臺灣博碩士論文加值系統

登革熱是由埃及斑蚊或白線斑蚊傳播的急性病毒性熱疾，又稱斷骨熱或天狗熱，其依嚴重程度可分為典型性登革熱(classical dengue fever, DF)、登革出血熱(dengue hemorrhagic fever, DHF)以及登革休克綜合症(dengue shock syndrome, DSS)，死亡約15~50%。本實驗採用人類大主動脈內皮細胞，並以登革熱核心蛋白做轉染，並在CD55抗體處理後，觀察受到感染之內皮細胞其一氧化氮(nitric oxide, NO)釋放量及細胞凋亡之程度。實驗結果發現，內皮細胞受到核心蛋白轉染後，登革熱核心蛋白造成細胞NO釋放量增加主要是由於iNOS (inducible NO synthase)蛋白表現增加所造成。此外CD55抗體存在下會造成細胞NO釋放量的增加，然而在僅轉染載體之組別及轉染登革熱核心蛋白之組別其NO釋放量相當，而隨著時間增加，eNOS及iNOS蛋白表現量均下降。在細胞凋亡試驗中，我們也觀察到CD55抗體處理24小時時，可以促使未轉染及僅轉染載體細胞凋亡增加，並不會使登革熱核心蛋白轉染之細胞細胞凋亡加劇。

Dengue fever caused by flavivirus forms a wide range of syndromes from classical dengue fever (DF) to severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). It becomes an important mosquito-borne disease affecting humans after malaria. In this study, we investigated the effect of dengue type 2 virus core protein on nitric oxide (NO) release and cell apoptosis in human aortic endothelial cells in the presence and absence of CD55 antibody. We found that the NO release from the core protein transfected cells is increased as compared to the vector-only control, which results from the protein expression of inducible NO synthase (iNOS). CD55 antibody increases the NO release through the activation of endothelial NO synthase (eNOS) but causes reduction of iNOS and eNOS protein expression after 24 h treatment. Cell apoptosis is enhanced by 24 h treatment of CD55 antibody in the untransfected and vector-only cells but not the core protein-transfected cells. In summary, the cause of the early increased permeability of blood vessels in the secondary infection of dengue virus may be due mostly to the activation of eNOS by NS1 antibody

目錄

摘要 I
Abstract II
目錄 III
圖目錄 VIII
符號 IX
第一章 緒論 1
1.1 前言 1
1.2 登革熱 2
1.3 病媒 6
1.4 登革熱病毒 7
1.5 Vector 8
1.6 內皮細胞 11
1.7 一氧化氮(NO) 13
1.8 多胺類(polyamine) 16
1.9 非病毒性載體(non-viral vector) 18
1.10 細胞凋亡(apoptosis) 20
第二章 材料 24
2.1 儀器 24
2.2 耗材 27
2.3 藥品 28
2.3.1細胞培養 28
2.3.2細胞實驗 29
2.3.3 Lysis Buffer 配置 29
2.3.4蛋白質濃度測定 29
2.3.5 LDL製備 29
2.3.6 DNA純化 29
2.3.7載體製備 30
2.3.8細胞存活率分析 30
2.3.9一氧化氮測量 30
2.3.10 Western blotting 30
第三章 實驗步驟與原理 33
3.1 人類大主動血管內皮細胞培養 33
3.1.1 藥品配置 33
3.1.2 細胞繼代培養 34
3.1.2.1 Gelatin-coating 34
3.1.2.2 細胞解凍 34
3.1.2.3 細胞分盤 34
3.1.2.4 細胞保存 35
3.2 低密度脂蛋白之純化 35
3.2.1 藥品配置 35
3.2.2 密度確認 36
3.2.3 LDL分離 36
3.2.4 LDL濃度測量 37
3.2.5 LDL保存 38
3.2.6 LDL的使用 38
3.3 DNA載體製備 38
3.3.1 載體合成 38
3.3.2 載體製備 38
3.4 細胞分離 39
3.4.1 藥品配置 39
3.4.2 實驗步驟 39
3.5 蛋白質濃度測定-比色法 39
3.5.1 藥品配置 40
3.5.2 實驗步驟 40
3.6 細胞存活率分析 40
3.6.1 藥品配置 40
3.6.2 WST-8 assay實驗步驟 41
3.6.3 MTT assay 實驗步驟 41
3.7 ㄧ氧化氮濃度之測量 42
3.7.1 藥品配置 42
3.7.2 NO測量 42
3.8 西方點墨技術(Western Blot) 43
3.8.1 藥品配置 43
3.8.2 實驗步驟 45
3.8.2.1 Gel的製備 45
3.8.2.2 SDS-PAGE分析 46
3.8.2.3 轉漬至PVDF薄膜 46
3.8.2.4 Blocking 46
3.8.2.5雜交反應 (Hybridization) 47
3.8.2.6 呈色、壓片 47
第四章 實驗結果與討論 48
4.1 細胞轉染率測定 48
4.2 轉染登革熱核心蛋白染對內皮細胞的影響 48
4.2.1 細胞凋亡分析 48
4.2.2 NO釋放量測試 49
4.3 CD55抗體下轉染登革熱核心蛋白染對內皮細胞的影響 50
4.3.1 細胞凋亡分析 50
4.3.2 NO之釋放量測試 51
第五章 結論與未來展望 65
5.1 結論 65
5.2 未來展望 66
參考文獻 67

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