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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 | Kun-Yi Lai | en |
dc.contributor.author | 賴坤詣 | zh_TW |
dc.date.accessioned | 2021-05-15T17:50:43Z | - |
dc.date.available | 2017-10-09 | |
dc.date.available | 2021-05-15T17:50:43Z | - |
dc.date.copyright | 2014-10-09 | |
dc.date.issued | 2014 | |
dc.date.submitted | 2014-08-19 | |
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/4981 | - |
dc.description.abstract | EB 病毒是一具有致癌能力的人類皰疹病毒,能夠感染人類初代 B 細胞 (primary B cell) ,並使其不朽化成淋巴芽母細胞株 (lymphoblastoid cell line,LCL)。過去許多研究證實,轉形成LCL後細胞中會有許多細胞激素及細胞介素被誘導表現,而這些細胞激素主要被認為和LCL細胞增生及不朽化有關。本實驗室先前在 cDNA 微陣列結果中發現,其中一個細胞激素,白細胞介素32 (interleukin 32,IL-32) 在 EB病毒感染後的 B 細胞有明顯增加的現象。IL-32 在近年來被報導的功能為促發炎細胞激素的一員,能在不同的細胞內誘發各種細胞激素及細胞介素的產生,甚至具有影響細胞生長的能力。本研究目的是為了瞭解 IL-32 於 EB 病毒感染 B 細胞中之調控機制及細胞功能。實驗研究發現,隨著EB病毒感染B細胞的天數增加,IL-32的表現量也有隨之上升的現象。另外在四位健康捐血者的B細胞以EB病毒感染後不朽化成的LCL中也能看到IL-32皆有大量表現,顯示EB病毒具有活化IL-32表現的能力且此一現象是存在所有EB病毒感染後的LCL中。以免疫螢光染色法及細胞核質分離法發現在LCL內的IL-32 主要位在細胞質而非細胞核。進一步發現 EB 病毒的潛伏膜蛋白1 ( Latent membrane protein 1, LMP1)及溶裂期極早期基因Rta對誘發 IL-32 的 mRNA 及 蛋白質表現是重要的,另外,隨著表現越多的LMP1能以正相關的現象誘導越多IL-32表現,且在 IL-32 大量表現的 LCL 中,以shRNA抑制LMP1的表現後也可以看到 IL-32 表現有隨之下降的現象,以上結果顯示 LMP1 具有活化 IL-32 表現的能力。在 LMP1 分子調控機制上,我們發現 LMP1 能透過 C 端活化區域 CTAR1 以及 CTAR2 活化 IL-32 表現。以螢光酵素報導基因分析發現 IL-32 基因的啟動子上 -8至+2的核甘酸鹼基對之NF-kB結合位對於基因表現是重要的,進一步我們利用染色質免疫沉澱法,發現NF-kB p65 能夠結合在 IL-32 啟動子上,在LMP1 表現的 Akata 細胞中,另外在 LCL 及LMP1 表現的Akata細胞中以 shRNA 使 p65 基因受抑制後也可以看到 IL-32 表現有下降的現象,顯示LMP1活化IL-32是透過NFkB訊息傳遞路徑。在細胞功能研究部分,我們在LCL中以shRNA抑制IL-32表現後,觀察到EB病毒溶裂期基因的表現量皆有上升的現象,顯示LCL內EB病毒進入溶裂期,接著進一步發現溶裂期極早期基因Zta的啟動子(Zp)的活性,在經過Zta、TSA藥物刺激及蛋白激酶C (protein kinase C, PKC)的大量表現後會有增加的現象,重要的是這些Zp活化的現象會受到IL-32的抑制,顯示IL-32可能藉由抑制Zp的方式進而抑制EB病毒進入溶裂期,另外以共軛焦顯微鏡可以觀察到IL-32具有抑制PKC進入細胞核的現象,暗示IL-32確實能夠抑制PKC所造成的活化現象。除此之外,在LCL中以shRNA抑制IL-32表現後也觀察到IL-6有活化的現象,但IL-10及TNFa則沒有太大的影響,顯示IL-32具有影響細胞激素表現的能力。同樣在LCL中以shRNA抑制IL-32表現後則發現IL-32不會影響細胞增生。以上對生理功能的研究顯示,IL-32可能主要功能為抑制LCL中的EB病毒進入溶裂期及IL-6的表現。由於IL-6對LCL的生長及不朽化是重要的,且進入溶裂期有助於EB病毒的擴散及形成EB病毒相關之惡性腫瘤,幫助EB病毒形成適合存在的環境,IL-32可能扮演宿主抑制EB病毒入侵的角色。 | zh_TW |
dc.description.abstract | Epstein-Barr virus (EBV) is a human oncogenic herpesvirus, which has the potential to immortalize primary B cells into unlimitedly proliferating lymphoblastoid cell lines (LCLs). Previous study shows that several cytokines are induced during immortalization and these cytokines are required for cell proliferation and B cell immortalization. Our previous cDNA microarray results show that interleukin 32 (IL-32), a newly discovered proinflammatory cytokine, is upregulated after EBV infection. Recent studies show that IL-32 is a proinflammatory cytokine which can induce other cytokines and effect cell proliferation. So, we wonder what is the role of IL-32 in EBV-infected cells. The expression of IL-32 is up-regulated during EBV infection. B cells purified from four different donors are infected by EBV for 28 days (defined as LCL), and also, IL-32 is up-regulated in these LCLs. The cellular localization of IL-32 is mainly in the cytoplasm of LCLs. Furthermore, the EBV latent membrane protein 1 (LMP1) and Rta are responsible for inducing IL-32 expression in both mRNA and protein levels. LMP1 also induces IL-32 expression in a dose-dependent manner. Knockdown of endogenous LMP1 in LCLs with shRNA approach suppresses the IL-32 expression. These data suggest that LMP1 is critical for induction of IL-32 expression. We also demonstrated that the carboxy-terminal activating region (CTAR) 1 and CTAR2 of LMP1 are required to induce IL-32. The NF-kB site located at the -8 to +2 nucleotide locus of IL-32 promoter is crucial for activating IL-32 in the reporter assays. Moreover, using ChIP assay, we demonstrate that NF-kB p65 binds to IL-32 promoter region in LMP1-expressing Akata cells. Knockdown of p65 with shRNA suppresses IL-32 expression. These data show that NF-kB p65 is critical for IL-32 expression. In biological function study, knockdown of IL-32 in LCL induces EBV lytic protein expression. Moreover, the promoter acitivity of immediately early gene Zta is induces via the expressing of Zta, treating TSA, and PKC stimulation. However, these activations on Zta promoter (Zp) activity are suppressed by IL-32 expression. Also, overexpressing IL-32 in Hela cells blocks the translocation of PKC into nuclear. These data indicate that IL-32 suppresses EBV lytic cycle by suppressing Zp activity. Furthermore, depletion of IL-32 in LCL also induces the expression of IL-6 instead of IL-10 or TNFa. This result shows that IL-32 can affect particular cytokine production in LCL, which may be different from other cell types. However, depletion of IL-32 dose not have impact on cell proliferation. Overall, IL-32 inhibits the lytic cycle of EBV and partial IL-6 expression. IL-6 is critical for the proliferation and immortalization of LCL. EBV lytic cycle progression is important for EBV evation and formation of EBV-associated malignancies. These concepts indicate that IL-32 might play a role in EBV life cycle. | en |
dc.description.provenance | Made available in DSpace on 2021-05-15T17:50:43Z (GMT). No. of bitstreams: 1 ntu-103-R01445114-1.pdf: 3087830 bytes, checksum: bda805bd8ee053d69a40c79d765b0e03 (MD5) Previous issue date: 2014 | en |
dc.description.tableofcontents | 中文摘要……………………………………………………………II
英文摘要……………………………………………………………IV 第一章 序論 ………………………………………………1 第二章 實驗材料與方法……………………………12 第三章 實驗結果…………………………………………31 第四章 討論…………………………………………………42 第五章 圖表…………………………………………………48 第六章 參考文獻…………………………………………85 | |
dc.language.iso | zh-TW | |
dc.title | 探討白細胞介素 IL-32 於 EB 病毒感染 B 細胞中之調控機制及細胞功能 | zh_TW |
dc.title | The regulatory and biological functions of IL-32 gene in EBV-infected cells | en |
dc.type | Thesis | |
dc.date.schoolyear | 102-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 董馨蓮(Shin-Lian, Doong),林素珍(Sue-Jane Lin) | |
dc.subject.keyword | EB病毒,LMP1,細胞激素,白細胞介素-32, | zh_TW |
dc.subject.keyword | EBV,LMP1,cytokine,IL-32, | en |
dc.relation.page | 95 | |
dc.rights.note | 同意授權(全球公開) | |
dc.date.accepted | 2014-08-19 | |
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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