動脈粥狀硬化一直是世界上主要的死亡原因之一，其產生的起因為血管內皮細胞的功能失調。內皮細胞的功能失調常發生在血管彎曲或分岔處附近，伴隨著危險因子的作用下，例如高膽固醇血症和高血壓等，常導致內皮細胞受損並加速動脈硬化斑的形成。進一步，這些好發動脈粥狀硬化區域之內皮細胞，其功能異常 (如：發炎、細胞屏障的完整性和凝血功能) 與代謝失衡息息相關，故內皮細胞的代謝已逐漸成為調節功能性生物學的關鍵角色。然而，對於高膽固醇血症與好發動脈硬化區域對內皮細胞代謝的作用和影響仍然是未知。迄今為止，研究內皮細胞代謝的研究也僅限於使用培養的內皮細胞，但由於體外條件不能完全概括體內環境。於是，我們分別對高膽固醇血症模型豬中的動脈粥狀硬化抵制區和動脈粥狀硬化好發區的血管內皮細胞，進行了大規模的代謝物質譜組學分析。從高膽固醇血症模型豬的好發動脈粥狀硬化區獲得內皮細胞代謝物分佈情形，我們觀察到參與在穀胱甘肽循環的中間代謝物5-oxoproline (5-OP) 大量累積在動脈粥狀硬化好發區的血管內皮細胞上，並證實使5-oxoprolinase (OPLAH) 水解酶的表達下降而導致5-OP在內皮細胞中的過度累積。為了進一步研究OPLAH水解酶在動脈硬化中的角色，我們藉由致動脈粥狀硬化的載脂蛋白E基因剔除鼠 (ApoE-/-) 產出以四環素轉錄調控系統調控的內皮細胞特異性OPLAH轉基因小鼠 (ECOplahDDKApoE-/-) 模型。在高膽固醇血症的條件下，動脈硬化好發位區域上的內皮細胞，藉由誘導轉殖基因 OPLAH 的表達，降低5-OP的積累並增加抗氧化活性，減少細胞過氧化物的生成，減緩內皮細胞凋亡的發生，也可抑制動脈硬化好發區的發炎因子ICAM-1表現。此外在高膽固醇飼料誘發動脈硬化病理過程中，ECOplahDDKApoE-/-轉基因小鼠也抑制動脈粥狀硬化早期的血管通透性和發炎反應，減少巨噬細胞的浸潤並延緩晚期動脈粥狀硬化脂質斑塊形成。綜合以上，我們的研究表明由高膽固醇血症與好發動脈硬化區域對內皮細胞的OPLAH水解酶調控，參與了動脈粥狀硬化進程的內皮細胞失調。

Atherosclerosis was one of the main causes of mortality in the world, and its cause is the dysfunction of Endothelial cells (ECs). The dysfunction of ECs is often occur near the bent or bifurcation of blood vessel, lead to the damage of ECs and accelerated the development of plaque formation under the stimulation effect of cardiovascular risk factors such as systemic hypercholesterolemia (HC) and hypertension. Furthermore, the dysfunctions in these athero-susceptible endothelium (such as inflammation, integrity of cell barriers and vascular hemostasis), are highly associated with metabolic imbalances, the endothelial metabolism has gradually become a crucial regulators in functional biology. However, the stimulation effects of HC and athero-susceptible endothelium on the endothelial metabolism remain unknown. To date, studies investigating ECs metabolism have been limited to using cultured ECs, whose metabolism may be rewired because of in vitro conditions that do not fully recapitulate the in vivo environment. We firstly performed the large-scale lipid chromatography tandem mass spectrometry-based metabolomics analysis on the athero-resistant and athero-susceptible endothelium in the hypercholesterolemia porcine model. According the metabolite profiles in athero-resistant and athero-susceptible ECs of hypercholesterolemia porcine, we observed that 5-oxoproline (5-OP), an intermediate metabolite involved in the gamma-glutamyl cycle, highly accumulate in athero-susceptible ECs. It has been proved that down-regulating the expression of hydrolytic enzyme 5-oxoprolinase (OPLAH) resulted in the excessive accumulation of 5-OP in ECs. To further study the pathological roles of OPLAH, we generated ECs-specific transgenic OPLAH mice (ECOplahDDKApoE-/-) with tetracycline-on system on atherogenic apolipoprotein E knock-out (ApoE-/-) mice. In the athero-susceptible ECs of vascular region under HC condition, transgenic OPLAH expression decreased the 5-OP accumulation and increased the anti-oxidative activity to reduce the occurrence of oxidative stress. It also inhibited the expression of the inflammatory factor ICAM-1 and lessened the occurrence of apoptosis. Furthermore, ECOplahDDKApoE-/- mice in the pathological process of high cholesterol diet-inducible atherosclerosis inhibited the vascular permeability and inflammation in the early phase of atherosclerosis, lessened the infiltration of macrophages and retarded the formation of plaques in the lesion development of atherosclerosis. Taken together, our study showed that HC and athero-susceptible ECs mediated OPLAH involved in the endothelial dysfunction of atherosclerosis.

摘要----------------------------------------------------------------i
Abstract----------------------------------------------------------iii
致謝----------------------------------------------------------------v
目錄----------------------------------------------------------------vi
圖目錄--------------------------------------------------------------ix
表目錄--------------------------------------------------------------xi
第一章　緒論----------------------------------------------------------1
一、 研究背景------------------------------------------------------1
1.1 動脈粥狀硬化---------------------------------------------------1
1.2 血管內皮細胞與動脈粥狀硬化的好發位之關聯----------------------------2
1.3 血管內皮細胞功能異常：氧化壓力、通透性與細胞凋亡---------------------4
1.4 血管內皮細胞代謝失衡與動脈粥狀硬化區域性之關聯-----------------------6
1.5 γ-穀胺酸循環---------------------------------------------------7
1.6 γ-穀胺酸循環與動脈粥狀硬化之作用關係------------------------------10
1.7 動脈粥狀硬化之實驗動物模型：ApoE基因剔除鼠-------------------------10
二、 研究動機與目的------------------------------------------------12
第二章　實驗材料與方法-------------------------------------------------14
2.1 產出OPLAH轉基因小鼠--------------------------------------------14
2.2 小鼠基因分型--------------------------------------------------14
2.3 聚合酶連鎖反應-------------------------------------------------15
2.4 製備誘導外送OPLAH基因表現之誘導藥物------------------------------17
2.5 高膽固醇飼料--------------------------------------------------17
2.6 檢驗小鼠血清生化值---------------------------------------------18
2.7 人類臍帶靜脈內皮細胞培養----------------------------------------19
2.8 細胞轉染------------------------------------------------------20
2.9 蛋白質定量----------------------------------------------------21
2.10 西方墨點法----------------------------------------------------22
2.11 組織切片免疫螢光染色--------------------------------------------24
2.12 En face免疫螢光染色-------------------------------------------25
2.13 TUNEL組織化學染色---------------------------------------------26
2.14 油紅組織化學染色-----------------------------------------------27
2.15 伊文斯藍組織化學染色--------------------------------------------27
2.16 統計分析------------------------------------------------------27
第三章　實驗結果------------------------------------------------------28
3.1 好發動脈粥狀硬化血管區域之內皮細胞發生5-oxoproline過量累積，且其下游水解酵素5-oxoprolinase表現量同時下降。-------------------------------------28
3.2 建立OPLAH轉基因實驗小鼠模型。------------------------------------35
3.3 在體外環境下，確立以腹腔注射Doxycycline誘導ECOplahDDK表現，並顯示ECOplahDDK無劑量依賴性。----------------------------------------------39
3.4 誘導血管內皮細胞ECOplahDDK過量表現可抑制5-oxoproline累積量。-------45
3.5 ECOplahDDK的過量表現，降低細胞中ROS的過量累積，減少內皮細胞的發炎反應，抑制動脈粥狀硬化早期階段的血管通透性變化。----------------------------------47
3.6 調控內皮ECOplahDDK過表達，可減少動脈粥狀硬化斑塊內的巨噬細胞，進而減少動脈粥狀硬化斑塊的產生。-------------------------------------------------53
3.7 ECOplahDDK過表現，增加細胞中的抗氧化活性，抑制細胞凋亡的產生。-------58
3.8 OPLAH在不同時間點表達都具預防動脈粥狀硬化的效果。-------------------62
第四章　討論----------------------------------------------------------65
4.1 穀胱甘肽循環在動脈粥狀硬化過程中扮演相當重要的角色-------------------65
4.2 OPLAH的表現量下降之可能機制與原因--------------------------------66
4.3 在動脈粥狀硬化好發區內皮細胞內代謝物5-oxoproline的來源--------------67
4.4 5-oxoproline在細胞反應中所扮演的角色-----------------------------69
4.5 OPLAH降低了因5-oxoproline導致的陰離子間隙代謝性酸中毒--------------70
4.6 ECOplahDDKApoE-/-基因轉殖鼠的體重差異---------------------------73
第五章　結論----------------------------------------------------------74
第六章　參考文獻------------------------------------------------------76

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