受體與miRNA-10a的關係 9
1.8 GATA轉錄因子與血管黏著分子的關係對內皮細胞發炎的影響 10
1.9 研究目的 11
貳、材料與方法 12
2.1 初級臍帶靜脈內皮細胞培養 12
2.2 小片段干擾RNA細胞轉染 13
2.3 給予內皮細胞賀爾蒙受體活化劑實驗 13
2.4 RNA的萃取與定量 14
2.5 反轉錄反應 14
2.6 聚合酶鏈鎖反應 15
2.7 即時定量聚合酶鏈鎖反應 15
2.8 西方墨點法 16
2.9 Luciferase活性測定 18
2.10染色質免疫沉澱 18
2.11動物實驗 20
2.12原位雜合 20
2.13體外流體系統 22
參、實驗結果 23
3.1 脈衝型剪應力促進內皮細胞miRNA-10a表現，而振盪型剪應力則具有抑制內皮細胞miRNA-10a表現的能力 23
3.2 視黃酸受體-α與視黃醇X受體-α協同調控miRNA-10a的表現 23
3.3 第六型GATA為miRNA-10a下游調控的目標基因 25
3.4 第六型GATA及血管細胞黏著分子-1為miRNA-10a下游調控基因 26
3.5 受不同剪應力調控的miRNA-10a會藉由第六型GATA影響血管細胞黏著分子-1的表現 27
3.6 視黃酸受體-α和視黃醇X受體-α協同調控miRNA-10a而影響第六型GATA及血管細胞黏著分子-1的表現 29
肆、討論 31
4.1 不同血液流動型態為影響內皮細胞功能之重要因素 31
4.2 MicroRNA-10a為連接血液動力與內皮細胞發炎反應之重要橋梁 32
4.3 視黃酸受體與視黃醇X受體協同調控miRNA-10a表現 34
4.4 第六型GATA為不同流體剪應力調控血管細胞黏著分子-1表現的重要轉錄因子 35
伍、結論 37
參考文獻 39
圖表 45

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