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研究生:蔡宜靜
研究生(外文):Tsai, Yi-Jing
論文名稱:探討抗發炎藥物對登革病毒之影響
論文名稱(外文):To Investigate the Effect of Anti-inflammatory Drugs on Dengue Virus
指導教授:顏莉蓁
指導教授(外文):Yen, Li-Chen
口試委員:翁國峰林谷峻
口試委員(外文):Weng, Kuo-FengLin, Gu-Jiun
口試日期:2017-05-09
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2017
畢業學年度:105
語文別:中文
論文頁數:82
中文關鍵詞:登革病毒抗發炎細胞激素
外文關鍵詞:Dengue virusCOX-2PGE2CytokinesAnti-viral drugs
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登革病毒(DENV)是最普遍的蚊媒病毒之一。當宿主被登革病毒感染時,大多數的人會傾向發展成輕微的症狀,像是登革熱;但是少部分的人感染後會引發COX-2的表現以及增加相關下游產物PGE2的產生,並且也會誘發與調控細胞內的cytokines,造成細胞傷害,並增加血管擴張及提高血管通透性,造成嚴重的疾病,例如登革出血熱或登革休克症候群,而一般治療登革熱的方式只能在感染後進行支持性治療,目前僅有一個疫苗順利在墨西哥、巴西等國家上市,臺灣尚未許可上市,且目前並無直接抗登革病毒的藥物。2d是一種具有強效及長效抗發炎效果的化合物,此研究主要測試2d的抗發炎反應是否可以抑制登革病毒感染所造成的嚴重發炎疾病。實驗結果顯示在HEK-293細胞和Raw264.7細胞加入2d之後,發現可以抑制登革病毒感染所引發的COX-2之表現和PGE2的產生,以及抑制IL-8、IP-10、MIF等等Cytokines,並且使登革病毒蛋白質表現減少,降低登革病毒的生產量。在動物實驗結果中,2d提升感染登革小鼠存活率和降低病毒血症程度,根據實驗結果證實2d有效的抑制登革病毒以及減少登革病毒引起的發炎相關因子,具有可作為新穎抗登革病毒藥物的潛力。
Dengue Virus (DENV) infection is one of the most mosquito-borne infectious diseases. DENV infection may develop severe syndromes like dengue hemorrhagic fever or dengue septic shock. Those severe diseases, are caused by DENV-induced lead to the performance of COX-2 and improve the production of downstream products related to PGE2, and can also induce and regulate intracellular cytokines to increase blood vessels to dilate, improve vascular permeability and cause white blood cells migrate to the inflammatory site, causing cell damage. Till to date only one dengue vaccine has been succeeded in final stage of clinical trails, and no anti-dengue viral drugs are developed in clinical use. The only way for treatment of dengue patients is the supporting care. We hypothesized that the anti-inflammation acting of 2d may inhibit DENV-induced inflammation and sever disease. It is known that 2d have potent and long-lasting anti-inflammatory activity. The results showed that the 2d can inhibit expression of COX-2 and the production of PGE2 and the inhibition of IL-8, IP-10, MIF and so on were inhibited by dengue virus infection with 2d, and the effect of virus replication was reduced. In Animal study, to test 2d efficacy in vivo. 2d can enhance the survival rate and reduce viremia level. In conclusion, this data collectively suggests that 2d has a great potential in anti-viral drugs development.
目錄 I
圖目錄 III
中文摘要 IV
英文摘要 V
縮寫表 1
前言 2
一、 登革病毒的簡介 3
二、 登革病毒蛋白簡介 4
三、 登革病毒的傳播途徑 8
四、 登革病毒流行病學簡介 9
五、 登革病毒之生活史 9
六、 登革臨床疾病 10
七、 登革熱病毒感染與細胞激素的產生 12
八、 對抗登革病毒相關疾病的策略藥物與現有疫苗之發展 12
九、 抗發炎藥物2d介紹 14
十、 實驗目的 14
研究方法 16
一、 細胞培養 (Cell culture) 17
二、 細胞增生實驗 (Cell Proliferation assay) 18
三、 西方墨點法 (Western blot) 18
四、 病毒斑測試 (Plaque assay) 22
五、 病毒斑點形成實驗 (Focus forming assay) 22
六、 間接免疫螢光染色法 (Indirect immunofluorescent assay) 23
七、 萃取細胞之RNA (RNA Preparation) 24
八、 反轉錄聚合酵素連鎖反應 (RT-PCR) 25
九、 即時聚合酶鏈式反應 (Real-Time PCR,qPCR) 25
十、 酵素免疫分析法 (Enzyme-Linked Immunosorbent Assay,ELISA) 26
十一、 動物實驗 (Animal study) 26
十二、 統計分析 (Statistical analysis) 27
實驗結果 28
壹、 探討2d的細胞存活率之影響 29
貳、 2d抑制登革病毒1~4型病毒蛋白質表現及病毒複製 29
參、 探討2d是否能抑制由登革病毒誘發的發炎反應 33
肆、 探討2d保護感染登革小鼠效果 39
討論 41
壹、 探討2d抑制登革病毒1~4型感染效果 42
貳、 探討2d是否能抑制由登革病毒誘發的發炎反應 43
參、 探討2d保護感染登革小鼠效果 44
肆、 抗發炎藥物與登革疾病治療 45
參考文獻 48
圖示 53
附錄 74

圖目錄

圖一、2d之結構 54
圖二、2d對於HEK-293、Raw264.7細胞株存活率之影響 55
圖三、2d處理對感染登革第1~4型的HEK-293細胞株表現病毒蛋白質及病毒複製效果 56
圖四、2d處理對感染登革第1~4型的Raw264.7細胞株表現病毒蛋白質及病毒複製效果 58
圖五、2d對受登革病毒第二型感染HEK-293細胞株表現NS3蛋白質之影響 60
圖六、2d對受登革病毒第二型感染Raw264.7細胞株表現NS3蛋白質之影響 61
圖七、2d治療對於登革病毒第1~4型感染HEK-293細胞株之TNF-α影響 62
圖八、2d治療對於登革病毒第1~4型感染HEK-293細胞株之IL-6影響 63
圖九、2d治療對於登革病毒第1~4型感染HEK-293細胞株之IL-8影響 64
圖十、2d治療對於登革病毒第1~4型感染HEK-293細胞株之PGE2影響 65
圖十一、2d治療對於登革病毒第1~4型感染Raw264.7細胞株之TNF-α影響 66
圖十二、2d治療對於登革病毒第1~4型感染Raw264.7細胞株之IL-6影響 67
圖十三、2d治療對於登革病毒第2型感染HEK-293細胞株之mRNA影響 68
圖十四、2d治療對於登革病毒第2型感染Raw264.7細胞株之mRNA影響 70
圖十五、2d治療對於感染登革病毒第2型小鼠生存率之影響 72
圖十六、2d治療對於登革病毒第2型造成的viremia效果 73
附錄一、本實驗所使用之引子 (primer) 74
附錄二、測試2d藥物的細胞毒性 75
附錄三、測試2d治療減少登革病毒RNA百分比 76






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