臺灣博碩士論文加值系統

心腦血管疾病是導致人類死亡重要病因，而動脈粥樣硬化又是大多數心腦血管疾病發生前的早期病理症狀，不僅是一種慢性炎症疾病，也有著十分複雜的病理過程。 根據流行病學家和臨床學者的研究推測，低密度脂蛋白是造成心血管疾病的一個重要原因。血液中若含有高量低密度脂蛋白，滲透進入人類動脈內皮細胞下方的水平會升高，而內皮下的低密度脂蛋白可以被動脈壁中內皮細胞、平滑肌細胞、巨噬細胞產生的氧自由基所氧化修飾，形成氧化的低密度脂蛋白，進而引起了一連串的免疫反應機制造成動脈粥樣硬化症。最近的研究指出，C反應蛋白參予了這樣的發炎過程，我們可以在患有心血管疾病病人的血液中發現這項物質，藉由濃度的表現評估心血管疾病的風險。雖然該蛋白主要是由肝臟產生，但也有研究指出疾病位置組織本身也會分泌產生C反應蛋白，因此，氧化低密度脂蛋白與內皮細胞是否存在這樣的關係，是我們所關注的。因此本研究是利用氧化低密度脂蛋刺激動脈內皮細胞，以酵素免疫分析、聚丙烯醯胺膠體電泳，和毛細管區帶電泳對C反應蛋白做偵測鑑定。根據酵素免疫分析的結果得知，動脈內皮細胞會受氧化低密度脂蛋白的刺激分泌C反應蛋白。

Abstract
Cardiovascular disease is one of the most important cause of human death.
Atherosclerosis is the early pathology symptom before the disease takes place. It is
not only a chronic inflammation, but also pathology course is very complicated.
According to epidemic and clinic studies, low density lipoprotein is an important
factor to cause the cardiovascular disease. High concentration of low density
lipoprotein will cause the level below human aortic endothelial cells to rise. Low
density lipoprotein oxidized by oxygen free radical from endothelial cells, smooth
muscle cells, macrophages to form oxidized low density lipoprotein (ox-LDL). It
caused the immune response and atherosclerosis. Recent research showing that
C-reactive protein (CRP) participates the inflammation. C-reactive protein can be find
in patient’s blood, it’s concentration correlates with the risk of the cardiovascular
disease. Although CRP is primarily produced by liver, new finding have shown that
CRP is also produced by diseased tissues. We are interested in the relationship
between oxidized low density lipoprotein and endothelial cells. In this study, in vitro
ox-LDL was used to stimulate human aortic endothelial cells (HAEC). Then,
SDS-PAGE, enzyme-linked immunosorbent assay, capillary zone electrophoresis we
used to detect CRP. From the result of ELISA , it was found that HAEC synthesized
CRP under the influence of ox-LDL.

中文摘要 i
Abstract ii
目次 iii
圖次 vi
表次 ix
第一章、緒論 1
第一節、前言 1
第二節、動脈粥樣硬化（Atherosclerosis） 2
1.2.1、動脈粥樣硬化與心腦血管疾病 2
1.2.2、動脈血管組成及功能 2
1.2.3、動脈粥樣硬化症的形成【5】 3
第三節、C反應蛋白 8
1.3.1、什麼是CRP 8
1.3.2、CRP之特性與來源 8
第四節、C反應蛋白偵測 10
1.4.1、酵素免疫分析 10
1.4.2、聚丙烯醯胺膠體電泳 11
1.4.3、毛細管電泳 16
1.4.3.2、電滲現象與電滲流 18
1.4.3.3、影響分離效率的因素—譜帶展寬（band broadening） 21
第五節、研究目的 22
第二章、實驗部分 23
第一節、藥品 23
第二節、native low density lipoprotein（n-LDL）之分離製備與體外修飾 26
2.2.1、超高速離心分離血漿中n-LDL【29】【30】 26
2.2.2、體外氧化n-LDL【32】 27
第三節、細胞培養 29
2.3.1、Human Aortic Endothelial Cells之體外培養 29
2.3.2、Human Aortic Endothelial Cells繼代冷凍細胞之活化 30
2.3.4、Human Aortic Endothelial Cells添加LDL培養 30
第四節、PC Gel 和DEAE Resin純化 31
2.4.1、Immobilized p-Aminophenyl Phosphoryl Choline Gel（PC Gel） 31
2.4.2、Diethylaminoethyl Resin（DEAE Resin） 31
第五節、Enzyme-Linked Immunosorbent Assay（ELISA） 33
第六節、Polyacrylamide Gel Electrophoresis（PAGE） 34
第七節、CE參數設定 36
第三章、結果與討論 37
第一節、LDL純化與修飾 37
3.1.1、血漿高速離心分離LDL 37
3.1.2、n-LDL與ox-LDL製備 37
第二節、HAEC體外培養 41
第三節、純化 55
3.3.1、Diethylaminoethyl Resin 55
3.3.2、Immobilized p-Aminophenyl Phosphoryl Choline Gel 62
第四節、ELISA偵測 65
第五節、梯度膠體電泳 69
第六節、CE分析 73
3.6.1、偵測條件優化 73
3.6.2、CRP抗體分析 83
3.6.3、純化樣品分析 86
第四章、結論 88
參考文獻 89

圖次
圖1.2-1 動脈血管構造.. 3
圖1.2-2 動脈硬化形成過程【5】.. 4
圖1.2-3 不飽和脂肪酸被自由基攻擊形成過氧化氫脂質【11】.. 6
圖1.3-1 CRP 晶體結構【17】.. 9
圖1.4-1 三明治免疫分析法流程.. 10
圖1.4-2 biotinylation system..11
圖1.4-3 環境pH 影響蛋白質所帶電荷.. 12
圖1.4-4 膠體聚合示意圖.. 13
圖1.4-5 SDS-PAGE 流程簡圖.. 14
圖1.4-6 戊二醛與胺基作用簡示圖.. 15
圖1.4-7 簡易CE 基本裝置.. 17
圖1.4-8 電滲和電滲流示意圖.. 18
圖1.4-9 CE 與HPLC 電滲流的流動形式.. 19
圖3.1-1 血漿離心前後差異圖.. 37
圖3.1-2 離心式濾膜.. 38
圖3.1-3 LDL 氧化前後CE 圖。(a) n-LDL ; (b) ox-LDL.. 40
圖3.2-1 正常細胞5 天培養狀況。(a) 10x；(b) 40x 物鏡觀察.. 41
圖3.2-2 加入200μL EGM-2 培養0 到6 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 43
圖3.2-3 加入200μL PBS 培養0 到6 h (hour)。(a) 10x；(b) 40x 物觀察.. 44
圖3.2-4 加入200μL n-LDL 培養0 到6 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 45
圖3.2-5 加入200μL ox-LDL 培養0 到6 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 46
圖3.2-6 加入200μL EGM-2 培養9 到15 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 47
圖3.2-7 加入200μL PBS 培養9 到15 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 48
圖3.2-8 加入200μL n-LDL 培養9 到15 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 49

圖3.2-9 加入200μL ox-LDL 培養9 到15 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 50
圖3.2-10 加入200μL EGM-2 培養18 到24 h (hour)。(a) 10x；(b) 40x 物鏡觀察
.. 51
圖3.2-11 加入200μL PBS 培養18 到24 h (hour)。(a) 10x；(b) 40x 物鏡觀察.. 52
圖3.2-12 加入200μL n-LDL 培養18 到24 h (hour)。(a) 10x；(b) 40x 物鏡觀察..53
圖3.2-13 加入200μL n-LDL 培養18 到24 h (hour)。(a) 10x；(b) 40x 物鏡觀察..54
圖3.3-1 Elution Buffer pH 4.5，NaCl 濃度0、0.5、2、2M 下沖提EGM-2 .. 56
圖3.3-2 Elution Buffer pH 4.5，NaCl 濃度0、0.5、2、2M 下沖提BSA .. 57
圖3.3-3 Elution Buffer pH 4.5，NaCl 濃度0、0.5、2、2M 下沖提CRP standard..58
圖3.3-4 Elution Buffer pH 6.5 含2M NaCl 沖提EGM-2、BSA、CRP .. 59
圖3.3-5 Elution Buffer pH 8.1 含2M NaCl 沖提EGM-2、BSA、CRP .. 60
圖3.3-6 HAEC Cell Culture Supernant 以DEAE 純化.. 61
圖3.3-7 EGM-2 以PC Gel 純化.. 62
圖3.3-8 BSA 以PC Gel 純化.. 63
圖3.3-9 CRP stndard 以PC Gel 純化.. 63
圖3.3-10 以PC Gel 純化HAEC Cell Culture Supernatant.. 64
圖3.5-1 12.5％ SDS-PAGE 測試.. 69
圖3.5-2 4-20％ Gel 測試.. 70
圖3.5-3 DEAE Resin 純化之樣品跑4-20％ Gel 後銀染.. 71
圖3.5-4 PC Gel 純化之樣品跑4-20％ Gel 後銀染.. 72
圖3.6-1 pH 7.6 緩衝溶液分離 (a) CRP；(b) BSA .. 74
圖3.6-2 pH 7.6 緩衝溶液分離CRP+ BSA.. 75
圖3.6-3 pH 8.1 緩衝溶液分離 (a) CRP；(b) BSA .. 76
圖3.6-4 pH 8.1 緩衝溶液分離CRP+ BSA.. 77
圖3.6-5 pH 8.6 緩衝溶液分離 (a) CRP；(b) BSA .. 78
圖3.6-6 pH 8.6 緩衝溶液分離CRP+ BSA.. 79
viii
圖3.6-7 pH 9.1 緩衝溶液分離 (a) CRP；(b) BSA .. 80
圖3.6-8 pH 9.1 緩衝溶液分離CRP+ BSA.. 81
圖3.6-9 CE 圖之blank (樣品溶液).. 82
圖3.6-10 CRP 抗體以CE 分析.. 84
圖3.6-11 以CE 分析抗體與CRP 之結合.. 85

表次
表3.4-1 DEAE Resin 純化後，elution 樣品ELISA 分析數值.. 66
表3.4-2 PC Gel 純化後，elution 樣品ELISA 分析數值.. 67
表3.4-3 PC Gel 純化後，wash 樣品ELISA 分析數值.. 68

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