中文摘要
已知氧化態低密度脂蛋白(oxidized low density lipoprotein; ox-LDL) 在動脈粥狀硬化病症中扮演很重要角色，惟有關LDL (low density lipoprotein)如何進入內皮下層促成LDL的堆積、氧化，還有ox-LDL對於血管內皮細胞的生長抑制機制則仍待探究，本論文乃就以上兩主題提出研究報告。
首先以人的LDL於抗氧化劑存在下製備，然後以銅離子氧化成不同程度，以來比較未氧化LDL和氧化態LDL 對血管內皮細胞(EC)形成壓力纖維( stress fiber）、 細胞膜波浪狀和胞飲能力的變化。研究顯示在濃度 100至 200 ug cholesterol per ml protein時，不論未氧化LDL和氧化LDL都會促使EC拉長和產生壓力纖維, 惟後者比較之明顯；此外， ox-LDL也誘導 EC 細胞膜產生波浪狀和增加胞飲能力，且與LDL氧化程度和ox-LDL劑量相關。若以抗氧化劑先處理EC，則抑制 ox-LDL誘導 EC 細胞膜產生波浪狀和增加胞飲的能力，卻不能抑制壓力纖維的形成。至於以未氧化LDL處理之EC雖也增加胞飲能力，但所需劑量較高，顯然不同於處理ox-LDL者之表現，惟促進胞飲作用並未伴隨細胞膜波浪狀的產生， 因此LDL和ox-LDL的這些作用，可能是經由不同機制產生，但皆能提高胞飲活性，增加內皮層的穿透細胞能力，導致血漿的成份（如LDL）流入內皮層下腔。
其次，為了了解ox-LDL抑制 EC生長之機制，本研究進一步檢視ox-LDL處理細胞Ras訊息路徑被干擾的情形。結果顯示 EC經 ox-LDL處理會導致細胞全部的 Ras蛋白質和細胞膜上的 Ras蛋白質明顯的下降，後者並與下降43% 的 HMG-CoA reductase活性符合；EC經 ox-LDL處理後，以Northern blot分析其 Ras mRNA的穩定性並無明顯改變；此結果顯示ox-LDL處理後，可能會造成EC的Ras蛋白質拋錨於細胞膜上的量減少到不足，使其不能傳導bFGF的刺激細胞分裂訊息，由於這種作用可被血清蛋白混合氧化態膽固醇的複合物所模擬，所以ox-LDL抑制EC Ras farnesylation的作用，可能源自於ox-LDL本身所含的氧化態固醇類成份。

ABSTRACT
Oxidized low density lipoprotein (ox-LDL) has thus been considered to be a major culprit that induces activation, or dysfunction of the endothelial cells associated with the initiation of the atherosclerotic lesions. The alterations of the structural and/or functional integrity of the endothelial barrier allowed a net influx of lipoproteins from the circulating plasma into the subendothelium. The mechanisms underlying the observed endothelial dysfunction elicited by ox-LDL have not been completely elucidated. In this study, how hypercholesterolemia promoted subendothelial LDL retention/oxidation and the mechanism underlying the antiproliferative effect of ox-LDL on vascular EC have been detected.
Human LDLwas prepared in the presence of antioxidants and was oxidized to different levels (measured by thiobarbituric acid reactive substance) with copper ion. The effects of unoxidized LDL and oxidized-LDL (ox-LDL) on stress fiber formation, cell membrane ruffling and pinocytosis (measured by [14C]-sucrose uptake) in cultured human umbilical cord vein endothelial cells (EC) were compared. We showed that at a concentration range of 100 to 200 ug cholesterol per ml, both unoxidized LDL and ox-LDL promoted EC elongation and stress fiber formation, however, the effect by the latter was more prominent when compared at the same dose range. In addition, ox-LDL also induced EC membrane ruffling and promoted pinocytosis. These effects were positively correlated with the extents of LDL oxidation and was dose dependent on ox-LDL. Ox-LDL- promoted membrane ruffling and pinocytosis were effectively blocked by brief preexposure of the cells to antioxidants. In contrast, the stress fiber formation was not affected by antioxidant pretreatment. Although native LDL also promoted [14C]-sucrose uptake, it was less potent than ox-LDL and required significantly higher concentrations to see the effect. Unlike ox-LDL, native LDL-enhanced pinocytosis was not accompanied with the appearance of membrane ruffling, it was, therefore, suggested that they might act via different mechanisms. Elevated pinocytosis might increase the transcytotic activity of the endothelium leading to an increased influx of plasma components, i.e., LDL, into the subendothelial space.
To understand the mechanisms ox-LDL exposure reversibly inhibited EC growth, we examined the possible interference of Ras signaling pathway in ox-LDL treated cells. By immunoprecipitation and Western blot analysis, we showed that exposure of EC to ox-LDL resulted in a marked reduction of total cellular Ras and membrane-associated Ras proteins. Reduction of membrane-associated Ras proteins was coincided with a 43% decrease in HMG-CoA reductase activity as determined by the conversion of [14C]HMG-CoA to [14C]mevalonate. Meanwhile, the steady-state levels of all three Ras mRNA species were not significantly changed as measured by Northern blot analysis. The reduction of Ras membrane anchoring was correlated with a retarded cell growth in serum and an impaired cell response to bFGF mitogenesis. The results suggested that in ox-LDL exposed EC, Ras farnesylation and its subsequent membrane anchoring might be reduced to an extent that it was no longer adequate (in amount) to convey the bFGF mitogenesis. Ox-LDL effect was due to its oxsterol content as these effects were mimicked by bovine serum albumin complexed oxycholesterols.

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