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研究生:吳思瑩
研究生(外文):Shih-YingWu
論文名稱:17-丙烯胺基-17-去甲氧基格爾德黴素對抗腸病毒七十一型之研究
論文名稱(外文):The effect of 17-allylamino-17-demethoxygeldanamycin on enterovirus 71 replication in vitro and in vivo
指導教授:余俊強
指導教授(外文):Chun-Keung Yu
學位類別:碩士
校院名稱:國立成功大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:61
中文關鍵詞:腸病毒七十一型17-丙烯胺基-17-去甲氧基格爾德黴素熱休克蛋白九十
外文關鍵詞:EV7117-AAGHsp90
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腸病毒七十一型是屬於微小病毒科的腸病毒屬,嬰幼兒童感染會發生手口足症,偶爾病發重症,例如腦膜腦炎、肺水腫、或是神經性疾病,更嚴重者會致死。現今在臨床上針對腸病毒七十一型感染的用藥僅限於米力農(Milrinone)、靜脈注射免疫球蛋白(IVIG)及一些支持性療法,因此希冀可以尋找更多其他有效治療用藥。熱休克蛋白九十(Heat shock protein 90;Hsp90)是宿主細胞內重要的伴隨蛋白,可以幫助胞內蛋白正確摺疊,執行功能。由於病毒是絕對寄生,可以利用宿主蛋白幫助自己複製,微小病毒科若干病毒已證實會利用Hsp90協助病毒蛋白成熟,而且其複製可被17-丙烯胺基-17-去甲氧基格爾德黴素 (17- allylamino- 17- demethoxygeldanamycin ;17-AAG)抑制,因此我們要探討是否可用17-AAG抑制腸病毒七十一型的感染。首先,利用噻唑藍溴化四唑方法和臺盼藍細胞存活率分析法確認17-AAG在1 µM的濃度以下,於24小時內不會對細胞產生即毒性。而後在溶斑檢定法及半數組織培養感染劑量確定可以有效抑制腸病毒七十一型的複製,並且可以減少病毒所造成的細胞病變情形。相關實驗也證實,在細胞感染腸病毒七十一型後,越早給予17-AAG處理,抑制病毒的效果越好。在動物實驗部分,我們證實感染腸病毒七十一型的三日齡仔鼠若給予17-AAG治療,會有較高的存活率。總的來說,在體內及體外試驗都證明17-AAG可以抑制腸病毒七十一型的複製及感染,可以提供腸病毒七十一型臨床用藥的一個選擇。
Enterovirus 71 (EV71) belongs to the Picornaviridae family. EV71 infection may cause hand, foot and mouth diseases (HFMD) in infants and young children, but sometimes result in severe complications, including acute encephalitis and some neurological diseases, even death. Nowadays, the clinical treatments are limited to Milrinone and supporting treatment. We hope to find a new treatment for EV71 infection. Heat shock protein 90 (HSP90) is a major chaperon in host cells. It often helps cellular protein fold correctly. Because viruses are intracellular obligate parasites, it may utilize host proteins for their replication. We hypothesized that EV71 may hijack HSP90 for its replication, and that we can use HSP90 inhibitor, 17-allyamino-17-demethoxygeldanamycin (17-AAG), to inhibit EV71 replication. We first checked the cytotoxicity of 17-AAG with MTT assay and trypan blue exclusion assay. We found that 17-AAG didn’t have acute cytotoxicity in 24 hrs at a dose lower than 1 µM. And then, we wanted to know whether 17-AAG could inhibit EV71 viral replication in vitro. RD cells were infected with EV71/4643, and 17-AAG was given in 1, 2, and 6 hrs. Viral titer of EV71 was decreased after treating 17-AAG. Finally, we wanted to test the effect of 17-AAG in vivo. The EV71/MP4-y5 infected ICR mice that treated with 2.5 or 5 mg/kg 17-AAG had higher survival rate than the DMSO control mice. In Sum, 17-AAG could inhibit EV71 replication in vitro and in vivo.
中文摘要.................................................Ⅰ
英文摘要.................................................Ⅱ
致謝.....................................................Ⅲ
目錄.....................................................Ⅳ
圖目錄...................................................Ⅷ
第一章 緒論
一、腸病毒七十一型之病毒學簡介..............................1
1.分類.................................................1
2.基因型與結構..........................................1
3.生命週期與複製........................................1
二、腸病毒七十一型之致病機轉................................3
三、腸病毒七十一型之疾病與流行病學...........................3
四、腸病毒七十一型之預防與治療..............................4
1.抑制病毒的附著、進入與脫殼..............................4
2.抑制病毒蛋白的合成 ....................................5
3.抑制病毒蛋白3C蛋白酶 ..................................5
4.抑制病毒蛋白 2C ...................................5
5.抑制病毒蛋白 3A ...................................6
6.抑制病毒蛋白 3D RNA polymerase .......................6
7.核苷酸類似物..........................................6
五、宿主蛋白與病毒之交互作用................................7
1.轉錄起始因子..........................................7
2.HnRNP A1和K .........................................7
3.FBP1和2 .............................................8
4.Cleavage stimulation factor (CstF) -64..............8
六、伴隨蛋白..............................................8
1.伴隨蛋白的簡介........................................8
2.Hsp90與神經退化性疾病.................................10
3.Hsp90與癌症 .........................................10
4.Hsp90與病毒感染症疾病 ................................10
七、17-丙烯胺基-17-去甲氧基格爾德黴素(17-Allylamino-17-demethoxygeldanamycin,17-AAG) 之簡介....................11
第二章 研究動機與特異目標..................................12
第三章 材料與方法
一、材料.................................................14
二、方法
1.細胞培養.............................................19
2.病毒培養.............................................20
3.病毒定量:病毒斑分析法.................................21
4.病毒株鑑定:間接免疫螢光染色法..........................21
5.細胞毒性試驗..........................................22
6.病毒抑制能力試驗......................................22
7.半數組織培養感染量 (TCID50)............................23
8.17-AAG對小鼠產生的藥物毒性測試.........................23
9.測試EV71/MP4-y5的80%致死率............................23
10.腸病毒七十一型小鼠模式中,17-AAG對病毒抑制能力試驗.......23
11.西方墨點法..........................................24
12.統計分析............................................26
第四章 結果
一、腸病毒71型之病毒株鑑定.................................27
二、17-AAG之細胞毒性測試..................................27
三、17-AAG對於腸病毒七十一型在體外實驗之抑制作用.............28
四、17-AAG對於腸病毒71型在小鼠體內實驗之抑制作用.............29
五、17-AAG合併使用Ribavirin對於腸病毒七十一型之抑制作用......30
六、腸病毒七十一型與熱休克蛋白九十..........................33
第五章 討論..............................................34
參考文獻.................................................39

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