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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 蔡錦華(Ching-Hwa Tsai) | |
| dc.contributor.author | Shu-Chun Tsai | en |
| dc.contributor.author | 蔡淑君 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:16:38Z | - |
| dc.date.available | 2009-09-15 | |
| dc.date.copyright | 2009-09-15 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-05-27 | |
| dc.identifier.citation | Adams, A. (1987). Replication of latent Epstein-Barr virus genomes in Raji cells. J Virol 61, 1743-1746.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41358 | - |
| dc.description.abstract | EB病毒感染會影響宿主細胞中細胞激素的表現，進而改變了宿主細胞的命運。過去，細胞激素介白質13 曾在EB病毒相關的何杰金氏淋巴癌切片檢體中被測出；且在自然殺手細胞型慢性活化EB病毒感染的病人血清中，介白質13的量也明顯的增加；然而介白質13在EB病毒相關的疾病中所扮演的角色及生理意義則仍未被探討過，而其表現又是如何被EB病毒所調控也仍是個未知的機制。利用細胞激素抗體陣列分析，我們發現EB病毒感染B細胞之後，B細胞中介白質13的表現量會上升。更進一步的研究發現，EB病毒的溶裂期蛋白質Zta可以促進介白質13的表現量增加；而Zta誘發介白質13表現的分子機制則是透過Zta直接結合在介白質13的啟動子，進而轉活化介白質13的表現。此外，EB病毒所感染的B細胞以及EB病毒所轉染的淋巴母細胞株的生長均會受到介白質13中和抗體的抑制，顯示介白質13對這些細胞生長的重要性。根據上述的實驗結果，我們推測EB病毒溶裂期蛋白質Zta所誘發的介白質13可以幫助B細胞的增生，進而促進了何杰金氏淋巴癌的生長以及一些EB病毒相關的淋巴增生疾病的產生。 | zh_TW |
| dc.description.abstract | Epstein-Barr virus (EBV) infection can alter the cytokine expression profiles of host cells and determine the fate of those cells. Of note, interleukin-13 (IL-13) was detected in EBV-associated Hodgkin’s lymphoma biopsies. The amounts of IL-13 in the plasma of NK cell type chronic active EBV-infection patients are also elevated, but its biological role and regulatory mechanism are not understood. Using cytokine antibody arrays, we found that IL-13 production is induced in B cells after EBV infection. Further investigation indicates that EBV lytic protein, Zta, which is a transactivator, directly activates IL-13 expression following transfection. The mechanism underlying Zta-mediated induction of IL-13 is through direct binding of Zta to the IL-13 promoter, via a consensus AP-1 binding site to transactivate IL-13 expression. Blockade of IL-13 by its neutralization antibody shows an inhibition of cell proliferation in EBV-infected primary B cells and EBV-immortalized lymphoblastoid cell lines (LCLs), indicating the importance of IL-13 in the early stage of EBV-induced cell proliferation and in long-term maintenance of cell growth. Our data suggest Zta-induced IL-13 production facilitates B-cell proliferation and may contribute to the pathogenesis of Hodgkin’s lymphoma and EBV-associated lymphoproliferative diseases (LPD). | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:16:38Z (GMT). No. of bitstreams: 1 ntu-98-F92445121-1.pdf: 4317529 bytes, checksum: c15c7aaa8ca1f1c47562544898116022 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 口試委員會審定書………………………………………………………...…….I
中文摘要…………………………………………………………..……………II Abstract……………………………………………...………………………….III Contents………………………………...………………………………………IV 1. Introduction…………………………………………………………………...1 1.1 Discovery of EBV……………………………………………………………………...1 1.2 Classification, viral structure and genome of EBV…………………………………….1 1.3 EBV life cycle………………………………………………………………………….2 1.4 Latent and lytic gene products of EBV……………………………………………..….2 1.5 EBV infection and EBV-associated diseases…………………………………………..4 1.6 Cytokines induced by EBV…………………………………………………………….6 1.7 Interleukin-13 (IL-13)………………………………………………………………….7 1.8 Aims of this study…………………………………………………………………….10 2. Materials and methods…………………………………………………….…11 2.1 Preparation of EBV virion…………………………………………………………….11 2.2 Purification of peripheral blood mononuclear cells and B cells for EBV infection….11 2.3 Flow cytometry analysis……………………………………………………………...12 2.4 Cell lines……………………………………………………………………………...13 2.5 Electroporation of BJAB cells………………………………………………………..14 2.6 RNA extraction and reverse transcription…………………………………………….14 2.7 Quantitative polymerase chain reaction (Q-PCR)……………………………………15 2.8 Western blot analysis and antibodies…………………………………………………16 2.9 Cytokine antibody array and enzyme-linked immunosorbent assay (ELISA)……….17 2.10 Plasmids……………………………………………………………………………..18 2.11 Preparation of infectious lentiviruses………………………………………………..19 2.12 Reporter assay……………………………………………………………………….20 2.13 Electromobility shift assay (EMSA)………………………………………………...21 2.14 Chromatin immunoprecipitation assay (ChIP) ……………………………………..21 2.15 DNA extraction and bisulfite sequencing…………………………………………...24 2.16 Treatment of cells with 5-azacytidine……………………………………………….24 2.17 IL-13 neutralization assay………………………………………………………..….24 3. Results……………………………………………………………………….26 3.1 IL-13 is induced in EBV-infected peripheral mononuclear cells……………………..26 3.2 IL-13 is induced in EBV-infected B cells…………………………………………….28 3.3 EBV lytic cycle progression enhances IL-13 expression..............................................29 3.4 EBV lytic protein, Zta, plays a key role in induction of IL-13 ....................................29 3.5 Induction of IL-13 expression is mediated by Zta transactivation activity...................31 3.6 Zta directly binds to IL-13 promoter.............................................................................32 3.7 Overexpression of Zta does not affect methylation status of IL-13 promoter..............33 3.8 De-methylation of IL-13 promoter induces IL-13 production in LCLs........................34 3.9 IL-13 is required for the growth of EBV-infected B cells and LCLs............................35 3.10 Zta induces IL-13 expression in epithelial cells..........................................................36 4. Discussion……………………………………………………………………38 4.1 EBV lytic reactivation and disease progression…………………………....................38 4.2 Zta and tumor formation……………………………………………………………...38 4.3 Function of Zta……...………………………………………………………………...39 4.4 Promoter methylation in Zta-mediated induction of IL-13…………..……………….40 4.5 IL-4 and IL-13 in EBV-associated diseases…………...………...……………………42 4.6 IL-13 and cell proliferation…………………………………………………………...44 4.7 Expression of IL-13 in epithelial cells………………………………………..………46 4.8 Conclusion……………………………………………………………………………46 Tables…………………………………………………………………………...48 Table 1. Nucleotide sequences used in Q-PCR analysis…………………………………48 Figures………………………………………………………………………….49 Figure 1. Comparison of cytokine expression profiles of EBV-infected and uninfected PBMCs…………………………………..……………………………………49 Figure 2. Purification of primary B cells for EBV infection…………………………….50 Figure 3. Detection of EB viral products in EBV-infected B cells……………………...51 Figure 4. Confirmation of the cytokines expression in EBV-infected B cells by RT-Q-PCR……………………………………………………………….……..52 Figure 5. Quantification of cytokines expression in EBV-infected B cells by ELISA …53 Figure 6. Detection of IL-13 expression level in different LCLs……………………….54 Figure 7. EBV lytic cycle progression enhances IL-13 expression..................................55 Figure 8. EBV lytic protein, Zta, contributes to the induction of IL-13………………...56 Figure 9. Zta induces IL-13 expression in a dose-dependent manner…………………..57 Figure10. Zta induces IL-13 expression in different B cell lines………………………..58 Figure 11. IL-13 expression is decreased after knockdown of Zta……………………...59 Figure 12. Zta-knockout EBV impairs induction of IL-13……………………………...60 Figure 13. Illustration of IL-13 promoter region nucleotide -1160 to +65……………...61 Figure 14. Zta increases IL-13 promoter activity……………………………………….62 Figure 15. Zta binds to IL-13 promoter in vitro……………63 Figure 16. Zta binds to IL-13 promoter in vivo……………64 Figure 17. Methylation status of IL-13 promoter region………………………………..65 Figure 18. De-methylation of IL-13 promoter induces IL-13 production in EBV-positive cells……………………………………………………………………66 Figure 19. IL-13 is crucial for triggering cell growth of EBV-infected primary B cells..67 Figure 20. IL-13 is important for maintaining cell growth of LCLs…………………….68 Figure 21. Overexpression of IL-13 enhances proliferation of LCLs…………………...69 Figure 22. Zta induces IL-13 expression in epithelial cells……………………………..70 Figure 23. The model of Zta-mediated induction of IL-13 and promotion of cell proliferation…………………………………71 Reference………………………………………………………………72 Appendix I………………………………………………………………82 Figure 1. Illustration of the lentivirus-based expression plasmid, pSIN vector…………82 Figure 2. Illustration of the expression plasmid, pSIN-Zta……………………………...83 Figure 3. Illustration of the expression plasmid, pSIN-LMP1…………………………..84 Figure 4. Illustration of the expression plasmid, pSIN-LMP2A…………………………………85 Figure 5. Illustration of the expression plasmid, pSIN-IL-13……………………………………86 Figure 6. Illustration of the reporter plasmids, pGL2-pIL-13, driven by IL-13 promoter fragments……………………87 Figure 7. Illustration of the expression plasmids, pLKO-siLuciferase and pLKO-siZta..88 Appendix II……………………………………………………………89 Appendix III……………………………………………………90 | |
| dc.language.iso | en | |
| dc.subject | 介白質13 | zh_TW |
| dc.subject | 淋巴增生疾病 | zh_TW |
| dc.subject | 何杰金氏淋巴癌 | zh_TW |
| dc.subject | 淋巴母細胞株 | zh_TW |
| dc.subject | 溶裂期蛋白質Zta | zh_TW |
| dc.subject | EB病毒 | zh_TW |
| dc.subject | lymphoproliferative disease | en |
| dc.subject | Epstein-Barr virus (EBV) | en |
| dc.subject | lymphoblastoid cell line (LCL) | en |
| dc.subject | interleukin-13 (IL-13) | en |
| dc.subject | Zta | en |
| dc.title | 探討EB病毒對細胞激素介白質13表現之調控機制 | zh_TW |
| dc.title | Regulation of interleukin-13 expression by Epstein-Barr virus | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-2 | |
| dc.description.degree | 博士 | |
| dc.contributor.oralexamcommittee | 陳美如,李建國,王學偉,林素芳,呂仁 | |
| dc.subject.keyword | EB病毒,介白質13,溶裂期蛋白質Zta,淋巴母細胞株,何杰金氏淋巴癌,淋巴增生疾病, | zh_TW |
| dc.subject.keyword | Epstein-Barr virus (EBV),interleukin-13 (IL-13),Zta,lymphoblastoid cell line (LCL),lymphoproliferative disease, | en |
| dc.relation.page | 90 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-06-01 | |
| dc.contributor.author-college | 醫學院 | zh_TW |
| dc.contributor.author-dept | 微生物學研究所 | zh_TW |
| 顯示於系所單位： | 微生物學科所 | |
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