臺灣博碩士論文加值系統

中文摘要
低密度脂蛋白（low-density lipoprotein；LDL）的氧化修飾是動脈粥狀硬化（atherosclerosis）形成過程中的一個重要危險因子，細胞吞噬氧化LDL（oxidized LDL；oxLDL）亦是關鍵步驟之一。本研究主要在探討內在（endogenous）與外在（exogenous）物質對LDL氧化與脂質運送的影響。
首先，我將內生性的高密度脂蛋白（high-density lipoprotein；HDL），透過去脂，得到脂蛋白元apoHDL，藉由銅離子或細胞介入LDL氧化的實驗來瞭解apoHDL對LDL氧化的影響。從conjugated diene（CD），thiobarbituric acid reactive substances（TBARS）與agarose gel electrophoresis的結果中都顯示apoHDL能抑制LDL的被氧化修飾。在膽固醇運送中，隨著apoHDL濃度的增加，使oxLDL帶入巨噬細胞膽固醇的量（cholesterol influx）減少，膽固醇送出細胞（cholesterol efflux）的能力則是明顯增加。在oil red O染色實驗中，也顯示當apoHDL與oxLDL同時處理細胞時可減少脂質顆粒堆積在細胞中。
接著我以巨噬細胞（macrophages）為模式，探討脂肪酸對氧化LDL及脂質運送的影響。實驗中以各種脂肪酸先處理巨噬細胞 24小時後，再加入銅離子與LDL，結果只有linoleic acid，linolenic acid與docosahexaenoic acid處理的細胞，培養基中的TBARS會增加。另外，針對這些細胞同時給予維生素E或維生素C，結果看到當個別給予細胞1 mM維生素E或640 M維生素C時，培養基中產生的TBARS有下降。若脂肪酸先處理巨噬細胞24小時後，再加入維生素C與LDL，40 M維生素C即可顯著抑制培養基中TBARS的產生。若將維生素E與維生素C按照不同比例混合，同時與脂肪酸共同處理細胞，結果看到當160 M維生素E與160 M維生素C先處理細胞24小時，再加入LDL組，TBARS形成之抑制效果優於其他混合；而當細胞先處理維生素E 24小時後，再加入LDL與維生素C，不管以何種比例混合，皆能達到很好的抑制效果。此外，以600 M linolenic acid處理的細胞可促進膽固醇的吸收，docosahexaenoic acid組則是減少膽固醇的吸收。
另外，也將各種脂肪酸製備成reconstituted LDL（rLDL）處理巨噬細胞，實驗結果顯示只有linolenic acid所構築的rLDL最易遭到氧化攻擊。針對rLDL（18:3）給予抗氧化劑處理，結果顯示當維生素E或維生素C先個別處理巨噬細胞24小時後再加入rLDL，1 mM維生素E，可使培養基中TBARS之產生下降，而維生素C卻是不影響TBARS之產生。至於維生素C與rLDL同時處理細胞時，亦增加培養基中TBARS的形成。另外，若將維生素E先處理細胞，再加入rLDL與維生素C，或是維生素E與維生素C先處理細胞24小時後，再加入rLDL的細胞組中，不管是何種混合，都沒有抑制作用。在rLDL實驗中，細胞對氧化rLDL（18:2，20:4）所帶入之膽固醇的吸收增加，而rLDL在細胞膽固醇的送出中不受影響。
本論文藉由對apoHDL具有抑制LDL的氧化，降低巨噬細胞對膽固醇的吸收與增加膽固醇運送出細胞外的能力；另外，也針對各種脂肪酸以及不同處理方式的維生素E與維生素C，在不同濃度之下對LDL氧化與脂質運送影響的研究，提供了不同內在、外在因子對LDL氧化與脂質運送中的影響。

Abstract
Oxidative modification of low-density lipoprotein (LDL) is an important risk factor and the uptake of oxidized LDL (oxLDL) by macrophages is also a critical step in atherogenesis. The purpose of this study is to delineate the effects of endogenous and exogenous components on oxidation of LDL and lipid transport.
The effect of apoHDL, prepared by delipidation of HDL, on Cu2+ or cell-mediated LDL oxidation was examined. The results from analyses of conjugated diene (CD), thiobarbituric acid reactive substance (TBARS) and agarose gel electrophoresis showed that apoHDL inhibited the oxidation of LDL. In addition, the cholesterol influx was decreased and cholesterol efflux was increased significantly by apoHDL in a dose-dependent manner. This result was in accordance with oil red O staining data, which showed that apoHDL could decreased the accumulation of lipid droplets within cells.
First, the macrophages were used to study the effects of fatty acids on the oxidation of LDL and lipid transport. Results showed that pretreatment of cells with linoleic acid (18:2), linolenic acid (18:3) or docosahexaenoic acid (22:6) increased the TBARS formation in the medium. Pretreatment of cells with fatty acids and antioxidants (vitamin E and vitamin C), the TBARS formation in the medium was decreased. The addition of vitamin C (40 M), followed by preincubation of cells with fatty acids increased the TBARS formation in the medium. Pretreatment of cells with fatty acids and a mixture of vitamin E and vitamin C in 1:1 ratio showed the best inhibition of TBARS formation. When preincubation of vitamin E followed by the addition of vitamin C, TBARS formation was inhibited in mixtures of vitamin E and vitamin C. Cholesterol influx was increased when treated cells with linoleic acid and decreased when treated with docosahexaenoic acid.
Different composition of reconstituted LDL (rLDL), prepared from different fatty acids, were incubated with macrophages. The results indicated that rLDL, prepared from linolenic acid could be oxidized easier than the other of rLDL. Pretreatment of macrophages with 1 mM vitamin E could inhibited the oxidation of rLDL but pretreatment with vitamin C exerted an opposite effect. When incubating cells with vitamin C and rLDL, TBARS formation was increased. Pretreatment of macrophages with vitamin E followed by the addition of vitamin C showed the best inhibition of TBARS formation at 240 M vitamin E and 80 M vitamin C. Inhibition of TBARS was not shown in the group of macrophages pretreated with mixtures of vitamins E and C. The cholesterol influx was decreased in macrophages incubated with oxidized-rLDL (18:2, 18:3 and 20:4), and the cholesterol efflux was increased in macrophages incubated with oxidized-rLDL (18:2).
The results from this study showed that apoHDL inhibited the oxidation of LDL and cholesterol influx and, in contrast, enhanced cholesterol efflux significantly. And the study of different fatty acids and antioxidants provide a useful information regarding the effects of endogenous and exogenous factors on LDL oxidation and lipid transport.
縮寫表I
中文摘要 III
Abstract VI
緒論 1
實驗材料 7
實驗藥品 7
放射性同位素 9
其他材料 9
儀器設備 10
實驗方法 11
血漿檢體之取樣製備 11
脂蛋白元的製備與定量 11
脂蛋白的分離 11
脂蛋白的去脂質作用 12
脂蛋白元的製備 12
脂蛋白元的定量 13
SDS-polyacrylamide gel electrophoresis 13
蛋白樣品的處理 16
電泳的操作方法 16
Coommasive brilliant Blue染色及去染 17
低密度脂蛋白氧化的測定 18
溶液的製備 18
LDL體外氧化反應 19
細飽介入LDL氧化反應 19
LDL中脂質過氧化程度的指標---TBARS測定 21
LDL中Conjugated diene生成的測定 23
LDL之agarose gel電泳分析 24
巨噬細胞中膽固醇運送及脂質堆積的測定 26
native LDL的[3H]CE標定與測定 26
LDL的氧化與[3H]Cholesteryl ester-oxLDL的標定與測定 27
Lipoprotein deficiency serum製備 27
膽固醇送入巨噬細胞的測定 28
膽固醇送出巨噬細胞的測定 29
脂質堆積的測定 30
脂肪酸與抗氧化劑的製備 32
0.15 M NaCl，pH 7.4(含12% BSA) 32
脂肪酸的製備 32
維生素C的配製 34
維生素E的配製 34
脂肪酸、抗氧化劑與巨噬細胞氧化LDL之作用 35
對native LDL之影響分析 35
recontituted LDL氧化的影響分析 36
統計分析 40
結果 41
脂蛋白元的種類分析 41
脂蛋白元對LDL氧化的影響 41
脂蛋白元對LDL體外氧化（in vitro）的影響 41
脂蛋白元對細胞介入（cell-mediated）LDL氧化的影響 42
脂蛋白元對膽固醇及脂質運送的影響 43
脂肪酸與抗氧化劑對細胞氧化LDL與脂質運送的影響 43
脂肪酸與抗氧化劑對rLDL的氧化與脂質運送的影響 45
討論 48
圖表 59
參考文獻 80

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