臺灣博碩士論文加值系統

登革熱(dengue fever)是好發於熱帶及亞熱帶國家的傳染性疾病，是由登革病毒(Dengue virus)所引起，登革熱屬於發病率高且傳播速度快之疾病，台灣是登革熱的疫情區之一，每年都有上千名患者感染登革熱，對台灣地區而言，登革熱是需與予重視的傳染病，為了抑止登革熱的傳播，我們需要更了解登革病毒。 根據過去實驗室Microarray的實驗結果顯示，當登革病毒感染樹突狀細胞後，會產生大量由干擾素誘導分泌的鳥甘酸結合蛋白4(GBP4)，GBP4在登革病毒的感染過程中扮演什麼角色，並不清楚。本論文第一部分主要探討GBP4在登革病毒感染過程中扮演的角色。若是能夠了解GBP4在登革病毒感染過程中的角色，可能可以提供抑制或治療登革病毒感染的標的。根據我們研究的結果發現當病毒感染到人類肺腺癌細胞株A549後，會誘導產生大量的GBP4，但GBP4對於病毒的複製能力並不會有什麼影響，但對於I型干擾素的IFNβ1有負調控的功能，同時GBP4會正調控III型干擾素中的IFNλ1。但是這部份結果與樹突狀細胞(dendritic cell)的結果並不相符，我們目前尚未找到方法解釋這個部分的問題，所以暫時暫停這部分的研究。 第二部分則是因為在我們實驗室過去的對Gal9的研究中，發現Gal9除了會誘導促發炎因子IL-12的產生外，也會對抗發炎因子 IL-10的表現具有正調控的功能，而在過去文獻中顯示，在登革重症的病患血清中IL-10的表現量會高於病情較輕者，但是IL-10在登革病毒感染過程中的分子機制，目前並不清楚，因此，在這部分的研究中想要去探討在登革病毒感染時，被誘導表現的Gal9是透過什麼機制以調控IL-10的表現量。結果發現登革病毒感染上調的Gal9在A549會對MAPK p38的磷酸化與NF-κB、STAT3的活化有正調控的現象。因此，推測登革病毒誘導產生的Gal9可能透過MAPK p38的磷酸化與NF-κB、STAT3的活化調控IL-10的產生。

Dengue fever is a tropical and subtropical disease caused by dengue virus infection and is transmitted by mosquitoes. It is a deadly viral disease and affects millions of people worldwide. Until now, there is no vaccines or medicines that can prevent the infection. Dengue virus is a positive-sense, single stranded RNA virus belongs to flaviviridae family. Its genomes is about 11000 bases that codes 3 structural protein and 7 types of non structural protein. There are four serotype of dengue virus referred as DENV1, DENV2, DENV3 and DENV4. IFNs are known potent in against viral propagation via induce several ISGs. GBP4, an IFN inducible gene, had been reported can negative regulate the production of type I IFN. From our lab’s microarray data, GBP4 was induced in dengue virus infected dendritic cells. Due to the function of GBP4 is not well understood in dengue virus infection, I want to investigate the role of GBP4 in DV infection. In my study, DV infection induced GBP4 not only in dendritic cell but also in A549 which I used it in my following experiments. Through knockdown experiment, dengue virus production was not influence in GBP4 deficient cell. Interesting, we found that GBP4 might negative regulate IFNβ1 but positive regulate IFNλ1 expression in A549 cell. However, these data weren’t comparable to what found in DC. Therefore, this project was temparatory pending and turned to the next one.
Interleukin-10(IL-10) is an anti-inflammatory cytokine that was defined as a TH2-type cytokine. Previous reports indicated IL-10 was robust induced in DV-infected patients . We had found that Gal9 positive regulate IL-10 in DV-infected DC. However, how IL-10 was regulated by Gal9 in DV infection is not well understand. Therefore, I plan to investigate how Gal9 regulate the production of IL-10 in DV-infected cell.
In my study, Gal9 positive regulate IL-10 in DV-infected A549 which is similar to what found in dendritic cell. I examined whether MAPK, NF-κB and STAT3 are the target signaling molecules activated by Gal9 and influence IL-10. Our data showed that the activation of NF-κB and STAT3 were decreased in Gal9 deficient cell after DV infection. These result indicated that Gal9 positive regulate the production of IL-10 might through NF-κB and STAT3. Subsequently, we plan to further confirm Gal9 regulate IL-10 and is through NF-κB and STAT3 via gain of function study. In addition, we will also explore which component of dengue virus responsible for induction of Gal9.

第一部分：鳥甘酸結合蛋白4在登革病毒感染過程扮演的角色
第一章 緒論
第一節 登革熱(dengue fever) 2
第二節 登革病毒 (Dengue virus) 3
第三節 干擾素 (Interferon，IFN) 4
第四節 鳥甘酸結合蛋白(guanylate-binding protein) 4
第五節 研究目的與策略 5
第二章 材料與方法
第一節 細胞培養 6
第二節 細胞轉染 (Cell transfection) 6
第三節 蛋白質電泳與西方墨點法 (Western Blot) 7
第四節 病毒斑試驗 (Plaque assay) 10
第五節 流式細胞術 (Flow assay) 11
第六節 即時定量聚合酶鏈式反應 (qPCR) 12
第七節 結果統計分析方法 14
第三章 結果
第一節 A549為能穩定表現GBP4的細胞株 15
第二節 GBP4不影響登革病毒生產 15
第三節 在A549登革病毒感染誘導產生I型及III型干擾素 16
第四節 在A549 GBP4可能負調控IFNβ1而正調控IFNλ1 16
第四章 討論 17

第二部分：半乳糖凝集素9調控介白素10的分子機制
第一章 緒論
第一節 半乳糖凝集素9 (Galectins9) 27
第二節 介白質10 (Interlukin 10) 28
第三節 研究目的與策略 29
第二章 材料與方法
第一節 電泳速度變動分析 (Electrophoretic moblilty shift assay, EMSA) 31
第二節 酵素結合免疫吸附法 (enzyme-linked immunosobent assay, ELISA) 34
第三章 結果
第一節 登革病毒感染A549細胞株上調Gal9與IL-10 36
第二節 在A549中登革病毒誘導產生之Gal9正調控IL-10 36
第三節 登革病毒促進MAPK的磷酸化增加 37
第四節 Gal9正調控MAPK p38的磷酸化 38
第五節 登革病毒活化IL-10的上游轉錄因子NF-κB、STAT3及AP-1 38
第六節 Gal9正調控IL-10的上游轉錄因子NF-κB與STAT3 39
第四章 討論 40
第五章 參考文獻 59

1. Maria G. Guzman , Peeling RW et al., “Dengue: a continuing global threat,” Nat Rev Microbiol. Vol.8, no.12, pp.S7-16, Dec.2010
2. MacMicking JD, “IFN-inducible GTPases and immunity to intracellular pathogens,” Trends Immunol. Vol.25, no.11, pp.601-609, Nov.2004
3. Vestal DJ, Jeyaratnam JA., “The Guanylate-Binding Proteins: Emerging Insights into the Biochemical Properties and Functions of This Family of Large Interferon-Induced Guanosine Triphosphatase,” J Interferon Cytokine Res. Vol.31, no.1, pp.89–97, Jan.2011
4. Kim BH1, Shenoy AR, Kumar P, Das R, Tiwari S, MacMicking JD., “A family of IFN-γ-inducible 65-kD GTPases protects against bacterial infection,” Science. Vol.332, no.6030, pp.717-721, May.2011
5. Daniel Degrandi , Pfeffer K. et al., “Extensive Characterization of IFN-Induced GTPases mGBP1 to mGBP10 Involved in Host Defense,” J Immunol. Vol.179, no.11, pp.7729-7740, Dec.2007
6. Itsui Y , Watanabe M. et al., “Antiviral effects of the interferon-induced protein guanylate binding protein 1 and its interaction with the hepatitis C virus NS5B protein,” Hepatology. Vol.50, no.6, pp.1727-1737, Dec.2009
7. Wen Pan, Xiangyang Zuo, Tingting Feng, Xiaohong Shi,Jianfeng Daicor, “Guanylate-binding protein 1 participates in cellular antiviral response to dengue virus,” Virol J. Vol.9, pp.292, Nov.2012
8. Anderson SL1, Carton JM, Lou J, Xing L, Rubin BY., “Interferon-induced guanylate binding protein-1 (GBP-1) mediates an antiviral effect against vesicular stomatitis virus and encephalomyocarditis virus,” Virology. Vol.256, no.1, pp.8-14, Mar.1999
9. Hu Y , Sun B. et al., “Guanylate binding protein 4 negatively regulates virus-induced type I IFN and antiviral response by targeting IFN regulatory factor 7.” J Immunol. Vol.187, no.12, pp.6456-6462, Dec.2011
10. Chen Zhu, Kuchroo VK et al., “The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity,” Nature Immunology. Vol.6, pp.1245-1252, Nov.2005
11. Lucy Golden Mason , Rosen HR et al., “Galectin-9 Functionally Impairs Natural Killer Cells in Humans and Mice,” J Virol. Vol.87, no.9, pp.4835–4845, May.2013
12. Hsu YL, Wang MY, Ho LJ, Huang CY, Lai JH, “Up-regulation of galectin-9 induces cell migration in human dendritic cells infected with dengue virus,” Journal of Cellular and Molecular Medicine. Vol.19, no.5, pp.1065-1076, May.2015
13. Hema Bashyam, “Th1/Th2 cross-regulation and the discovery of IL-10,” J Exp Med. Vol.204, no.2 pp.237, Feb.2007
14. C. M. Hawrylowicz1 & A. O'Garra, “Potential role of interleukin-10-secreting regulatory T cells in allergy and asthma,” Nature Reviews Immunology. Vol.5, pp.271-283, Apr.2005
15. Asadullah K1, Sterry W, Volk HD, “Interleukin-10 therapy--review of a new approach,” Pharmacol Rev. Vol.55, no.2, pp.241-269, Jun.2003
16. Margarida Saraiva & Anne O'Garr, “The regulation of IL-10 production by immune cells,” Nature Reviews Immunology. Vol.10, pp.170-181, Mar.2010
17. Gathsaurie Neelika Malavige, Graham S Ogg et al., “Serum IL-10 as a marker of severe dengue infection,” BMC Infectious Diseases. Vol.13, no.1, pp.341, Jul.2013,
18. Tsai TT1, Chuang YJ, Lin YS, Wan SW, Chen CL, Lin CF., “An emerging role for the anti-inflammatory cytokine interleukin-10 in dengue virus infection,” J Biomed Sci. Vol.20, pp.40, Jun.2013
19. Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG, “Regulation and Functions of the IL-10 Family of Cytokines in Inflammation and Disease,” Annual Review of Immunology. Vol.29, pp.71-109, Apr.2011
20. Gabryšová L, Howes A, Saraiva M, O'Garra A, “The Regulation of IL-10 Expression,” Current Topics in Microbiology and Immunology. Vol.380, pp.157-190 Jul.2014,
21. Méndez-Samperio P1, Trejo A, Pérez A, “Mycobacterium bovis Bacillus Calmette-Guérin (BCG) stimulates IL-10 production via the PI3K/Akt and p38 MAPK pathways in human lung epithelial cells.” Cell Immunol. Vol.251, no.1, pp.37-42, Jan.2008