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博碩士論文 etd-0215105-121303 詳細資訊
Title page for etd-0215105-121303
論文名稱
Title
家族性高膽固醇血症病患之低密度脂蛋白分離物對血管新生的抑制效應
The Anti-angiogenic Functions of Low Density Lipoproteins Subfractions from Patients with Familial Hypercholestrolemia
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
55
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2005-01-19
繳交日期
Date of Submission
2005-02-15
關鍵字
Keywords
基質分解酵素、粥狀硬化、血管新生作用、氧化態彽密度脂蛋白
oxidatively modified LDL (oxLDL), matrix metalloproteinases (MMPs), angiogenesis, atherosclerosis, apoptosis
統計
Statistics
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The thesis/dissertation has been browsed 5649 times, has been downloaded 9 times.
中文摘要
低密度脂蛋白的氧化修飾、高葡萄糖含量及過氧化物會促使內皮細胞的凋亡,因此參與早期動脈血管粥狀硬化的發生。經由其他研究結果發現,存在於家族性高膽固醇血症(FH)的病人血液中之低密度脂蛋白可分成五個次片段(FHL1-L5),其中以L5片段抑制DNA的合成及刺激單核細胞與內皮細胞黏著的效應最強。由於抑制血管新生作用會促使內皮細胞失去功能進而造成血管粥狀硬化的發生,因此本研究將探討存在於家族性高膽固醇血症族群之低密度脂蛋白的抑制血管新生效應。首先,將不同劑量之低密度脂蛋白的五個次片段作用於已培養的內皮細胞中,進行細胞增生等不同實驗來決定其抑制血管新生之效應。由實驗結果發現,在細胞增生及移行方面,其中L4和L5兩個次片段具有抑制效應。再者,此兩片段亦會造成不同程度的細胞凋亡現象及降低管狀結構的形成。對於內皮細胞分泌基質分解酵素促使細胞外基質崩解之作用,L4和L5同樣具有抑制的功能。然而,存在於家族性高膽固醇血症族群中的L1-L3三個次片段及正常膽固醇血症族群中的所有次片段在上述不同實驗中均不具有抑制效應。综合我們的結果可證明相較於FH-L4及其他次片段,FH-L5在血管新生之過程中具有顯著的抑制效應,且此體外之抑制效應與體內粥狀硬化過程中經過氧化修飾之低密度脂蛋白造成內皮細胞傷害的效應具有一致性。
Abstract
Compelling evidence indicated that major risk factors for atherosclerosis such as oxidatively modified low density lipoprotein (oxLDL), high glucose, and reactive oxygen species promote endothelial cell apoptosis and thereby may contribute to the initiation of atherosclerotic lesion formation. Using fast protein liquid chromatography (FPLC), plasma LDL from familial hypercholesterolemic (FH) patients were separated into five subfractions, L1–L5. Among them, L5 subfraction was highly electronegative and suppressed DNA synthesis in cultured bovine aortic endothelial cells (BAEC) and stimulated mononuclear cell adhesion to cultured endothelial cells in vitro. Because impaired angiogenesis plays an important role in the pathogenesis of atherosclerosis, the anti-angiogenic functions of LDL subfractions from FH subjects were examined. Subconfluent BAEC (6 to 10 passages) maintained in DMEM containing 10% serum were treated with LDL subfractions at a dose of 20 μg/ml, and the effects on anti-angiogenic functions, including cell proliferation, migration, apoptosis, tube formation, and secretion of matrix metalloproteinase (MMP) were determined. Similar to Cu2+ ox-LDL, FH-L4 and FH-L5 inhibited cell proliferation to 80.9±2.4% (p<0.05) and 58.5±4.3% of control (p<0.001), respectively, while the other FH (L1-L3) and all subfractions isolated from normocholesterolemic (N) subjects had negligible effects. Similarly, FH-L4 and -L5, but not FH-L1 to -L3, retarded cell migration to 326.9 ± 19.4 (p<0.05) and 215±16 cells (p<0.001 with the control values of 402±34 cells), respectively. FH-L5 induced almost 20% of BAEC to undergo apoptosis; FH-L4 caused very mild effects, and other subfractions did not affect apoptosis In addition, FH-L4 and -L5 perturbed tube formation by BAEC in culture (5.8±0.2 and 3.4±0.4, respectively, versus control 8.5±1.5 tubes). Finally, FH-L4 and -L5 inhibited secretion of MMP-2 by BAEC (72.7±6.9 and 18.9±4.8% of control, respectively). The results demonstrate that FH-L5 potently affects multiple processes that are vital to normal angiogenesis, FH-L4 had milder effects, and other FH and N subfractions had negligible effects. In turn, these effects in vitro on processes pivotal to angiogenesis are consistent with potential effects of ox-LDL on endothelial dysfunction during atherogenesis in vivo.
目次 Table of Contents
Index
Page
Contents i
Abstract in Chinese iv
Abstract in English v


Introduction…………………………………………………...........1
Materials and Methods
Reagents………………………………….…………………......10
LDL preparations and LDL subfractions……………………….10
Cell culture………………………….………..............................10
Cell proliferation assay………………….……….......................11
Apoptosis assay………………………….………......................12
Cell migration assay………………………….………..............12
Tube formation assay………………………….……….............13
Matrix metalloproteinases (MMPs) zymography…...................14
Western blot analysis………………………….……….............14
qRT-PCR………………………………………….…................15

Statistical analysis……………………………………………..16
Results
Electronegative FH-LDL subfractions reduce proliferation and increased apoptosis in BAEC……………………………........17
Electronegative FH-LDL subfractions reduce BAEC migration………………………………………………………18
Electronegative FH-LDL subfractions reduce tube formation by BAEC………………………………………………………….19
Electronegative FH-LDL subfractions inhibit MMP-2 secretion
by BAEC…………………………………………………........19
Discussion………………………………………………………..21
Future Perspectives………………………………………............28
References………………………………………………………..29
Tables…………………………………………………………….35
Figures……………………………………………………….......37
參考文獻 References
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