臺灣博碩士論文加值系統

　　腸病毒71型 (EV71)是一正向、單股的RNA病毒，屬於小RNA病毒科、腸病毒屬。1969年EV71第一次被分離出來後，全世界也陸續爆發大規模的流行；然而，目前並無有效的疫苗用於對抗EV71。薑黃素被認為具有多種有益的生物特性，如抗發炎、抗氧化與抑制蛋白質激酶C的活性。許多文獻也指出薑黃素具有抗病毒的特性，其能抑制B型肝炎病毒 (hepatitis B virus ;HBV)、HCV與coxsackievirus B3生長。在我們的研究中發現薑黃素能有效地減少病毒蛋白質生成與病毒效價上升。因已有文獻指出原兒茶酸等具抗氧化功能之化合物能藉由抑制病毒感染所引發的氧化壓力而抑制病毒生長，而薑黃素也被認為是一天然的抗氧化物質，根據我們的實驗結果發現薑黃素並未明顯減少由EV71感染所引發的氧化壓力。除此之外，薑黃素也被認為能抑制蛋白質激酶C (Protein kinase C; PKC) 的活性，我們發現感染時加入PKC inhibitor，能減低病毒蛋白質生成。但目前並沒有文獻證明PKC與EV71之間的關係，而根據我們的研究結果顯示薑黃素可能是透過影響PKC以調控EV71，未來能再藉由實驗證明薑黃素是否是透過抑制PKC的活性而影響EV71。

　　Enterovirus 71 (EV71), a single positive-strand RNA genome of approximately 7400 nucleotides, belongs to the genus Enterovirus within the family of Piconaviridae. Since the initial identification of EV71 in California in 1969, outbreaks of EV71 infection have occurred periodically throughout the world. However, there is no specific and effective vaccine for EV71. Curcumin, derived from eastern traditional medicines, Curcuma longa, has been found to have a variety of beneficial properties, such as anti-inflammatory, antioxidant, and regulating protein kinase C (PKC) activities. Reports have indicated that curcumin showed anti-viral activity against the coxsackie virus B3, the hepatitis B virus (HBV) and HCV. In our report, we found that EV71 viral protein synthesis and virus titer were significantly reduced when treating with curcumin. To understand how curcumin regulate EV71 replication, we first verified that curcumin treatment could not decrease EV71-induced ROS generation. Additionally, studies have shown that curcumin has the ability to inhibit PKC activity. We used PKC inhibitor (Ro-31-8220) to treat EV71-infected cell, and the data showed that viral protein synthesis were also decreased. The results indicated that curcumin may regulate EV71 replication through regulating PKC activity.

指導教授推薦書 iii
口試委員會審定書 iv
國家圖書館 博碩士論文電子檔案上網授權書 iii
長庚大學博碩士論文著作授權書 iv
誌謝 v
摘要 vi
Abstract vii
目錄 viii
圖表目錄 xi
第一章 緒論 1
1.1 腸病毒的流行病學 1
1.2 腸病毒的分類 1
1.3 腸病毒的一般特性 2
1.4 腸病毒的顆粒結構 2
1.5 腸病毒的基因體 2
1.6 腸病毒的生活史 3
1.7 薑黃素成分與結構 3
1.8 薑黃素的生物特性 4
1.9 氧化壓力 5
1.10 病毒與氧化壓力 5
1.11 薑黃素與抗氧化 6
1.12 蛋白質激酶Ｃ 6
1.13 薑黃素與蛋白質激酶Ｃ 7
1.14 研究目的 7
第二章 材料與方法 9
2.1 細胞培養 9
2.2 病毒株 9
2.3 薑黃素配製 9
2.4細胞毒性測試 9
2.5 病毒感染與薑黃素處理之方法 10
2.6西方墨點法（Western Blot） 10
2.7 病毒的生長曲線 11
2.8 病毒斑試驗 11
2.9 轉染（transfection）試驗 12
2.10 in vitro transcription assay 12
2.11活性氧物質的測定 12
第三章 結果 14
3.1薑黃素對於HT-29細胞之細胞型態與存活率之影響 14
3.2不同濃度薑黃素抑制病毒蛋白質表現之影響 14
3.3不同時間加入薑黃素對於抑制病毒蛋白質表現之影響 15
3.4薑黃素抑制病毒蛋白質表現之影響 15
3.5薑黃素抑制腸病毒71型複製 16
3.6薑黃素抑制腸病毒71型在細胞內的複製 16
3.7薑黃素對於腸病毒71型IRES活性之影響 16
3.8薑黃素抑制腸病毒71型感染所引發氧化壓力之生成 17
3.9薑黃素與蛋白質激酶C 18
第四章 討論 19
參考文獻 22
圖表 25


圖表目錄
圖一：細胞毒性測試 25
圖二：不同濃度薑黃素抑制病毒蛋白質表現之影響 26
圖三：不同時間加入薑黃素對於抑制病毒蛋白質表現之影響 28
圖四：薑黃素抑制病毒蛋白質生成之影響 29
圖五：薑黃素抑制腸病毒71型複製 30
圖六：薑黃素抑制腸病毒71型在細胞內的複製 31
圖七：薑黃素對於腸病毒71型IRES活性之影響 32
圖八：薑黃素抑制腸病毒71型感染所產生之活性氧生成 33
圖九：薑黃素與蛋白質激酶C抑制劑抑制EV71病毒蛋白質生成 34

Ammon, H. P. and M. A. Wahl (1991). "Pharmacology of Curcuma longa." Planta Med 57(1): 1-7.
Anand, P., A. B. Kunnumakkara, et al. (2007). "Bioavailability of curcumin: problems and promises." Mol Pharm 4(6): 807-818.
Ansardi, D. C., D. C. Porter, et al. (1996). "Poliovirus assembly and encapsidation of genomic RNA." Adv Virus Res 46: 1-68.
Bailly, J. L., M. C. Cardoso, et al. (2004). "Isolation and identification of an enterovirus 77 recovered from a refugee child from Kosovo, and characterization of the complete virus genome." Virus Res 99(2): 147-155.
Brouet, I. and H. Ohshima (1995). "Curcumin, an anti-tumour promoter and anti-inflammatory agent, inhibits induction of nitric oxide synthase in activated macrophages." Biochem Biophys Res Commun 206(2): 533-540.
Giachetti, C. and B. L. Semler (1991). "Role of a viral membrane polypeptide in strand-specific initiation of poliovirus RNA synthesis." J Virol 65(5): 2647-2654.
Grynkiewicz, G. and P. Slifirski (2012). "Curcumin and curcuminoids in quest for medicinal status." Acta Biochim Pol 59(2): 201-212.
Ho, M. (2000). "Enterovirus 71: the virus, its infections and outbreaks." J Microbiol Immunol Infect 33(4): 205-216.
Kim, K., K. H. Kim, et al. (2010). "Curcumin inhibits hepatitis C virus replication via suppressing the Akt-SREBP-1 pathway." FEBS Lett 584(4): 707-712.
Korenaga, M., M. Okuda, et al. (2005). "Mitochondrial dysfunction in hepatitis C." J Clin Gastroenterol 39(4 Suppl 2): S162-166.
Koundouris, A., G. E. Kass, et al. (2000). "Poliovirus induces an early impairment of mitochondrial function by inhibiting succinate dehydrogenase activity." Biochem Biophys Res Commun 271(3): 610-614.
Krausslich, H. G., M. J. Nicklin, et al. (1987). "Poliovirus proteinase 2A induces cleavage of eucaryotic initiation factor 4F polypeptide p220." J Virol 61(9): 2711-2718.
Lee, J., Y. H. Im, et al. (2005). "Curcumin inhibits interferon-alpha induced NF-kappaB and COX-2 in human A549 non-small cell lung cancer cells." Biochem Biophys Res Commun 334(2): 313-318.
Liu, J. Y., S. J. Lin, et al. (1993). "Inhibitory effects of curcumin on protein kinase C activity induced by 12-O-tetradecanoyl-phorbol-13-acetate in NIH 3T3 cells." Carcinogenesis 14(5): 857-861.
Lloyd, R. E., H. G. Jense, et al. (1987). "Restriction of translation of capped mRNA in vitro as a model for poliovirus-induced inhibition of host cell protein synthesis: relationship to p220 cleavage." J Virol 61(8): 2480-2488.
Mahmmoud, Y. A. (2007). "Modulation of protein kinase C by curcumin; inhibition and activation switched by calcium ions." Br J Pharmacol 150(2): 200-208.
McMinn, P. C. (2002). "An overview of the evolution of enterovirus 71 and its clinical and public health significance." FEMS Microbiol Rev 26(1): 91-107.
Melnick, J. L., V. I. Agol, et al. (1974). "Picornaviridae." Intervirology 4(5): 303-316.
Minor, P. D., M. Ferguson, et al. (1986). "Antigenic structure of polioviruses of serotypes 1, 2 and 3." J Gen Virol 67 ( Pt 7): 1283-1291.
Minor, P. D., G. C. Schild, et al. (1983). "Location and primary structure of a major antigenic site for poliovirus neutralization." Nature 301(5902): 674-679.
Neufeld, K. L., O. C. Richards, et al. (1991). "Purification, characterization, and comparison of poliovirus RNA polymerase from native and recombinant sources." J Biol Chem 266(35): 24212-24219.
Newton, A. C. (2003). "Regulation of the ABC kinases by phosphorylation: protein kinase C as a paradigm." Biochem J 370(Pt 2): 361-371.
Novak, J. E. and K. Kirkegaard (1991). "Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells." J Virol 65(6): 3384-3387.
Shih, S. R., M. S. Ho, et al. (2000). "Genetic analysis of enterovirus 71 isolated from fatal and non-fatal cases of hand, foot and mouth disease during an epidemic in Taiwan, 1998." Virus Res 68(2): 127-136.
Shih, S. R., V. Stollar, et al. (2011). "Host factors in enterovirus 71 replication." J Virol 85(19): 9658-9666.
Si, X., Y. Wang, et al. (2007). "Dysregulation of the ubiquitin-proteasome system by curcumin suppresses coxsackievirus B3 replication." J Virol 81(7): 3142-3150.
Sies, H. (1997). "Oxidative stress: oxidants and antioxidants." Exp Physiol 82(2): 291-295.
Thangapazham, R. L., A. Sharma, et al. (2006). "Multiple molecular targets in cancer chemoprevention by curcumin." AAPS J 8(3): E443-449.
Vajragupta, O., P. Boonchoong, et al. (2005). "Active site binding modes of curcumin in HIV-1 protease and integrase." Bioorg Med Chem Lett 15(14): 3364-3368.