臺灣博碩士論文加值系統

蚊子是許多傳染病的主要病媒，舉凡瘧疾、絲蟲病、日本腦炎、登革熱、屈躬病，乃至近年爆發的茲卡病毒，都是經由不同種類的病媒蚊感染人類。其中，登革熱是一種具威脅性、不容小覷的傳染病，其主要病媒為埃及斑蚊與白線斑蚊。近年來，台灣的登革熱病例猛然攀升，根據疾管署的統計資料，2015年全國本土病例高達四萬三千多人。然而，目前尚未有可用的疫苗及藥物問世，對於登革熱病患只能採取支持性療法。因此，控制登革熱較實際可行的方法便是病媒的控制。過去在哺乳類的研究發現，當登革病毒感染宿主細胞時，病毒會在內質網進行複製與包裹，此時大量產生的病毒蛋白質可以藉由內質網的未摺疊蛋白質反應 (unfolded protein response, UPR) 以維持病毒蛋白質之穩定。此外，登革病毒的感染也會活化細胞中的自噬作用 (autophagy)。先前在哺乳類的研究中指出，未摺疊蛋白質反應與自噬作用皆有助於登革病毒的複製。不過，上述現象在埃及斑蚊中的研究則付之闕如。故本研究想進一步探討登革病毒感染埃及斑蚊後，未摺疊蛋白質反應與自噬作用對於病毒在病媒蚊體內複製的影響。我們初步以q-PCR偵測吸血後埃及斑蚊之未摺疊蛋白質反應相關基因表現。結果發現其表現量明顯提升。若以RNAi抑制埃及斑蚊未摺疊蛋白質反應相關基因後再感染登革病毒，其病毒之基因及蛋白質表現量皆明顯下降。另外，Western blot的結果顯示當埃及斑蚊吸血感染登革病毒，中腸在吸血後早期的自噬作用比正常吸血組明顯增多。在免疫螢光染色實驗中，則發現自噬作用相關蛋白與登革病毒產生共位現象。這些結果顯示埃及斑蚊之未摺疊蛋白質反應與自噬作用可能參與登革病毒的複製。往後我們將深入瞭解抑制登革病毒在埃及斑蚊體內複製的機制，這些結果對於未來制訂防治登革熱之策略將有重大助益。

The mosquito is the main vector of several important arthropod-borne diseases, such as dengue fever, Zika fever, Chikungunya fever and malaria. Among these infectious diseases, dengue fever caused great concern in Taiwan in recent years. According to the record from Taiwan CDC, there are more than 43 thousand infected cases in 2015. Until now, there is no effective dengue vaccine or drug available. Therefore, vector control becomes a potential alternative strategy for dengue control. In previous studies, dengue virus (DENV) was demonstrated to be replicated on the ER membrane of the mammalian cells. Unfolded protein response (UPR) was then activated in response to ER stress. Several reports indicated that dengue virus infection was associated with activation of autophagy in mammalian cells. However, the activation of UPR and autophagy by dengue virus in the mosquito remain largely unknown. In our preliminary results, we showed that UPR associated genes were highly expressed in blood-feeding mosquitoes. Silencing of UPR-associated genes with reverse genetic approach resulted in the inhibition of DENV genome and protein expression. Interestingly, activation of autophagy was exhibited in the midgut of DENV-infected mosquitoes. Also we found colocolization of DENV and autophagy-associated protein. Our results suggest that UPR and autophagy may participate in the regulation of DENV replication in mosquito. We will further investigate the detailed mechanisms underlying the regulation of viral replication in the mosquito. Results from this study may pave the way for the development of novel disease control strategies.

口試委員會審定書 i
序言 ii
摘要 iii
Abstract iv
目錄 v
圖目錄 viii
表目錄 ix
第一章 緒論 1
1.1. 病媒蚊傳播疾病 (Mosquito-borne disease) 1
1.1.1. 瘧疾 3
1.1.2. 茲卡病毒感染症 4
1.1.3. 登革熱 4
1.2. 埃及斑蚊生活史 6
1.3. 未摺疊蛋白質反應 (unfolded protein response, UPR) 6
1.4. 自噬作用 (autophagy) 8
1.5. 未摺疊蛋白質反應與自噬作用在內質網壓力下的交互作用 9
1.6. 實驗動機與假說 10
第二章 實驗材料與方法 11
2.1. 實驗步驟與流程 11
2.1.1. 埃及斑蚊之飼養與繼代 11
2.1.2. 病毒製備 11
2.1.3. 病毒注射 (Virus injection) 12
2.1.4. 埃及斑蚊之餵血 12
2.1.5. 雙股RNA (double-stranded RNA, dsRNA) 製備 12
2.1.6. RNAi-mediated silencing 與 knock-down efficiency test 14
2.1.7. RNA萃取 (RNA extraction) 14
2.1.8. 反轉錄作用 (reverse transcription, RT) 14
2.1.9. 聚合酶連鎖反應 (polymerase chain reaction, PCR) 15
2.1.10. 即時定量聚合酶連鎖反應 (real-time PCR, Quantitative PCR, q-PCR) 15
2.1.11. 埃及斑蚊唾液 (saliva) 之收取 15
2.1.12. Cell-based ELISA 16
2.1.13. Transmission assay 17
2.1.14. 溶斑試驗 (Plaque assay) 17
2.1.15. 西方點墨法 (Western blot) 17
2.1.16. Lysotracker staining 18
2.1.17. 免疫螢光染色法 (Immunofluorescence assay, IFA) 18
2.2. 實驗試劑之配置 19
第三章 結果 22
3.1. 埃及斑蚊感染登革病毒之RNA表現情形 22
3.2. UPR相關基因對於登革病毒在埃及斑蚊體內複製之影響 22
3.3. UPR及autophagy對於登革病毒在吸血後埃及斑蚊體內複製之影響 23
3.4. UPR相關基因對於埃及斑蚊唾液內登革病毒感染力之影響 24
3.5. Autophagy相關基因對於埃及斑蚊唾液內登革病毒感染力之影響 25
3.6. UPR相關蛋白在埃及斑蚊吸血感染登革病毒後不同組織之表現情形 25
3.7. Autophagy相關蛋白在埃及斑蚊吸血感染登革病毒後不同組織之表現情形 26
3.8. 埃及斑蚊於吸血後其中腸內autophagy之表現情形 26
3.9. 探討登革病毒於埃及斑蚊中腸內與自噬體之共位情形 27
3.10. UPR對於埃及斑蚊吸血後其autophagy在中腸表現之影響 27
3.11. 登革病毒對於埃及斑蚊體內JNK磷酸化之影響 28
第四章 討論 29
4.1. UPR與autophagy在埃及斑蚊吸血後之表現 29
4.2. UPR參與登革病毒於埃及斑蚊體內之複製 30
4.3. Autophagy參與登革病毒於埃及斑蚊體內之複製 31
4.4. UPR及autophagy於埃及斑蚊吸血後之交互關係 32
附圖 34
附表 50
參考文獻 54

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