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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 伍安怡(Betty A. Wu-Hsieh) | |
dc.contributor.author | Shih-Ching Lin | en |
dc.contributor.author | 林世青 | zh_TW |
dc.date.accessioned | 2021-06-16T16:42:22Z | - |
dc.date.available | 2017-09-18 | |
dc.date.copyright | 2012-09-18 | |
dc.date.issued | 2012 | |
dc.date.submitted | 2012-08-24 | |
dc.identifier.citation | References
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/63447 | - |
dc.description.abstract | 登革是重要的傳染疾病,先前指出引發出血的原因為登革病毒與內皮細胞交互作用,進而導致內皮細胞死亡。已知登革病毒蛋白酶可引發內皮細胞凋亡,故本研究目的為探討登革病毒 NS3 蛋白酶造成人類微血管內皮細胞 (HMEC-1) 凋亡的分子機制。首先我建構帶有登革病毒 NS2B-蛋白酶、蛋白酶、NS2B 與失去活性 H51A 點突變 NS2B-蛋白酶之慢病毒載體,再分別導入 HMEC-1 中。Annexin V 與 propidium iodide 染色結果顯示,登革病毒 NS2B-蛋白酶或蛋白酶會引發 HMEC-1 細胞凋亡,但 NS2B 與 H51A 點突變 NS2B-蛋白酶則無法引發凋亡,顯示蛋白酶活性為 NS3 蛋白酶造成之 HMEC-1 細胞凋亡所必需。
藉由使用 L-NAME 與 apocynin,證明活性氧與活性氮並未參與蛋白酶造成之細胞凋亡。同時發現登革病毒 NS2B-蛋白酶會引發 HMEC-1 caspase-8、caspase-3 但非 caspase-9 活化,給予細胞 caspase-8、caspase-3 或 pan-caspase 抑制劑後可有效減少蛋白酶造成之細胞凋亡。而利用 BAY 11-7082 抑制 NF-κB 活化即可阻斷 NS2B-蛋白酶造成之 HMEC-1 細胞凋亡,證明 NF-κB 訊息傳遞路徑與 NS2B-蛋白酶造成之細胞凋亡有關。 本篇研究結論為登革病毒 NS3 蛋白酶透過活化細胞內 NF-κB,造成 caspase-8 與 caspase-3 活化,進而引發 HMEC-1 凋亡。 | zh_TW |
dc.description.abstract | Dengue is an important infectious disease. We reported previously that dengue virus interaction with endothelial cells resulting in endothelial cell death is key to hemorrhage development. Dengue viral protease has been shown to cause endothelial cell apoptosis. The goal of this study is to investigate the mechanism of how DENV NS3 protease mediates human microvascular endothelial cell line (HMEC-1) apoptosis. First of all, I generated lentiviral constructs that carry dengue viral components NS2B-protease, protease, 2B and protease activity-dead mutant 2B-protease H51A. HMEC-1 cells were transduced by these components separately. The results of Annexin V and propidium iodide staining showed that transduction of 2B-protease or protease but not 2B or 2B-protease H51A induced HMEC-1 apoptosis, indicating that NS3 protease mediated-HMEC-1 apoptosis is protease activity-dependent.
By use of L-NAME and apocynin, I ruled out the involvement of ROS/RNS production in protease-induced apoptosis. In the mean time, I found that DENV 2B-protease induced caspase-8 and caspase-3 but not caspase-9 activation in HMEC-1 cells. Treatment by caspase-8, caspase-3 and pan-caspase but not caspase-9 inhibitors ameliorated protease-induced apoptosis. Interestingly, inhibition of NF-κB activation by BAY 11-7082 blocked 2B-protease-induced HMEC-1 apoptosis, indicating that NF-κB signaling is involved in 2B-protease-induced apoptosis. In summary, these findings revealed that DENV NS3 protease induces cellular caspase-8 and caspase-3 activation, triggering HMEC-1 apoptosis, and NF-κB signaling is required for the triggering of the caspase pathway. | en |
dc.description.provenance | Made available in DSpace on 2021-06-16T16:42:22Z (GMT). No. of bitstreams: 1 ntu-101-R99449005-1.pdf: 5301192 bytes, checksum: 50795274be4ea51064f0b77f4d02ff16 (MD5) Previous issue date: 2012 | en |
dc.description.tableofcontents | Table of Contents
Abstract i Abstract (Chinese) ii Table of Contents iii List of Figures and Appendix vi CHAPTER I Introduction 1 The epidemic of dengue virus 2 1. Hemorrhage development in DENV infection 3 1.1. Antibody-dependent enhancement 3 1.2. Original antigenic sin 4 1.3. Increased vascular permeability for hemorrhage development 4 2. The contribution of host TNF-α in endothelium apoptosis 5 2.1. Virus infections that affect host caspase-dependent apoptotic pathway 7 2.2. Viral conponents that influence host caspase-dependent apoptotic pathway 9 2.3. Viruses that interfere host NF-κB pathway 11 3. The composition of DENV genome and the dialysis of DENV NS3 protease 13 3.1. The investigation of NS3 protease activity 14 3.2. The interactome of DENV NS3 to host proteins 15 4. The relationship of ROS/RNS production and vascular permeability 15 4.1. The induction of ROS and RNS production by an viral protein 17 5. Adeno-associated viral vector 18 5.1. rAAV-mediated in vitro gene delivery 19 5.2. Tissue or cell type specificity of rAAVs 19 CHAPTER II Aims of The Study 22 1. To validate NS3 protease-induced apoptosis in HMEC-1 by using lentiviral transduction system 23 2. To identify the molecular mechanism of DENV protease-induced apoptosis and to clarify the relationship between NF-κB and caspase activation 24 3. To generate endothelium-specific rAAV-9 for DENV NS3 protease in vivo transduction 25 CHAPTER III Materials & Methods 27 Part 1 Experimental procedures 28 1.1 Virus stock 28 1.2 Cell cultures 28 1.3 Apoptosis assay of DENV-2-infected HMEC-1 29 1.3.1 DENV-2 16681 infection of HMEC-1 29 1.3.2 Annexin V staining 29 1.3.3 Annexin V and PI double staining 29 1.4 Plasmid construction 30 1.5 In vitro validation of lentiviral or rAAV constructs 30 1.5.1 Transfection into 293T cells and protein extraction 30 1.5.2 Western blot analysis 31 1.6 Lentivirus preparation 31 1.6.1 Lentivirus generation 31 1.6.2 Lentivirus titer determination 32 1.7 Apoptosis assay of Lentivirus-transduced HMEC-1 32 1.7.1 Lentivirus transduction of HMEC-1 32 1.7.2 Annexin V and PI double staining 33 1.7.3 In vitro treatment with inhibitors 33 1.8. Intracellular staining of lentivirus transduced HMEC-1 34 Part 2 Medium and buffer solutions 35 2.1 Antibodies for Western blot analysis 35 2.2 Solutions 35 2.3 Chemicals and reagents 38 2.4 Equipments 39 2.5 Disposable plastics 40 2.6 Primer sequences 41 CHAPTER IV Results 43 Part 1. DENV NS3 protease-mediated HMEC-1 apoptosis is protease activity-dependent 44 1-1. DENV infection induces HMEC-1 apoptosis 44 1-2. DENV NS3 protease induces HMEC-1 apoptosis 45 1-3. DENV protease-mediated HMEC-1 apoptosis depends on protease activity 46 Part 2. The molecular mechanism of protease-induced apoptosis in HMEC-1 47 2-1. NS3 protease-induced HMEC-1 apoptosis is ROS/RNS-independent 47 2-2. DENV NS3 protease-mediated HMEC-1 apoptosis is caspase-8- and caspase-3-dependent 47 2-3. Blocking NF-κB activation inhibits caspase-8 and caspase-3 cleavage 49 Part 3. The production of endothelium-specific rAAV-9 50 3-1. DENV protease constructs for rAAV packaging 50 3-2. Quantification of rAAV-9 titer by RT-qPCR 50 CHAPTER V Discussion 52 Caspase-8 and caspase-3 are involved in DENV-induced HMEC-1 apoptosis 53 DENV NS3 protease induces caspase-8 and caspase-3 activation 54 NF-κB activation is prior to caspase cleavage in HMEC-1 cells overexpressing DENV 2B-Pro 55 The role of NF-κB activation in cell survival and death 56 ROS/RNS may be not involved in 2B-Pro-induced HMEC-1 apoptosis 57 DENV NS3 protease-mediated HMEC-1 apoptosis is independent of NS2B 58 DENV NS3 protease-mediated HMEC-1 apoptosis depends on protease activity 59 References 61 Figures 79 Appendix 106 List of Figures and Appendix Fig 1. DENV infection induces HMEC-1 apoptosis in a time- and dose-dependent manner 80 Fig 2. NS3 protein is expressed in HMEC-1 during DENV infection 83 Fig 3. DENV protease constructs for lentivirus packaging 85 Fig 4. DENV NS3 protease induces HMEC-1 apoptosis 88 Fig 5. DENV NS3 protease-mediated HMEC-1 apoptosis depends on protease activity 90 Fig 6. NS3 protease-induced HMEC-1 apoptosis is independent of ROS/RNS 92 Fig 7. Cleaved caspase-8 and caspase-3 are observed in DENV protease-expressed HMEC-1 94 Fig 8. Titration of caspase inhibitors in HMEC-1 96 Fig 9. DENV NS3 protease-mediated HMEC-1 apoptosis is caspase-8- and caspase-3-dependent 98 Fig 10. NF-κB activation is critical for protease-induced caspase-8 and caspase-3 cleavage 101 Fig 11. DENV protease constructs for rAAV packaging 103 Appendix 1. Quantification of rAAV-9 genomic titer by RT-qPCR 107 | |
dc.language.iso | en | |
dc.title | 探討登革病毒蛋白酶造成微血管內皮細胞凋亡的分子機制 | zh_TW |
dc.title | To investigate the molecular mechanism of DENV protease-induced microvascular endothelial cell apoptosis | en |
dc.type | Thesis | |
dc.date.schoolyear | 100-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | ?希椿(Shi-Chuen Miaw),顧家綺(Chia-Chi Ku),鄧述諄(Shu-Chun Teng) | |
dc.subject.keyword | 登革病毒,NS3蛋白酶,微血管內皮細胞,細胞凋亡,半胱氨酸天冬氨酸酶, | zh_TW |
dc.subject.keyword | Dengue virus,DENV,NS3 protease,HMEC-1,apoptosis,caspase, | en |
dc.relation.page | 108 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2012-08-27 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
顯示於系所單位: | 免疫學研究所 |
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