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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 張?仁 | |
| dc.contributor.author | Yu-Wun Jiang | en |
| dc.contributor.author | 江玉雯 | zh_TW |
| dc.date.accessioned | 2021-06-15T00:28:59Z | - |
| dc.date.available | 2009-02-03 | |
| dc.date.copyright | 2009-02-03 | |
| dc.date.issued | 2009 | |
| dc.date.submitted | 2009-01-20 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/41723 | - |
| dc.description.abstract | 腫瘤壞死因子(Tumor necrosis factor-α, TNF-α)是一個免疫系統中的細胞激素，此細胞激素也能夠刺激急性反應的發生。我們研究了二個被TNF-α所誘導表現的基因的調控機制：環氧化酵素(cyclooxygenase-2, COX-2)以及Tristetraprolin (TTP)。我們觀察到在NIH3T3細胞中，COX-2 mRNA可以被TNF-α所誘導表現，並且TNF-α的誘導效果能夠被組蛋白去乙醯化酵素抑制劑TSA所抑制。除此之外，NFκB抑制劑BAY也具有和TSA相同的抑制效果，因此我們推測NFκB signaling pathway可能對COX-2的調控有重要影響。但是實驗結果卻發現NFκB 在細胞核與質的分佈或者與DNA的結合能力都不受TSA影響。最後藉由chromatin immunoprecipitation的方法知道TSA是作用在抑制polymerase II進行cox-2基因轉錄的elongation階段，至於詳細的分子機制仍需進一步的探討。另一個研究的主題是TTP mRNA穩定性的調控機制:當細胞受到TNF-α刺激時，TTP mRNA的半衰期會短暫地增長使mRNA能夠累積表現，進一步的研究後知道這個變化是在post-transcriptional level受到3’ untranslated region上AU-rich element的調控。我們會再進一步了解這個調控方式是透過何種訊息傳導來達成。 | zh_TW |
| dc.description.abstract | Tumor necrosis factor (TNF)-α is a cytokine involved in systemic inflammation, and is a member of a group of cytokines that stimulate the acute phase reaction. In this study, we focus on the regulatory mechanism of two TNF-α induced genes, cyclooxygenase-2 (COX-2) and Tristetraprolin (TTP). Using NIH3T3 cell line as a model, we found that COX-2 mRNA was activated by TNF-α treatment, and histone deacetylase inhibitor (HDACi) TSA could significantly block COX-2 activation. In addition, TNFα induced COX-2 expression could be inhibited by NFκB inhibitor BAY to a similar level as TSA. This indicated that NFκB signaling pathway may play an important role in modulating COX-2 expression. However, effects of TSA were not on NFκB nucleocytoplasmic distribution or DNA-binding ability. Results of chromatin immunoprecipitation (ChIP) assay revealed that TSA impaired COX-2 mRNA production by suppressing polymerase II elongation on the cox-2 gene. Further investigation on the molecular mechanism of this action would help to understand how HDACi suppressed gene expression. Another focus of this study is about the transient stabilization of TTP mRNA in response to TNF-α stimulation. We investigated the role of 3’untranslated region (UTR) in the regulation mechanism of TTP, and we found that the AU-rich element (ARE) was crucial for TTP expression modulation in the post-transcriptional level. Nevertheless, related works are still ongoing to explore the signaling cascade involved in transient TTP mRNA accumulation. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-15T00:28:59Z (GMT). No. of bitstreams: 1 ntu-98-R95b46036-1.pdf: 3249737 bytes, checksum: 53062c12e7fe3ee705603d002382e603 (MD5) Previous issue date: 2009 | en |
| dc.description.tableofcontents | 口試委員會審定書
誌謝 中文摘要 英文摘要 I. INTRODUCTION Tumor necrosis factor signaling...1 NFκB...3 HDAC inhibitors...5 Cyclooxygenase-2 (COX-2)...6 AU-rich element (ARE)-mediated mRNA stability regulation...7 Tristetraprolin (TTP)...9 II. MATERIALS AND METHODS Cell culture...13 Reagents for cell treatment...13 Plasmids and constructs...14 Site-directed mutagenesis...15 RNA isolation...16 Real-time PCR...17 Transient transfection, luciferase and galactosidase assays...17 Preparation of cytosolic and nuclear extracts and Western blotting assay...18 Electrophoretic mobility shift assay (EMSA)...19 Chromatin-immunoprecipitation (ChIP)...20 RNA pull-down assay...22 III. RESULTS Part I TSA suppressed TNF-α induced COX-2 mRNA expression in a cell type-specific manner...23 Both HDAC inhibitors, TSA and sodium butyrate impaired COX-2 production...24 NFκB inhibitor had a similar effect as TSA in suppressing COX-2 mRNA expression...25 cox-2 promoter contained a functional NFκB element...26 TSA did not alter NFκB expression and nucleocytoplasmic shuttling during TNF-α activation...27 TSA did not alter NFκB DNA binding ability during TNF-α activation...28 TSA suppressed COX-2 activation by inhibiting polymerase II enlongation...29 Suppression of polymerase elongation by TSA was not due to alternation of the interaction between NFκB and P-TEFb...31 Part II The expression profile of TTP during TNF-α stimulation of RAW264.7...32 Rapid change of TTP mRNA stability in the course of TNF-α stimulation...33 ARE as an essential element on ttp 3’UTR in regulating TTP expression...34 F118N mutant was created to monitor the possibility of TTP negative autoregulation...36 TTP AREs mediated the reduction of reporter activity...38 Detection of TTP ARE-binding proteins...39 IV. DISCUSSION Part I Reduction of COX-2 expression was due to TSA-induced pre-mature transcription...40 TSA may modulate signaling transduction pathways...41 TSA suppressed TNF-α induced COX-2 mRNA expression in a cell type-specific manner...42 TSA-induced initiation of cox-2 transcription is cell type-specific...43 TSA did not alter NFκB activity in TNF-α stimulated NIH3T3 cells...44 Inhibition of COX-2 basal level expression by TSA...46 Impaired COX-2 production by TSA and sodium butyrate but not by Apicidin or Scriptaid...46 Part II The stability of TTP mRNA changes rapidly in response to TNF-α stimulation...48 TTP ARE as a sufficient regulatory target of mRNA stabilization...48 Possible signaling transduction pathways involved in the stability of TTP mRNA...49 Detection of destabilizing or stabilizing proteins associated with TTP AREs...51 V. FIGURES Figure 1. TNF-α and COX-2 mRNA expression profile in response to TNF-α and TSA...52 Figure 2. Effects of various HDAC inhibitors on TNF-α-induced COX-2 mRNA expression in NIH3T3 cells...54 Figure 3. Effects of BAY on TNF-α-induced COX-2 mRNA expression in NIH3T3 cells...55 Figure 4. Analysis of cox-2 promoter activity in TSA-treated and TNF-α-stimulated NIH3T3 cells...56 Figure 5. NFκB expression and subcellular localization of TSA treated and TNF-α-stimulated NIH3T3 cells...57 Figure 6. DNA binding activity of NFκB on murine cox-2 promoter...58 Figure 7. Association of pol II on exon 1, 2, and 10 of the cox-2 gene...60 Figure 8. NFκB and P-TEFb association in TSA-treated and TNF-α-stimulated NIH3T3 cells...61 Figure 9. TTP mRNA and protein expression of TNF-α stimulated RAW264.7...62 Figure 10. TTP mRNA stability in TNF-α-stimulated RAW264.7 cells...64 Figure 11. Ectopic expression of TTP constructs...65 Figure 12. RNA pull-down assay...69 Figure 13. Functional characterization of TTP AREs...70 Figure 14. Identification of TTP ARE-associated protein by RNA pull-down...71 VI. SUPPLEMENTAL DATA AND TABLES Supplemental data 1. Subcellular localization of HuR...72 Supplemental data 2. COX-2 mRNA stability in response to TSA in NIH3T3 cells...73 Table 1. Histone deacetylase inhibitors...74 VII. REFERENCES...75 VIII. ABBREVIAION AND CHEMICAL SYMBOLS...82 | |
| dc.language.iso | en | |
| dc.subject | AU富含區 | zh_TW |
| dc.subject | 腫瘤壞死因子 | zh_TW |
| dc.subject | 環氧化酵素 | zh_TW |
| dc.subject | 核酸結合蛋白TTP | zh_TW |
| dc.subject | 組蛋白去乙醯化酵素抑制劑 | zh_TW |
| dc.subject | tristetraprolin | en |
| dc.subject | TNF-α | en |
| dc.subject | AU-rich element | en |
| dc.subject | cyclooxygenase-2 | en |
| dc.subject | histone deacetylase inhibitor | en |
| dc.title | 腫瘤壞死因子TNFα誘發基因表現之機轉
Part I: 組蛋白去乙醯化酵素抑制劑trichostatin A對環氧化酵素COX-2基因表現的抑制作用 Part II: RNA結合蛋白tristetraprolin的後轉錄調節 | zh_TW |
| dc.title | The mechanism of TNFα-induced gene expression
Part I: HDAC inhibitor trichostatin A-mediated inhibition of cyclooxygenase 2 gene expression Part II: The post-transcriptional regulation of RNA-binding protein tristetraprolin | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 97-1 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂勝春,果伽蘭,譚賢明 | |
| dc.subject.keyword | 腫瘤壞死因子,環氧化酵素,核酸結合蛋白TTP,組蛋白去乙醯化酵素抑制劑,AU富含區, | zh_TW |
| dc.subject.keyword | TNF-α,cyclooxygenase-2,tristetraprolin,histone deacetylase inhibitor,AU-rich element, | en |
| dc.relation.page | 82 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2009-01-20 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科學研究所 | zh_TW |
| 顯示於系所單位： | 生化科學研究所 | |
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