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研究生:李堯泰
研究生(外文):Yao-Tai Li
論文名稱:C反應蛋白/氧化低密度脂蛋白對人類主動脈內皮細胞釋放一氧化氮之影響
論文名稱(外文):Effect of C reactive protein / oxidized low density lipoprotein on nitric oxide production from human aortic endothelial cells
指導教授:黃光策
指導教授(外文):Kuang-Tse Huang
學位類別:碩士
校院名稱:國立中正大學
系所名稱:化學工程研究所
學門:工程學門
學類:化學工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
中文關鍵詞:內皮細胞一氧化氮氧化低密度脂蛋白C反應蛋白
外文關鍵詞:endothelial cellnitric oxideoxidized low density lipoproteinC reactive protein
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摘 要
動脈硬化的過程中可發現是由於嗜菌細胞與T淋巴球長期的累積,故可視為一種緩慢的發炎現象。氧化低密度脂蛋白 (oxidized low density lipoprotein, oxLDL) 是導致動脈硬化的危險因子,然而膽固醇並不是一個造成動脈硬化惡化的良好指標。最近的研究發現,C反應蛋白 (C reactive protein, CRP) 可視為動脈硬化產生的指標;且CRP會與oxLDL上的oxidized phosphocholine (oxPC) 結合,因此,同時參考oxLDL與CRP於體內的濃度可成為預測心血管疾病發生的重要指標。實驗發現oxLDL與CRP皆會減少一氧化氮 (NO) 的產生量與內皮一氧化氮合成酶 (eNOS) 的表現量,oxLDL會造成eNOS由細胞膜上的caveolae轉移至細胞質中,但CRP並不會。當oxLDL與CRP分子數比約為4:1時,會顯著地減少NO的產生量,但分子數比例不是4:1時,其對細胞產生的影響變小,原因推測為此比例之複合體並不足以活化CRP接受器。anti-CD36與anti-CRP皆可抑制oxLDL/CRP對細胞的影響,而使得NO產生量的提升。另一方面,oxLDL會使得細胞內caspase-3活性的增加,其影響效果比CRP來的顯著。

目 錄
中文摘要………………………………………………………………..Ⅰ
英文摘要………………………………………………………………..Ⅱ
目錄……………………………………………………………………..Ⅲ
圖目錄…………………………………………………………………..Ⅵ
表目錄…………………………………………………………………..Ⅷ
第一章 緒論…………………………………………………………..1
1.1 前言………………………………………………………….1
1.2 文獻回顧…………………………………………………….2
1.3 研究動機與目的…………………………………………...13
第二章 實驗儀器、器材與藥品……………………………………15
2.1 實驗儀器…………………………………………………...15
2.2 實驗器材…………………………………………………...17
2.3 實驗藥品…………………………………………………...18
2.3.1 細胞培養………………………………………………19
2.3.2 LDL的分離…………………………………………….19
2.3.3 oxLDL的製備………………………………………….20
2.3.4 CRP的製備…………………………………………….20
2.3.5 Limulus analyst (LAL) test…………………………….20
2.3.6 Lysis buffer contents……………………………………21
2.3.7 蛋白質濃度測定………………………………………21
2.3.8 oxLDL/CRP不同濃度組合變化及測量NO………….21
2.3.9 內外膜分離及萃取eNOS……………………………..22
2.3.10 SDS PAGE…………………………………………….22
2.3.11 Western blotting……………………………………….23
2.3.12 Caspase 活性測量……………………………………24
第三章 實驗步驟與分析方法………………………………………25
3.1 人類主動脈血管內皮細胞培養…………………………...25
3.2 LDL的分離­密度梯度超高速離心法……………………..27
3.3 oxLDL的製備……………………………………………....30
3.4 LDL以及oxLDL濃度測定………………………………..31
3.5 Crp的製備…………………………………………………..33
3.6 LAL Test…………………………………………………….34
3.7 細胞蛋白質濃度的測定以及CRP濃度測定……………..35
3.8 oxLDL/CRP不同濃度組合變化及測量NO……………….36
3.9內外膜分離及萃取eNOS…………………………………..40
3.10 SDS PAGE及western blotting…………………………….43
3.11 膜分離部分及SDS PAGE、western blotting………………49
3.12 oxLDL/CRP對Caspase活性之影響………………………..50
第四章 實驗結果與討論………………………………………………52
4.1 低密度脂蛋白(LDL)之氧化………………………………….52
4.2 oxLDL對內皮細胞釋放NO之影響…………………………53
4.3 CRP對內皮細胞釋放NO之影響………………………….…55
4.4 oxLDL/CRP對內皮細胞釋放NO之影響……………………57
4.5 抗體及oxLDL/CRP影響內皮細胞釋放NO之影響………..60
4.6 eNOS Western blotting與NO之關係………………………...63
4.7 oxLDL/CRP對eNOS分佈造成的影響………………………68
4.8 oxLDL/CRP對內皮細胞caspase-3活性之影響……………..70
第五章 結論與未來望…………………………………………………73
參考文獻………………………………………………………………..75
圖 目 錄
圖1.1 oxLDL 結構圖……………………………………………………3
圖1.2 CRP結構圖……………………………………..…………………5
圖1.3 oxLDL與CRP結合模擬圖………………………………………8
圖1.4 內皮細胞、白血球及oxLDL/CRP相互關係圖…………………9
圖1.5 NO在內皮細胞中訊息傳遞路徑……………………………….11
圖3.1 測量NO裝置圖…………………………………………………39
圖3.2 silica gel 分離細胞質與細胞膜示意圖……...…………………42
圖4.1 LDL於15 μM 氯化銅中隨時間之氧化情形…..……………52
圖4.2 oxLDL處理四小時影響內皮細胞Nitrite之產生量…………..54
圖4.3 oxLDL處理四小時後,第五小時影響內皮細胞Nitrite產生量.55
圖4.4 CRP處理四個小時影響內皮細胞Nitrite產生量………………56
圖4.5 CRP處理四小時後,第五小時影響內皮細胞Nitrite產生量…57
圖4.6 oxLDL/CRP處理四個小時影響內皮細胞Nitrite產生量……..59
圖4.7 oxLDL/CRP處理四小時後,第五小時影響內皮細胞Nitrite產生量……………………………………………………………..60
圖4.8 前四個小時抗體及oxLDL/CRP影響內皮細胞Nitrite釋放量.62
圖4.9 抗體及oxLDL/CRP處理四小時後,第五小時影響內皮細胞Nitrite產生量…...………………………………………………63
圖4.10 (a) eNOS分析圖 (b) eNOS定量圖…………………………....66
圖4.11 eNOS/βactin v.s Nitrite對比圖………………………………..67
圖4.12 eNOS在細胞膜與細胞質western blotting分析圖…….…….69
圖4.13 p-NA標準曲線…………………………………………………71
圖4.14 細胞處理oxLDL/CRP 24小時後caspase-3活性變化………72
表 目 錄
表3.1 超高速離心密度梯度分佈圖………………………………….28
表3.2 配製7.5 % 下層膠體溶液之配方…………………………….48
表3.3 配製4 % 上層膠體溶液之配方………………………………49

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