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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 蔡錦華(Ching-Hwa Tsai) | |
dc.contributor.author | Pei-Fang Tsai | en |
dc.contributor.author | 蔡佩芳 | zh_TW |
dc.date.accessioned | 2021-06-15T01:15:21Z | - |
dc.date.available | 2014-09-15 | |
dc.date.copyright | 2009-09-15 | |
dc.date.issued | 2009 | |
dc.date.submitted | 2009-07-28 | |
dc.identifier.citation | Abdelrahim, M. & Safe, S. (2005) Cyclooxygenase-2 inhibitors decrease vascular endothelial growth factor expression in colon cancer cells by enhanced degradation of Sp1 and Sp4 proteins. Mol Pharmacol 68(2), 317-29.
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/42519 | - |
dc.description.abstract | EB 病毒(Epstein-Barr Virus)為人類gamma herpesvirus的一員,主要感染人類的表皮細胞和淋巴細胞。EB病毒與其它已知的疱疹病毒同樣存在有潛伏期與溶裂期兩種生活型態,目前已知,EB病毒由潛伏期進入溶裂期時首先會活化BZLF1及BRLF1基因表現。在in vitro的細胞培養中,可使用一些刺激物,例如:抗免疫球蛋白(anti-immunoglobulin)、 12-O-tetradecanoylphorbol-13-acetate (TPA)、 組織蛋白去乙醯酶抑制劑(Histone deacetylase inhibitor, HDACi) 誘發潛伏期性感染細胞的EB病毒再活化進入溶裂期。本實驗室先前對於HDACi如何誘發病毒再活化產生的研究中發現,PKCδ在其中扮演重要的角色,因此本研究將進一步探討PKCδ透過何種機制調控EB病毒再活化。
首先利用螢光報導基因檢測發現短暫轉染表現PKCδ就足以活化EB病毒BZLF1啟動子(Zp)但BRLF1啟動子(Rp)則否。構築不同Zp 5’端序列刪除株報導質體尋找PKCδ所調控的位置,結果顯示位於Zp-99至-80的區域,主要包含ZID element,對於PKCδ調控Zp活性很重要。在構築ZID element突變株報導質體分析後發現位於Sp1可能結合的位置對於PKCδ調控Zp的活性十分重要。更進一步以電泳位移分析、 DNA 親和力沉澱分析及染色質免疫沉澱分析分別證實ZID element上確實有Sp1的結合。 接著利用免疫沉澱法證實TSA會造成Sp1絲胺酸磷酸化並且此現象需仰賴PKCδ蛋白質激酶活性而存在。TSA也會增強PKCδ和Sp1之間的結合藉此形成複合體結合於Zp上。除此之外,利用了訊息傳遞路徑的抑制劑處理後,發現PI3K訊息傳遞扮演PKCδ活化之上游路徑同樣參與在TSA誘發病毒溶裂期產生過程中。 最後,透過免疫沉澱法發現細胞內Sp1與HDAC2的結合情形,並且TSA會影響兩者之結合。染色質免疫沉澱分析同樣觀察到在TSA處理後HDAC2會離開Zp,此現象在PKCδ活性受抑制後則消失。以上實驗結果顯示,TSA透過PI3K訊息傳遞路徑活化PKCδ。PKCδ在HDACi誘發病毒再活化過程會導致Sp1磷酸化及HDAC2這個轉錄抑制者離開Zp,藉此活化BZLF1基因表現。 | zh_TW |
dc.description.abstract | Epstein-Barr virus (EBV) is a human gamma herpesvirus that infects epithelial and lymphoid cells. Like other human herpesviruses, EBV contains latency and lytic replication life cycles and its reactivation from latency is initiated by the expression of the two viral immediate-early genes, BZLF1 and BRLF1. In in vitro culture system, reactivation of EBV in latently infected cells can be induced by various agents, including anti-immunoglobulin、12-O-tetradecanoylphorbol-13-acetate (TPA) and histone deacetylase inhibitor (HDACi). In our previous study, we found that protein kinase C delta (PKCδ) is required for induction of EBV lytic cycle by HDACi. So, this thesis is aimed to further investigate the regulatory mechanism of PKCδ in HDACi-induced EBV lytic cycle progression.
Firstly, the results of luciferase reporter assay demonstrated that overexpression PKCδ is sufficient to induce the activities of BZLF1 promoter (Zp) but not BRLF1 promoter (Rp). Moreover, deletion analysis of Zp revealed that the region between -99 and -80, which contains ZID element, is important for the activation of Zp by PKCδ. The mutagenesis analysis of ZID element revealed that the Sp1 putative sequence on ZID is essential for PKCδ to induce Zp activity. In addition, the binding of Sp1 on ZID was proved by in vitor electrophoretic mobility shift assay (EMSA), DNA-affinity precipitation assay (DAPA) and in vivo chromatin immunoprecipitation (ChIP) approaches. Furthermore, upon treatment of TSA, data from immunoprecipitation assay indicated that Sp1 can be phosphorylated at seine residues(s), which is PKCδ kinase activity dependent. TSA also stimulation promotes physical interactions between PKCδ and Sp1, resulting in the association of PKCδwith Sp1 on BZLF1 promoter. In addition, we screened the effect of kinase inhibitors on blocking the EBV reactivation and the results indicated that PI3K signaling pathway act as the upstream of PKCδ and also play the crucial role in TSA-mediated EBV reactivation. Finally, the co-immunoprecipitation assay revealed that the physically interaction was also observed between Sp1 and Histone deacetylase 2 (HDAC2) and that TSA treatment interfered with this association. ChIP assay also hinted that HDAC2 repressor dissociated from BZLF1 promoter after TSA treated, which was inhibited by PKCδ blockage. So, we hypothesize that TSA induces PKCδactivation through PI3K signaling pathway, then the active PKCδcan cause the phosphorylation of Sp1, which resulting in the release of HDAC2 repressor from BZLF1 promoter, and then turn on BZLF1 gene expression. | en |
dc.description.provenance | Made available in DSpace on 2021-06-15T01:15:21Z (GMT). No. of bitstreams: 1 ntu-98-R96445103-1.pdf: 4160309 bytes, checksum: 592412deea818800323de6203528f04b (MD5) Previous issue date: 2009 | en |
dc.description.tableofcontents | 口試委員審定書…………………………………………………… I
致謝…………………………………………………………………Ⅱ 中文摘要……………………………………………………………Ⅲ Abstract…………………………………………………………… Ⅴ 目錄…………………………………………………………………Ⅶ 縮寫檢索表…………………………………………………………Ⅸ 序論…………………………………………………………………1 一、EB病毒(Epstein-Barr virus, EBV) ………………………………… 1 1.1 EB病毒發現史 1.2 EB病毒的結構與遺傳物質 1.3 EB病毒的致病性 1.4 EB病毒生活史 1.5 EB病毒再活化 二、BZLF1啟動子(Zp) ……………………………………………… 6 2.1 正向順式調控片段 2.2 負向順式調控片段 2.3 Zp上的結合轉錄因子 2.4 Zp活化之機制 三、蛋白質激酶C (Protein kinase C, PKC) …………………………… 8 3.1 蛋白質激酶C的發現 3.2 蛋白質激酶C的結構與分類 3.3 蛋白質激酶C的活化 3.4 蛋白質激酶Cδ 3.5 蛋白質激酶Cδ抑制劑-Rottlerin 四、實驗目的…………………………………………………………13 實驗材料……………………………………………………………14 一、 藥品 二、 套組試劑 三、 質體 四、 細胞株 五、 抗體 六、 酵素 七、 引子 八、 寡聚核苷酸 實驗方法……………………………………………………………19 一、質體構築 (Plasmid construct) 二、轉型反應 (Transformation) 三、大量質體DNA抽取(Maxi-preparation of plasmid DNA) 四、細胞培養 (Cell culture) 五、細胞暫時轉染 (Transient transfection) 六、EB病毒溶裂循環的誘導 ( Induce EB lytic cycle) 七、抑制劑的處理 ( Inhibitor treatment) 八、西方墨點法(Western blot) 九、螢光酵素-報告基因活性分析 (Luciferase repoter assay) 十、電泳位移分析(Electrophoretic mobility shift assay,EMSA) 十一、染色質免疫沉澱分析 (Chromatin immunoprecipitation ,ChIP) 十二、DNA親和力沉澱 (DNA affinity precipitation assay, DAPA) 十三、細胞質核分離萃取 (Cytosotic &Nuclear fractionation) 十四、免疫沈澱法 (Immunoprecipitation assay) 實驗結果……………………………………………………………35 討論…………………………………………………………………46 圖表…………………………………………………………………51 附錄…………………………………………………………………79 參考文獻……………………………………………………………83 | |
dc.language.iso | zh-TW | |
dc.title | 探討蛋白質激酶Cδ調控BZLF1啟動子在組蛋白去乙醯酶抑制劑誘發EB病毒再活化之機制 | zh_TW |
dc.title | Regulation of BZLF1 promoter through Protein Kinase C Delta in HDAC Inhibitor-induced Epstein-Barr Virus Reactivation | en |
dc.type | Thesis | |
dc.date.schoolyear | 97-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 董馨蓮(Shin-Lian Doong),張麗冠(Li-Kwan Chang) | |
dc.subject.keyword | EB病毒,蛋白質激酶,Cδ,BZLF1啟動子,Sp1,HDAC2, | zh_TW |
dc.subject.keyword | Epstein-Barr virus,Protein Kinase Cδ,BZLF1 promoter,Sp1,HDAC2, | en |
dc.relation.page | 93 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2009-07-28 | |
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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