臺灣博碩士論文加值系統

誌謝 i
中文摘要 iii
Abstract v
目錄 vii
壹、 緒論 1
一、 Coronavirus disease 2019 (COVID-19) 1
1. COVID-19的簡介 1
2. COVID-19的流行病學 1
二、 COVID-19治療之策略及治療藥物 3
1. COVID-19治療之策略 3
2. COVID-19治療藥物 4
三、 冠狀病毒 (Coronavirus, CoV) 6
1. 冠狀病毒的簡介 6
2. 冠狀病毒的感染機制 7
3. 冠狀病毒的生命週期 8
四、 血管收縮素轉換酶2 9
1. 血管收縮素轉換酶2的簡介 9
2. 血管收縮素轉換酶2的生理功能 9
貳、 研究動機與目標 (Purpose) 12
一、 研究動機 12
二、 研究目標 12
參、 研究材料與方法 13
一、 細胞培養 13
1. NOK 13
2. SAS 13
3. OECM-1 14
4. OC4 15
5. OC5 16
6. YMOC-1 16
7. CAL27 17
8. HEK293 17
9. H1299 18
二、 細胞冷凍 19
三、 細胞解凍 19
四、 RNA萃取 20
五、 反轉錄聚合酶連鎖反應 20
六、 即時聚合酶連鎖反應 21
七、 西方墨點法 22
1. 蛋白質樣本萃取 22
2. 蛋白質樣本定量 22
3. SDS聚丙烯醯胺凝膠電泳 23
4. 蛋白質轉漬 24
5. 免疫呈色 25
八、 質體DNA製備 25
1. 建立過度表現ACE2質體之製備 25
九、 質體轉型 28
十、 少量質體DNA製備 29
十一、 大量質體DNA製備 30
十二、 轉染 31
十三、 免疫螢光染色 31
十四、 免疫螢光染色定量分析 32
十五、 蛋白質三維結構數據 33
肆、 結果 34
一、 探討人類口腔上皮細胞株ACE2蛋白及mRNA的表現情形 34
二、 探討模擬病毒之肽鏈VRBMP與人類口腔上皮細胞株的結合情形 35
1. 設計模擬病毒之肽鏈VRBMP 35
2. VRBMP-1/VRBMP-2會與人類口腔上皮細胞株結合 36
3. Neg-1/Neg-2不會與人類口腔上皮細胞株結合 37
三、 探討ACE2誘餌胜肽片段抑制VRBMP-1與人類口腔上皮細胞株的結合情形 38
1. ACE2誘餌胜肽片段之設計 38
2. ACE2誘餌胜肽片段能夠抑制VRBMP-1與人類口腔上皮細胞株的結合 38
3. 優化ACE2誘餌胜肽片段更有效抑制VRBMP-1與人類口腔上皮細胞株的結合 39
伍、 討論 42
陸、 圖例 45
圖一、人類口腔上皮細胞株的內生性ACE2表現情形 45
圖三、ACE2誘餌胜肽片段能夠抑制VRBMP-1與人類口腔上皮細胞株結合 54
圖四、優化ACE2誘餌胜肽片段更有效地抑制VRBMP-1與人類口腔上皮細胞株結合 58
柒、 附圖 60
附圖一、 pcDNA3.1(-)質體圖 60
捌、 附表 61
附表一、SARS-CoV-2-S蛋白之RBD與人類ACE2結合的關鍵殘基 61
附表二、模擬病毒之肽鏈序列 62
附表三、ACE2誘餌胜肽片段序列 63
附表三、西方墨點法所使用的初級抗體 64
附表四、西方墨點法所使用的二級抗體 64
玖、 各項溶液之製備 65
一、 細菌培養 65
1. LB培養液 65
2. LB培養基 65
二、 西方墨點法 66
1. 1 mM Phenylmethylsulfonyl Fluoride (PMSF) 66
2. 10X PBS 66
3. 4X Sample Buffer 67
4. Lysis Buffer 67
5. 4X Upper Buffer 68
6. 4X Lower Buffer 68
7. 10X Running Buffer 68
8. 1X Running Buffer 69
9. Stacking Gel Solution (Upper Gel) (3.45%, 3 ml) 69
10. Separating Gel Solution (Lower Gel) (8.5%, 10 ml) 70
11. 1X Transfer Buffer 70
12. PBST 70
13. Blocking Solution (5% milk) 71
拾、參考文獻 72

1. Ludwig, S. and A. Zarbock, Coronaviruses and SARS-CoV-2: A Brief Overview. Anesth Analg, 2020. 131(1): p. 93-96.
2. Li, Q., et al., Early Transmission Dynamics in Wuhan, China, of Novel Coronavirus-Infected Pneumonia. N Engl J Med, 2020. 382(13): p. 1199-1207.
3. Zhu, N., et al., A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl J Med, 2020. 382(8): p. 727-733.
4. Jin, Y., et al., Virology, Epidemiology, Pathogenesis, and Control of COVID-19. Viruses, 2020. 12(4).
5. Xu, Z., et al., Pathological findings of COVID-19 associated with acute respiratory distress syndrome. Lancet Respir Med, 2020. 8(4): p. 420-422.
6. Guan, W.J., et al., Clinical Characteristics of Coronavirus Disease 2019 in China. N Engl J Med, 2020. 382(18): p. 1708-1720.
7. Huang, C., et al., Clinical features of patients infected with 2019 novel coronavirus in Wuhan, China. Lancet, 2020. 395(10223): p. 497-506.
8. Khan, M., et al., COVID-19: A Global Challenge with Old History, Epidemiology and Progress So Far. Molecules, 2020. 26(1).
9. Xu, J., et al., Systematic Comparison of Two Animal-to-Human Transmitted Human Coronaviruses: SARS-CoV-2 and SARS-CoV. Viruses, 2020. 12(2).
10. Zhou, P., et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature, 2020. 579(7798): p. 270-273.
11. Zhao, J., W. Cui, and B.P. Tian, The Potential Intermediate Hosts for SARS-CoV-2. Front Microbiol, 2020. 11: p. 580137.
12. Frutos, R., et al., COVID-19: Time to exonerate the pangolin from the transmission of SARS-CoV-2 to humans. Infect Genet Evol, 2020. 84: p. 104493.
13. Asrani, P., et al., Guidelines and Safety Considerations in the Laboratory Diagnosis of SARS-CoV-2 Infection: A Prerequisite Study for Health Professionals. Risk Manag Healthc Policy, 2021. 14: p. 379-389.
14. Centers for Disease Control. Available from: https://www.cdc.gov/coronavirus/2019-ncov/prevent-getting-sick/how-covid-spreads.html.
15. World Health Organization. Available from: https://covid19.who.int/table.
16. Sokolowska, M., Outsmarting SARS-CoV-2 by empowering a decoy ACE2. Signal Transduct Target Ther, 2020. 5(1): p. 260.
17. Eastman, R.T., et al., Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19. ACS Cent Sci, 2020. 6(5): p. 672-683.
18. Rentsch, C.T., et al., Effect of pre-exposure use of hydroxychloroquine on COVID-19 mortality: a population-based cohort study in patients with rheumatoid arthritis or systemic lupus erythematosus using the OpenSAFELY platform. Lancet Rheumatol, 2021. 3(1): p. e19-e27.
19. Asrani, P., et al., Clinical features and mechanistic insights into drug repurposing for combating COVID-19. Int J Biochem Cell Biol, 2022. 142: p. 106114.
20. Khiali, S., E. Khani, and T. Entezari-Maleki, A Comprehensive Review of Tocilizumab in COVID-19 Acute Respiratory Distress Syndrome. J Clin Pharmacol, 2020. 60(9): p. 1131-1146.
21. Parums, D.V., Editorial: Tocilizumab, a Humanized Therapeutic IL-6 Receptor (IL-6R) Monoclonal Antibody, and Future Combination Therapies for Severe COVID-19. Med Sci Monit, 2021. 27: p. e933973.
22. Food & Drug administration. Available from: https://www.fda.gov/emergency-preparedness-and-response/counterterrorism-and-emerging-threats/coronavirus-disease-2019-covid-19.
23. Ashraf, M.U., et al., COVID-19 Vaccines (Revisited) and Oral-Mucosal Vector System as a Potential Vaccine Platform. Vaccines (Basel), 2021. 9(2).
24. van Doremalen, N., et al., ChAdOx1 nCoV-19 vaccine prevents SARS-CoV-2 pneumonia in rhesus macaques. Nature, 2020. 586(7830): p. 578-582.
25. Krammer, F., SARS-CoV-2 vaccines in development. Nature, 2020. 586(7830): p. 516-527.
26. Khan, S., et al., Emergence of a Novel Coronavirus, Severe Acute Respiratory Syndrome Coronavirus 2: Biology and Therapeutic Options. J Clin Microbiol, 2020. 58(5).
27. Cui, J., F. Li, and Z.L. Shi, Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol, 2019. 17(3): p. 181-192.
28. Woo, P.C., et al., Discovery of seven novel Mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus. J Virol, 2012. 86(7): p. 3995-4008.
29. Forni, D., et al., Molecular Evolution of Human Coronavirus Genomes. Trends Microbiol, 2017. 25(1): p. 35-48.
30. Meng, X., et al., Gordian Knot: Gastrointestinal lesions caused by three highly pathogenic coronaviruses from SARS-CoV and MERS-CoV to SARS-CoV-2. Eur J Pharmacol, 2021. 890: p. 173659.
31. Fehr, A.R. and S. Perlman, Coronaviruses: an overview of their replication and pathogenesis. Methods Mol Biol, 2015. 1282: p. 1-23.
32. Al-Horani, R.A., S. Kar, and K.F. Aliter, Potential Anti-COVID-19 Therapeutics that Block the Early Stage of the Viral Life Cycle: Structures, Mechanisms, and Clinical Trials. Int J Mol Sci, 2020. 21(15).
33. Pišlar, A., et al., The role of cysteine peptidases in coronavirus cell entry and replication: The therapeutic potential of cathepsin inhibitors. PLoS Pathog, 2020. 16(11): p. e1009013.
34. Hoffmann, M., et al., SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor. Cell, 2020. 181(2): p. 271-280.e8.
35. Matsuyama, S., et al., Enhanced isolation of SARS-CoV-2 by TMPRSS2-expressing cells. Proc Natl Acad Sci U S A, 2020. 117(13): p. 7001-7003.
36. Gomes, C.P., et al., Cathepsin L in COVID-19: From Pharmacological Evidences to Genetics. Front Cell Infect Microbiol, 2020. 10: p. 589505.
37. Peacock, T.P., et al., The furin cleavage site in the SARS-CoV-2 spike protein is required for transmission in ferrets. Nat Microbiol, 2021. 6(7): p. 899-909.
38. V'Kovski, P., et al., Coronavirus biology and replication: implications for SARS-CoV-2. Nat Rev Microbiol, 2021. 19(3): p. 155-170.
39. Harrison, A.G., T. Lin, and P. Wang, Mechanisms of SARS-CoV-2 Transmission and Pathogenesis. Trends Immunol, 2020. 41(12): p. 1100-1115.
40. Kirtipal, N., S. Bharadwaj, and S.G. Kang, From SARS to SARS-CoV-2, insights on structure, pathogenicity and immunity aspects of pandemic human coronaviruses. Infect Genet Evol, 2020. 85: p. 104502.
41. Yan, R., et al., Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science, 2020. 367(6485): p. 1444-1448.
42. Wrapp, D., et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science, 2020. 367(6483): p. 1260-1263.
43. Xu, H., et al., High expression of ACE2 receptor of 2019-nCoV on the epithelial cells of oral mucosa. Int J Oral Sci, 2020. 12(1): p. 8.
44. Li, M.Y., et al., Expression of the SARS-CoV-2 cell receptor gene ACE2 in a wide variety of human tissues. Infect Dis Poverty, 2020. 9(1): p. 45.
45. Huang, N., et al., SARS-CoV-2 infection of the oral cavity and saliva. Nat Med, 2021. 27(5): p. 892-903.
46. Drozdzik, A. and M. Drozdzik, Oral Pathology in COVID-19 and SARS-CoV-2 Infection-Molecular Aspects. Int J Mol Sci, 2022. 23(3).
47. Patel, S., et al., Renin-angiotensin-aldosterone (RAAS): The ubiquitous system for homeostasis and pathologies. Biomed Pharmacother, 2017. 94: p. 317-325.
48. Yan, T., R. Xiao, and G. Lin, Angiotensin-converting enzyme 2 in severe acute respiratory syndrome coronavirus and SARS-CoV-2: A double-edged sword? Faseb j, 2020. 34(5): p. 6017-6026.
49. Mirabito Colafella, K.M., D.M. Bovée, and A.H.J. Danser, The renin-angiotensin-aldosterone system and its therapeutic targets. Exp Eye Res, 2019. 186: p. 107680.
50. Medina-Enríquez, M.M., et al., ACE2: the molecular doorway to SARS-CoV-2. Cell Biosci, 2020. 10(1): p. 148.
51. Gong, J., Y. Sun, and L. Xie, ACEI/ARB Drug Therapy in COVID-19 Patients: Yes Or No? J Transl Int Med, 2021. 9(1): p. 8-11.
52. Verdecchia, P., et al., The pivotal link between ACE2 deficiency and SARS-CoV-2 infection. Eur J Intern Med, 2020. 76: p. 14-20.
53. Banu, N., et al., Protective role of ACE2 and its downregulation in SARS-CoV-2 infection leading to Macrophage Activation Syndrome: Therapeutic implications. Life Sci, 2020. 256: p. 117905.
54. Onweni, C.L., et al., ACEI/ARB therapy in COVID-19: the double-edged sword of ACE2 and SARS-CoV-2 viral docking. Crit Care, 2020. 24(1): p. 475.
55. Yang, G., et al., Effects of Angiotensin II Receptor Blockers and ACE (Angiotensin-Converting Enzyme) Inhibitors on Virus Infection, Inflammatory Status, and Clinical Outcomes in Patients With COVID-19 and Hypertension: A Single-Center Retrospective Study. Hypertension, 2020. 76(1): p. 51-58.
56. Fosbøl, E.L., et al., Association of Angiotensin-Converting Enzyme Inhibitor or Angiotensin Receptor Blocker Use With COVID-19 Diagnosis and Mortality. Jama, 2020. 324(2): p. 168-177.
57. Berman, H.M., et al., The Protein Data Bank. Nucleic Acids Res, 2000. 28(1): p. 235-42.
58. Protein Data Bank. Available from: https://www.rcsb.org/structure/7knb.
59. Mehdipour, A.R. and G. Hummer, Dual nature of human ACE2 glycosylation in binding to SARS-CoV-2 spike. Proc Natl Acad Sci U S A, 2021. 118(19).
60. Gong, Y., et al., The glycosylation in SARS-CoV-2 and its receptor ACE2. Signal Transduct Target Ther, 2021. 6(1): p. 396.
61. Grishin, A.M., et al., Disulfide Bonds Play a Critical Role in the Structure and Function of the Receptor-binding Domain of the SARS-CoV-2 Spike Antigen. J Mol Biol, 2022. 434(2): p. 167357.
62. Andersen, K.G., et al., The proximal origin of SARS-CoV-2. Nat Med, 2020. 26(4): p. 450-452.
63. Sahu, A.K., et al., SARS-CoV-2: phylogenetic origins, pathogenesis, modes of transmission, and the potential role of nanotechnology. Virusdisease, 2021. 32(1): p. 1-12.
64. Garcia-Beltran, W.F., et al., Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity. Cell, 2021. 184(9): p. 2372-2383.e9.
65. Hoffmann, M. and S. Pöhlmann, Novel SARS-CoV-2 receptors: ASGR1 and KREMEN1. Cell Res, 2022. 32(1): p. 1-2.
66. Gu, Y., et al., Receptome profiling identifies KREMEN1 and ASGR1 as alternative functional receptors of SARS-CoV-2. Cell Res, 2022. 32(1): p. 24-37.
67. Zhang, Q., et al., Molecular mechanism of interaction between SARS-CoV-2 and host cells and interventional therapy. Signal Transduct Target Ther, 2021. 6(1): p. 233.
68. Gu, Y., et al., Interaction network of SARS-CoV-2 with host receptome through spike protein. 2020: p. 2020.09.09.287508.
69. Xia, S., et al., Inhibition of SARS-CoV-2 (previously 2019-nCoV) infection by a highly potent pan-coronavirus fusion inhibitor targeting its spike protein that harbors a high capacity to mediate membrane fusion. Cell Res, 2020. 30(4): p. 343-355.
70. Yang, J., et al., Molecular interaction and inhibition of SARS-CoV-2 binding to the ACE2 receptor. Nat Commun, 2020. 11(1): p. 4541.