成功大學博碩士論文系統

簡易檢索 / 詳目顯示

回結果列表

研究生：	張博淳 Chang, Po-Chun
論文名稱：	探討integrin-linked kinase抑制物抗登革病毒的能力 Study on anti-dengue virus activity of integrin-linked kinase inhibitors
指導教授：	張志鵬 Chang, Chih-Peng
學位類別：	碩士 Master
系所名稱：	醫學院 - 微生物及免疫學研究所 Department of Microbiology & Immunology
論文出版年：	2017
畢業學年度：	105
語文別：	英文
論文頁數：	59
中文關鍵詞：	登革病毒、整合素連接性激酶抑制物、登革感染老鼠模式、促發炎性細胞激素
外文關鍵詞：	Dengue virus, integrin-linked kinase inhibitor, dengue mouse model, pro-inflammatory cytokines
相關次數：	點閱：197 下載：11
分享至:	分享至facebook 分享至twitter

查詢本校圖書館目錄查詢臺灣博碩士論文知識加值系統勘誤回報

登革熱是一個常見蟲媒性病毒感染疾病，主要藉由登革病毒所傳染。登革病毒屬於黃病毒科中黃病毒屬，是一個擁有正向單股RNA的病毒。病人在感染登革病毒後大多只會形成輕微的登革熱，但有些病人會轉變成致命的登革出血熱或是登革休克症狀，嚴重者可能死亡。然而很不幸的是目前臨床上尚未有一個有效的抗病毒藥物可以使用。因此，抗登革病毒藥物的研發是非常迫切的。許多研究已證實宿主因子對於登革病毒感染的重要性，且宿主因子一般被認為是一個較好用於發展抗登革病毒的藥物的標靶，因為具有比病毒因子更低的突變機率。我們在先前的研究中發現整合素連接性激酶 (integrin-linked kinase, ILK)此一絲胺酸/蘇胺酸激酶會促進登革病毒的複製。在細胞實驗中，以整合素連接性激酶抑制物可以降低登革病毒複製的能力；然而整合素連接性激酶抑制物在登革病毒感染的動物內的抗登革病毒能力尚未清楚。在本研究中我們利用登革病毒感染的仔鼠模式去探討T315和Tr83此兩整合素連接性激酶抑制物抗登革病毒的效果。由結果得知藉由T315和Tr83的治療可以有效的在登革病毒感染的仔鼠中，抑制包含登革病毒蛋白、感染性病毒顆粒、促發炎性細胞激素以及神經性病症的產生，甚至抑制了老鼠的死亡。接著利用T315和Tr83處理登革病毒感染的U937細胞，去探討整合素連接性激酶抑制物是否會直接影響登革病毒誘導產生的細胞激素。我們發現加入T315和Tr83可以降低登革病毒誘導的介白素-6 (IL-6) 、介白素-1b (IL-1b)和介白素-10 (IL-10)。我們進一步利用整合素連接性激酶表現量較低的U937細胞證實整合素連接性激酶對於登革病毒誘導產生介白素-6的重要性。綜合所有研究結果，我們提供了證據證明T315和Tr83在登革病毒感染的仔鼠體內抗登革病毒的能力以及整合素連接性激酶對於介白素-6產生的調控角色。

Dengue is a common arthropod-borne viral infectious disease caused by dengue virus (DENV), a positive-sense single-stranded RNA virus belonging to the genus Flavivirus and family Flaviviridae. Patients infected by DENV mostly have mild dengue fever, however, some patients may develop to server life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) and further leads to death. Unfortunately, there is still no specific anti-DENV drug for patients in clinical. Thus, it is urgent to develop anti-DENV drugs. Accumulated studies have revealed the importance of host factors in DENV infection. Host factors may serve as a better target for development of anti-DENV therapy because host factors have lower mutation probability than viral factors. We have previous found that integrin-linked kinase (ILK), a serine/threonine protein kinase, is able to facilitate DENV replication. Inhibition of ILK by ILK inhibitors can reduce DENV replication in culture cells. However, the anti-DENV activities of ILK inhibitors to DENV-infected animals are still unclear. In this study, we used a DENV-infected suckling mouse model to examine the anti-DENV effects of the ILK inhibitors, T315 and Tr83. According to the results obtained, treatment of T315 and Tr83 is able to significantly inhibit production of DENV proteins, infectious viral particles, pro-inflammatory cytokines, and development of neuropathological symptoms, and even mortality in DENV-infected suckling mice. To further investigate whether ILK inhibitors directly regulates DENV-induced cytokine production, the DENV-infected U937 cells were treated with T315 and Tr83. We found that treatment of T315 and Tr83 can reduce DENV-induced IL-6, IL-1b, and IL-10 production. We further revealed that ILK is important for DENV-induced IL-6 production by using ILK silencied U937 cells. In conclusion, we provided the evidences to show the the anti-DENV viral ability of T315 and Tr83 in DENV-infected suckling mice, and the regulatory role of ILK in IL-6 production.

中文摘要	I
Abstract	II
Acknowledgement	III
Table of contents	V
Abbreviations	VIII
I. Introduction	1
1.1 Epidemiology of dengue virus	1
1.2 Virology of DENV	1
1.3 Pathogenesis of DENV infection	4
1.4 DENV infection and host factors	6
1.5 Targeting host factors may help to control DENV infection	7
1.6 Integrin-linked kinase as a host target for anti-DENV therapy	8
1.7 Animal models of DENV infection	9
II. Objective and Specific Aims	11
III. Materials and methods	12
A. Materials	12
A-1. Mice	12
A-2. Cell lines	12
A-3. Viruses	13
A-4. Bacteria and plasmids	13
A-5. Antibody	14
A-6. ILK inhibitors	15
A-7. Reagents	16
B. Methods	23
B-1. Virus culture	23
B-2.1 Plaque assay	23
B-2.2 DENV infection	23
B-3. Western blotting	24
B-4. Lentiviral-based RNAi gene knockdown	24
B-5. Lactate dehydrogenase (LDH) releasing assay	25
B-6. WST-1 assay	25
B-7. Immunofluorescence assay (IFA)	26
B-8. Intracerebral and intraperitoneal infection dengue mouse model	26
B-9. Enzyme-linked immunosorbent assay (ELISA) for cytokine detection	27
B-10. Statistical analysis	27
IV. Results	28
IV-1. DENV is able to replicate and induce cytokine production in suckling mice	28
IV-2. T315 and Tr83 are able to inhibit DENV replication and mortality in ICR suckling mice	29
IV-3. Treatment of T315 and Tr83 reduces DENV-induced cytokine production in ICR suckling mice	30
IV-4. DENV is able to replicate and induces cytokine production in monocytes	31
IV-5. T315 and Tr83 are able to suppress DENV-induced cytokine production in monocytes	32
IV-6. ILK is able to facilitate replication and IL-6 production of DENV in monocytes	32
V. Conclusion	34
VI. Discussion	35
VII. References	40
VIII. Figures	46
Figure 1. DENV is able to replicate and causes death in ICR suckling mice.	46
Figure 2. DENV infection triggers cytokine production in DENV-infected suckling mice.	47
Figure 3. Toxicity test of T315 and Tr83 to ICR suckling mice.	48
Figure 4. T315 and Tr83 are able to suppress DENV replication in suckling mice.	49
Figure 5. DENV NS3 protein expression was reduced by T315 or Tr83 in brains of suckling mice.	50
Figure 6. Treatment of T315 and Tr83 are able to reduce mortality of DENV-infected suckling mice.	52
Figure 7. Treatment of T315 and Tr83 reduces pro-inflammatory cytokine production in DENV-infected suckling mice.	53
Figure 8. DENV is able to infect U937 cells and induce cytokine production.	54
Figure 9. T315 and Tr83 induce cell growth inhibition in U937 cells.	55
Figure 10. DENV-induced cytokine productions are down-regulated by T315 and Tr83 in U937 cells.	56
Figure 11. Knockdown of ILK in U937 cells suppresses DENV replication and IL-6 production.	57
Figure 12. A proposed model of ILK-regulated DENV infection.	58
IX. Appendix	59
Appendix 1. Information of ILK inhibitors.	59

                                    

1. Diamond, M.S. & Pierson, T.C. Molecular Insight into Dengue Virus Pathogenesis and Its Implications for Disease Control. Cell 162, 488-492 (2015).
2. Bhatt, S., et al. The global distribution and burden of dengue. Nature 496, 504-507 (2013).
3. Sinkins, S.P. & Gould, F. Gene drive systems for insect disease vectors. Nature reviews. Genetics 7, 427-435 (2006).
4. Lee, I.K., Liu, J.W. & Yang, K.D. Clinical characteristics, risk factors, and outcomes in adults experiencing dengue hemorrhagic fever complicated with acute renal failure. The American journal of tropical medicine and hygiene 80, 651-655 (2009).
5. Henchal, E.A. & Putnak, J.R. The dengue viruses. Clinical Microbiology reviews 3, 376-396 (1990).
6. Mustafa, M.S., Rasotgi, V., Jain, S. & Gupta, V. Discovery of fifth serotype of dengue virus (DENV-5): A new public health dilemma in dengue control. Medical journal, Armed Forces India 71, 67-70 (2015).
7. Guzman, M.G., et al. Dengue: a continuing global threat. Nature reviews. Microbiology 8, S7-16 (2010).
8. Guzman, M.G. & Vazquez, S. The complexity of antibody-dependent enhancement of dengue virus infection. Viruses 2, 2649-2662 (2010).
9. Mukhopadhyay, S., Kuhn, R.J. & Rossmann, M.G. A structural perspective of the flavivirus life cycle. Nature reviews. Microbiology 3, 13-22 (2005).
10. Nemesio, H. & Villalain, J. Membranotropic regions of the dengue virus prM protein. Biochemistry 53, 5280-5289 (2014).
11. Lai, C.Y., et al. Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II. Journal of Virology 82, 6631-6643 (2008).
12. Idrees, S. & Ashfaq, U.A. A brief review on dengue molecular virology, diagnosis, treatment and prevalence in Pakistan. Genetic Vaccines and Therapy 10, 6 (2012).
13. Xu, H., et al. Serotype 1-specific monoclonal antibody-based antigen capture immunoassay for detection of circulating nonstructural protein NS1: Implications for early diagnosis and serotyping of dengue virus infections. Journal of Clinical Microbiology 44, 2872-2878 (2006).
14. Munoz-Jordan, J.L., Sanchez-Burgos, G.G., Laurent-Rolle, M. & Garcia-Sastre, A. Inhibition of interferon signaling by dengue virus. Proceedings of the National Academy of Sciences of the United States of America 100, 14333-14338 (2003).
15. Falgout, B., Pethel, M., Zhang, Y.M. & Lai, C.J. Both nonstructural proteins NS2B and NS3 are required for the proteolytic processing of dengue virus nonstructural proteins. Journal of Virology 65, 2467-2475 (1991).
16. Benarroch, D., et al. The RNA helicase, nucleotide 5'-triphosphatase, and RNA 5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicase catalytic core. Virology 328, 208-218 (2004).
17. Stern, O., et al. An N-terminal amphipathic helix in dengue virus nonstructural protein 4A mediates oligomerization and is essential for replication. Journal of Virology 87, 4080-4085 (2013).
18. Dalrymple, N.A., Cimica, V. & Mackow, E.R. Dengue Virus NS Proteins Inhibit RIG-I/MAVS Signaling by Blocking TBK1/IRF3 Phosphorylation: Dengue Virus Serotype 1 NS4A Is a Unique Interferon-Regulating Virulence Determinant. mBio 6, e00553-00515 (2015).
19. Ackermann, M. & Padmanabhan, R. De novo synthesis of RNA by the dengue virus RNA-dependent RNA polymerase exhibits temperature dependence at the initiation but not elongation phase. Journal of Biological Chemistry 276, 39926-39937 (2001).
20. Su, C.I., Tseng, C.H., Yu, C.Y. & Lai, M.M. SUMO Modification Stabilizes Dengue Virus Nonstructural Protein 5 To Support Virus Replication. Journal of Virology 90, 4308-4319 (2016).
21. Morrison, J. & Garcia-Sastre, A. STAT2 signaling and dengue virus infection. Jak-stat 3, e27715 (2014).
22. Lee, Y.R., et al. Dengue viruses can infect human primary lung epithelia as well as lung carcinoma cells, and can also induce the secretion of IL-6 and RANTES. Virus Research 126, 216-225 (2007).
23. Lin, C.F., et al. Expression of cytokine, chemokine, and adhesion molecules during endothelial cell activation induced by antibodies against dengue virus nonstructural protein 1. Journal of Immunology (Baltimore, Md. : 1950) 174, 395-403 (2005).
24. Screaton, G., Mongkolsapaya, J., Yacoub, S. & Roberts, C. New insights into the immunopathology and control of dengue virus infection. Nature reviews. Immunology 15, 745-759 (2015).
25. Rodenhuis-Zybert, I.A., Wilschut, J. & Smit, J.M. Dengue virus life cycle: viral and host factors modulating infectivity. Cellular and Molecular Life Sciences : CMLS 67, 2773-2786 (2010).
26. Martina, B.E., Koraka, P. & Osterhaus, A.D. Dengue virus pathogenesis: an integrated view. Clinical Microbiology reviews 22, 564-581 (2009).
27. Godoi, I.P., et al. CYD-TDV dengue vaccine: systematic review and meta-analysis of efficacy, immunogenicity and safety. Journal of Comparative Effectiveness Research 6, 165-180 (2017).
28. Halstead, S.B. & Aguiar, M. Dengue vaccines: Are they safe for travelers? Travel Medicine and Infectious Disease 14, 378-383 (2016).
29. Wan, S.W., et al. Autoimmunity in dengue pathogenesis. Journal of the Formosan Medical Association = Taiwan yi zhi 112, 3-11 (2013).
30. Halstead, S.B., Mahalingam, S., Marovich, M.A., Ubol, S. & Mosser, D.M. Intrinsic antibody-dependent enhancement of microbial infection in macrophages: disease regulation by immune complexes. The Lancet. Infectious Diseases 10, 712-722 (2010).
31. Chuang, Y.C., Lin, J., Lin, Y.S., Wang, S. & Yeh, T.M. Dengue Virus Nonstructural Protein 1-Induced Antibodies Cross-React with Human Plasminogen and Enhance Its Activation. Journal of Immunology (Baltimore, Md. : 1950) 196, 1218-1226 (2016).
32. Falconar, A.K. Antibody responses are generated to immunodominant ELK/KLE-type motifs on the nonstructural-1 glycoprotein during live dengue virus infections in mice and humans: implications for diagnosis, pathogenesis, and vaccine design. Clinical and Vaccine Immunology : CVI 14, 493-504 (2007).
33. Rathakrishnan, A., et al. Cytokine expression profile of dengue patients at different phases of illness. PloS One 7, e52215 (2012).
34. Rothman, A.L. Immunity to dengue virus: a tale of original antigenic sin and tropical cytokine storms. Nature reviews. Immunology 11, 532-543 (2011).
35. Malavige, G.N., et al. HLA class I and class II associations in dengue viral infections in a Sri Lankan population. PloS One 6, e20581 (2011).
36. Jeewandara, C., et al. Platelet activating factor contributes to vascular leak in acute dengue infection. PLoS Neglected Tropical Diseases 9, e0003459 (2015).
37. Jindadamrongwech, S., Thepparit, C. & Smith, D.R. Identification of GRP 78 (BiP) as a liver cell expressed receptor element for dengue virus serotype 2. Archives of Virology 149, 915-927 (2004).
38. Reyes-Del Valle, J., Chavez-Salinas, S., Medina, F. & Del Angel, R.M. Heat shock protein 90 and heat shock protein 70 are components of dengue virus receptor complex in human cells. Journal of Virology 79, 4557-4567 (2005).
39. Pelkmans, L., et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78-86 (2005).
40. Ceballos-Olvera, I., Chavez-Salinas, S., Medina, F., Ludert, J.E. & del Angel, R.M. JNK phosphorylation, induced during dengue virus infection, is important for viral infection and requires the presence of cholesterol. Virology 396, 30-36 (2010).
41. Lee, C.J., Liao, C.L. & Lin, Y.L. Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection. Journal of Virology 79, 8388-8399 (2005).
42. Hussain, M., Galvin, H.D., Haw, T.Y., Nutsford, A.N. & Husain, M. Drug resistance in influenza A virus: the epidemiology and management. Infection and Drug Resistance 10, 121-134 (2017).
43. Souza, D.G., et al. Essential role of platelet-activating factor receptor in the pathogenesis of Dengue virus infection. Proceedings of the National Academy of Sciences of the United States of America 106, 14138-14143 (2009).
44. Chen, H.H., et al. AR-12 suppresses dengue virus replication by down-regulation of PI3K/AKT and GRP78. Antiviral Research 142, 158-168 (2017).
45. Howe, M.K., et al. An inducible heat shock protein 70 small molecule inhibitor demonstrates anti-dengue virus activity, validating Hsp70 as a host antiviral target. Antiviral Research 130, 81-92 (2016).
46. Yu, C.Y., et al. Dengue virus targets the adaptor protein MITA to subvert host innate immunity. PLoS Pathogens 8, e1002780 (2012).
47. Dedhar, S., Williams, B. & Hannigan, G. Integrin-linked kinase (ILK): a regulator of integrin and growth-factor signalling. Trends in Cell Biology 9, 319-323 (1999).
48. Hannigan, G., Troussard, A.A. & Dedhar, S. Integrin-linked kinase: a cancer therapeutic target unique among its ILK. Nature reviews. Cancer 5, 51-63 (2005).
49. Wu, C. & Dedhar, S. Integrin-linked kinase (ILK) and its interactors: a new paradigm for the coupling of extracellular matrix to actin cytoskeleton and signaling complexes. Journal of Cell Biology 155, 505-510 (2001).
50. Persad, S., et al. Regulation of protein kinase B/Akt-serine 473 phosphorylation by integrin-linked kinase: critical roles for kinase activity and amino acids arginine 211 and serine 343. Journal of Biological Chemistry 276, 27462-27469 (2001).
51. Delcommenne, M., et al. Phosphoinositide-3-OH kinase-dependent regulation of glycogen synthase kinase 3 and protein kinase B/AKT by the integrin-linked kinase. Proceedings of the National Academy of Sciences of the United States of America 95, 11211-11216 (1998).
52. Legate, K.R., Montanez, E., Kudlacek, O. & Fassler, R. ILK, PINCH and parvin: the tIPP of integrin signalling. Nature reviews. Molecular cell biology 7, 20-31 (2006).
53. Li, R., et al. Overexpression of integrin-linked kinase (ILK) is associated with tumor progression and an unfavorable prognosis in patients with colorectal cancer. Journal of Molecular Histology 44, 183-189 (2013).
54. Chan, J., Ko, F.C., Yeung, Y.S., Ng, I.O. & Yam, J.W. Integrin-linked kinase overexpression and its oncogenic role in promoting tumorigenicity of hepatocellular carcinoma. PloS One 6, e16984 (2011).
55. Dai, D.L., et al. Increased expression of integrin-linked kinase is correlated with melanoma progression and poor patient survival. Clinical cancer research : an official journal of the American Association for Cancer Research 9, 4409-4414 (2003).
56. Esfandiarei, M., et al. Novel role for integrin-linked kinase in modulation of coxsackievirus B3 replication and virus-induced cardiomyocyte injury. Circulation research 99, 354-361 (2006).
57. Lee, S.L., et al. Identification and characterization of a novel integrin-linked kinase inhibitor. Journal of Medicinal Chemistry 54, 6364-6374 (2011).
58. Mercado-Pimentel, M.E., et al. The Novel Small Molecule Inhibitor, OSU-T315, Suppresses Vestibular Schwannoma and Meningioma Growth by Inhibiting PDK2 Function in the AKT Pathway Activation. Austin journal of Medical Oncology 3(2016).
59. Chiu, C.F., et al. T315 Decreases Acute Myeloid Leukemia Cell Viability through a Combination of Apoptosis Induction and Autophagic Cell Death. International Journal of Molecular Sciences 17(2016).
60. Liu, T.M., et al. OSU-T315: a novel targeted therapeutic that antagonizes AKT membrane localization and activation of chronic lymphocytic leukemia cells. Blood 125, 284-295 (2015).
61. Shirley, L.A., et al. Integrin-linked kinase affects signaling pathways and migration in thyroid cancer cells and is a potential therapeutic target. Surgery 159, 163-170 (2016).
62. de la Puente, P., et al. Identification of ILK as a novel therapeutic target for acute and chronic myeloid leukemia. Leukemia research (2015).
63. Sabin, A.B. & Schlesinger, R.W. PRODUCTION OF IMMUNITY TO DENGUE WITH VIRUS MODIFIED BY PROPAGATION IN MICE. Science (New York, N.Y.) 101, 640-642 (1945).
64. Bente, D.A., Melkus, M.W., Garcia, J.V. & Rico-Hesse, R. Dengue fever in humanized NOD/SCID mice. Journal of Virology 79, 13797-13799 (2005).
65. Hotta, H., et al. Inoculation of dengue virus into nude mice. The Journal of General Virology 52, 71-76 (1981).
66. Shresta, S., et al. Interferon-dependent immunity is essential for resistance to primary dengue virus infection in mice, whereas T- and B-cell-dependent immunity are less critical. Journal of Virology 78, 2701-2710 (2004).
67. Amaral, D.C., et al. Intracerebral infection with dengue-3 virus induces meningoencephalitis and behavioral changes that precede lethality in mice. Journal of Neuroinflammation 8, 23 (2011).
68. Cam, B.V., et al. Prospective case-control study of encephalopathy in children with dengue hemorrhagic fever. The American Journal of Tropical Medicine and Hygiene 65, 848-851 (2001).
69. Garcia-Rivera, E.J. & Rigau-Perez, J.G. Encephalitis and dengue. Lancet (London, England) 360, 261 (2002).
70. Tsai, T.T., et al. Microglia retard dengue virus-induced acute viral encephalitis. Scientific Reports 6, 27670 (2016).
71. Chan, K.W., Watanabe, S., Kavishna, R., Alonso, S. & Vasudevan, S.G. Animal models for studying dengue pathogenesis and therapy. Antiviral Research 123, 5-14 (2015).
72. Hottz, E.D., et al. Dengue induces platelet activation, mitochondrial dysfunction and cell death through mechanisms that involve DC-SIGN and caspases. Journal of Thrombosis and Haemostasis : JTH 11, 951-962 (2013).
73. Lin, J.C., et al. Dengue viral protease interaction with NF-kappaB inhibitor alpha/beta results in endothelial cell apoptosis and hemorrhage development. Journal of Immunology (Baltimore, Md. : 1950) 193, 1258-1267 (2014).
74. Levy, A., et al. Increment of interleukin 6, tumour necrosis factor alpha, nitric oxide, C-reactive protein and apoptosis in dengue. Transactions of the Royal Society of Tropical Medicine and Hygiene 104, 16-23 (2010).
75. Cheng, Y.L., et al. Dengue Virus Infection Causes the Activation of Distinct NF-kappaB Pathways for Inducible Nitric Oxide Synthase and TNF-alpha Expression in RAW264.7 Cells. Mediators of Inflammation 2015, 274025 (2015).
76. John, D.V., Lin, Y.S. & Perng, G.C. Biomarkers of severe dengue disease - a review. Journal of Biomedical Science 22, 83 (2015).
77. Chen, S.T., et al. CLEC5A is critical for dengue-virus-induced lethal disease. Nature 453, 672-676 (2008).
78. Wani, A.A., Jafarnejad, S.M., Zhou, J. & Li, G. Integrin-linked kinase regulates melanoma angiogenesis by activating NF-kappaB/interleukin-6 signaling pathway. Oncogene 30, 2778-2788 (2011).
79. Chen, L., Tao, Y. & Jiang, Y. Apelin activates the expression of inflammatory cytokines in microglial BV2 cells via PI-3K/Akt and MEK/Erk pathways. Science China. Life Sciences 58, 531-540 (2015).

校內：2022-08-24公開
校外：不公開電子論文尚未授權公開，紙本請查館藏目錄

簡易檢索 / 詳目顯示

相關論文