臺灣博碩士論文加值系統

登革病毒(dengue virus; DV)是一種具脂套膜的單股RNA病毒，屬於黃病毒科中黃病毒屬的DV亞屬，經由節肢動物感染人類，主要以埃及斑蚊和白線斑蚊叮咬宿主的途徑進而傳播給感染者並引起疾病的發生；流行病學的統計顯示登革病毒好發於在熱帶與亞熱帶國家，由以東南亞為最，是目前台灣地區很重要的公共衛生問題。
登革病毒感染會造成兩種病症：較輕微的登革熱及會致命的登革出血熱/登革休克症狀。登革熱會造成發燒、血管浸潤、嗜中性球低下症及補體活化等臨床症狀。而嗜中性球是人體內最重要的第一線防禦細胞，在受到外來微生物的侵入後，嗜中性球會受吸引到發炎組織並清除病原。嗜中性球在清除外來微生物(細菌)，目前已研究的相當透澈，然而嗜中性球在病毒感染的角色至今還不是很清楚。因此在本篇的研究我們想了解嗜中性球在登革病毒感染的角色，我們利用純化出的嗜中性球和登革病毒在體外培養，進而去了解嗜中性球受登革病毒刺激後對於病毒及嗜中性球生物活性的影響。
實驗結果顯示活的登革病毒可以使嗜中性球的存活率下降並使IL-8 MIP-1a與MIP-1b等化學趨化激素和LDH的上升，而利用紫外光去活性的登革病毒只發現到MIP-1b的產生。此外利用MPO酵素來作為觀察嗜中性球去顆粒作用的指標，發現不論是登革病毒的存活與否皆可刺激MPO的產生，而在登革病人的血清中也可以偵測到大量的MPO與IL-8含量。
另一方面我們利用共軛焦顯微鏡發現登革病毒可以藉由嗜中性球的吞噬作用進入體內，並刺激使得嗜中性球細胞表面的CD11b與TLR4表現量增加，同時我們也觀測到登革病毒也可使內皮細胞表面表現ICAM-1黏著分子增加並使得嗜中性球較易和內皮細胞黏著。
綜而言之，嗜中性球可以經由吞噬作用殺死登革病毒並活化產生化學趨化激素、去顆粒酵素與細胞表面的黏著分子，這些變化可能導致嗜中性球壞死及較易和內皮細胞黏著而造成嗜中性球減少及相關病變。

Dengue viruses (DV) are lipid-enveloped RNA viruses, which are transmitted by the mosquito vectors, Aedes aegypti and Aedes albopictus. DV are the most important flaviviruses in tropical and subtropical countries. Infection with DV may cause mild dengue fever or life threatening diseases such as dengue hemorrhagic fever and dengue shock syndrome. Vascular leakage, hemorrhage, neutropenia and complement activation are the hallmarks of these diseases. Polymorphonuclear leukocytes (PMNs) or neutrophils are critical effectors cells of the innate immune system, which can protect hosts by migrating to inflammatory sites and killing pathogen. However, the role of PMNs in the pathogenesis of DV infection is unclear. In this study, human neutrophils were incubated with DV to understand the effect of DV on the biological activities of neutrophils. Live but not UV inactivated DV (UV-DV) decreased the viability of neutrophils and increased the release of LDH and chemokines such as IL-8, and macrophage inflammatory protein (MIP-1a) from neutrophils. However, the granule enzyme, myeloperoxidase (MPO), was released from both live and UV-DV-stimulated neutrophils. In addition, the levels of IL-8 and MPO in the sera of dengue patients were also increased. Using fluorescent confocal microscopy, we found that DV was phagocytosed into neutrophils and co-localized with FITC-beads in neutrophils. Furthermore, DV-stimulation also increased the expression of CD11b neutrophils and ICAM-1 of HMEC-1 endothelial cell line and increased the binding of neutrophils to endothelial cell. In addition, the signal transduction molecule Toll-like receptor 4 (TLR4) was also increased in DV stimulated neutrophils. Taken together, neutrophils can uptake DV through phagocytosis and kill the virus. In this process, adherence of neutrophils to endothelial cells and necrosis of neutrophils were increased along with the release of chemokines, granule enzymes, and the expression of surface molecule. Therefore neutrophils may play important roles in the innate immune response against DV infection and may also contribute to the pathogenesis of dengue virus infection.

總目錄….…………………………………………………………..……………………..I
授權書….…………………………………………………………..……………….…..... Ⅳ
口試合格證明….…………………………………………………………………..…. Ⅴ
中文摘要….…………………………………………………………..……………...…. Ⅵ
英文摘要….…………………………………………………………..……….…..…..... Ⅷ
縮寫索引……….……………………………………………………..………………..... Ⅹ
圖目錄……….……………………………………………………..…………………..... ⅩⅠ
致謝….…………………………………………………………..…………………..…..... ⅩⅡ
第一章、緒論 ………………………………..…………………..………………....1
第二章、材料與方法………………………..…………………..………………....11
一、儀器….……………………………………………………………………………..11
二、試劑 …………………………………….………………………………………… 13
三、抗體 …………………………………….……………………………………..……15
1-1、登革病毒之製備…………………………………………………………..……….16
1-2、定量病毒效價…………………….………….…….…..……………………….….16
1-3、分離嗜中性球（PMN）……………………….……..………………………..…17
1-4、以酵素免疫法測定 PMN 釋出化學趨化激素（chemokines）濃度..17
2-1、細胞表面抗原免疫螢光染色……………...……………………………....19
2-2、反轉錄聚合酵素連鎖反應（RT-PCR）……...……………………………....19
2-3、細胞表面抗原免疫螢光染色……………………………...…………………..21
3-1、HUVEC分離培養 ………………………..………………………………..…..21
3-2、扇形流體室系統 ………………………..………………………………..…..21
3-3、白血球的先處理 ………………………..………………………………..…..22
3-2、內皮細胞的先處理 ……….……..……………….……………………………..22
3-3、內皮細胞與白血球吸附反應…….……………….………………..…..22
4-1、DV對PMN吞噬能力的測定……………………………………….………..…..23
4-2、偵測細胞內病毒蛋白…………..……………………………………..…..23
4-3、細胞存活率的測定 ……..……………………………………..….……..…….24
4-4、XTT assay ……..……………………………………..….……..…….…..……24
4-5、Annexin-V染色分析法…..……………………………………..….……..………..24
4-6、TUNEL分析法……………………………..….……..………..………..………..…25
4-7、DNA萃取及瓊膠電泳偵測凋亡 (DNA fragmentation Assay) …..…………26
4-8、LDH assay ……………………………..….……..………………..….………….27
第三章、結果 …………………………………….………………………………29
一、登革病毒刺激嗜中性球表現發炎性細胞激素之機轉…………………..29
1-1、發炎性細胞激素產生的時間及產量之變化….....…………..…………….29
1-2、發炎性細胞激素產生與病毒的關係…...………………………….…...30
1-3、登革病毒對嗜中性球去顆粒作用的影響…..……...….……………………30
1-4、登革病毒對TLR4表現量的影響 ...….……………………………………31
二、登革病毒對嗜中性球和內皮細胞黏著的影響……………………………32
2-1、登革病毒對嗜中性球細胞表面附著因子表現量的影響……………... 33
2-2、登革病毒對內皮細胞表面附著因子的影響…………………….……….33
2-3、嗜中性球受登革病毒刺激後與內皮細胞黏著的關係…………………..33
三、登革病毒對於嗜中性球生物功能活性之探討………….………………...34
3-1、登革病毒刺激對於嗜中性球細胞形態的影響…………………………... 34
3-2、 登革病毒對於嗜中性球吞噬作用之影響………………………….…………. 35
3-3、登革病毒對於嗜中性球存活之影響.………………………………..……… 36
3-4、登革病毒對於嗜中性球細胞凋亡(apoptosis)之影響…...….………….… 36
3-5、登革病毒對於嗜中性球細胞壞死(necosis)之影響….……………………37
第四章、討論…………….………………………...……………………………..…..39
一、登革病毒刺激嗜中性球表現化學趨化激素之機轉……………………..40
二、登革病毒對於嗜中性球去顆粒作用的影響……………………………….42
三、登革病毒刺激嗜中性球和內皮細胞黏著分子的影響…………….….....43
四、登革病毒刺激嗜中性球對於表面分子TLR4的影響……………………45
五、登革病毒刺激對於嗜中性球細胞活性的影響……………………………46
參考文獻…………………….………………………...……………………………..…..50
圖附錄………………..………….……………………...……………………………..…..58
自述…………………..………….……………………...……………………………..…..74

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