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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 呂紹俊(Shao-Chun Lu) | |
dc.contributor.author | Hsiao-Wen Wu | en |
dc.contributor.author | 吳曉雯 | zh_TW |
dc.date.accessioned | 2021-06-13T01:48:13Z | - |
dc.date.available | 2009-07-12 | |
dc.date.copyright | 2007-07-12 | |
dc.date.issued | 2007 | |
dc.date.submitted | 2007-07-10 | |
dc.identifier.citation | 吳宗諭 (2006) 轉錄因子Oct-2參與在脂多醣誘發巨噬細胞中iNOS啟動子活化的研究。 國立臺灣大學醫學院生物化學暨分子生物學研究所碩士論文
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dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/30274 | - |
dc.description.abstract | 近年研究發現,LPS會透過調控HDAC (histone deacetylase) family的酵素來改變巨噬細胞中LPS-responsive基因的表現,顯示LPS調控基因表現與染色質結構有關。為了研究HDAC抑制劑─TSA (trichostatin A)對LPS所誘發的iNOS基因表現之影響,RAW264.7巨噬細胞先以TSA處理,之後再以LPS刺激,以探討TSA對LPS誘導iNOS基因表現的影響。實驗結果顯示TSA會造成RAW264.7細胞中的histone H3乙醯化程度上升現象,並且抑制LPS促使NO的生成。經由RT-PCR以及西方墨點法分析發現TSA會降低LPS所誘發的iNOS mRNA及蛋白質表現量。iNOS基因表現主要是在轉錄層次的調控,實驗發現在iNOS基因表現中扮演樞紐角色的NF-κB其轉錄活性未受TSA影響,在TSA存在的情況下,不影響NF-κB reporter的活性,同時LPS仍可促使細胞核中的NF-κB p50以及p65蛋白質表現量增加。新的發現為TSA會抑制受LPS誘導的Oct-2 mRNA及蛋白質含量,但Oct-1 mRNA及蛋白質卻不受TSA影響。Oct-2轉錄因子的活化對於LPS誘導巨噬細胞中iNOS基因表現似乎是必要的步驟,由染色質免疫沉澱分析法(ChIP assays)結果顯示,LPS會增加Oct-2蛋白質結合到染色質上iNOS promoter的能力,此結合會受到TSA的處理下而被抑制。因此我懷疑iNOS基因表現受到TSA所影響是因為Oct-2被抑制的關係,因此將pCG-Oct-2轉染至細胞後再以TSA及LPS處理,發現在RAW264.7細胞中適量表達Oct-2蛋白質的情況下,可以回復因TSA處理而抑制LPS所誘導的iNOS蛋白質含量。 總而言之,由實驗結果推測TSA可能是透過抑制Oct-2活化來降低LPS所誘導的iNOS表現,TSA具有阻止巨噬細胞產生NO的作用以及抗發炎的效果。Oct-2的活化作用對於iNOS基因表現是相當重要的一環,但是TSA抑制Oct-2的表現機制仍不清楚。本實驗的結果突顯出Oct2這個轉錄因子對於iNOS表現的重要性。 | zh_TW |
dc.description.abstract | We studied trichostatin A (TSA) effect on the LPS induction of the inducible nitric oxide synthase (iNOS) gene in RAW264.7 macrophages. TSA pretreatment potently inhibited the release of nitric oxide (NO) in the cells treated with lipopolysaccharide (LPS), and Western blot and RT-PCR analyses revealed that TSA inhibited the LPS-induced induction of iNOS gene. Moreover, it is known that the activation of nuclear factor-κB (NF-κB) is the key step in the signaling pathways mediating iNOS induction. Nuclear p50 and p65 protein levels increased within 30min after LPS treatment, and pretreatment of TSA did not affect this LPS-induced nuclear translocation of p50 and p65. Moreover, in reporter gene analysis, TSA has no effect on NF-κB reporter activity. These results suggest that NF-κB is not targeted by TSA to block iNOS. Here for the first time, we found that TSA suppressed Oct-2 expression, and that activation of Oct-2 is likely to be essential for iNOS induction in LPS-treated macrophages. Chromatin immunoprecipitation assays revealed that, under control conditions, LPS significantly increases Oct-2 binding to octamer site of iNOS promoter, which was attenuated by co-treatment with TSA. TSA decreases LPS induction of Oct-2 binding to iNOS promoter. Forced expression of Oct-2 by transfection of pCG-Oct-2 plasmid restored protein levels of iNOS induced by LPS under TSA treatment in
v RAW264.7 macrophages. In conclusion, this study demonstrated that TSA inhibited the LPS-induced production of NO in the macrophages by inhibiting Oct-2 activation. These results revealed that Oct-2 activation is a crucial step in the transcriptional activation of the iNOS gene. This presumed inhibitory effect on Oct-2 activation by TSA may be associated with its potent NO blocking and anti-inflammatory effects. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T01:48:13Z (GMT). No. of bitstreams: 1 ntu-96-R94442015-1.pdf: 1142795 bytes, checksum: d881fc2344a10a4522f5c39bb3dadfa8 (MD5) Previous issue date: 2007 | en |
dc.description.tableofcontents | 口試委員會審定書 i
誌謝 ii 中文摘要 iii 英文摘要 iv 縮寫對照表 vi 第一章 緒論 1 第一節 文獻回顧 2 第二節 研究動機與實驗目的 14 第二章 材料與方法 16 第一節 實驗材料 17 第二節 細胞培養 19 第三節 iNOS promoter活性分析 19 第四節 分析細胞中不同mRNA的表現量 20 第五節 分析細胞中不同蛋白質的表現量(西方墨點法分析) 24 第六節 NF-κB活化程度的測量 26 第七節 細胞中NO(nitric oxide)生成量的測量 26 第八節 細胞存活率的測量(MTT assay) 27 第九節 分析細胞內轉錄因子與iNOS promoter的結合 27 第十節 大量製備pCG、pCG-Oct2表現質體 29 第十一節 分析在大量表現Oct-2轉錄因子的RAW264.7細胞中蛋白 表現量 31 第十二節 分析細胞外轉錄因子與iNOS promoter的結合 32 第三章 實驗結果 35 第一節 TSA對於RAW264.7巨噬細胞存活率的影響 36 第二節 TSA對於RAW264.7巨噬細胞中acetylated H3的影響 36 第三節 TSA對於LPS誘導之發炎基因表現的影響 36 第四節 TSA影響RAW264.7細胞中受LPS誘導的iNOS蛋白質表現 以及NO的生成 37 第五節 TSA對於啟動iNOS基因表現之重要轉錄因子的影響 37 第六節 RAW264.7細胞中iNOS基因表現與轉錄因子Oct-2的研究 40 第四章 討論 42 第一節 RAW264.7巨噬細胞中的HDACs與iNOS基因表現之關係 43 第二節 轉錄因子NF-κB和AP-1在iNOS基因表現中扮演的角色 44 第三節 轉錄因子Oct-1與Oct-2在iNOS基因表現中扮演的角色 46 第四節 TSA對細胞造成的效應 47 第五節 總結 49 第五章 圖表 50 第六章 附錄 63 第七章 參考文獻 72 | |
dc.language.iso | zh-TW | |
dc.title | Oct-2在TSA降低脂多醣誘導巨噬細胞的iNOS基因表現中扮演的關鍵角色 | zh_TW |
dc.title | Trichostatin A down-regulates LPS-induced iNOS expression in macrophages: pivotal role of Oct-2 | en |
dc.type | Thesis | |
dc.date.schoolyear | 95-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 姜安娜(An-Na Chiang),張淑芬(Shu-Fen Zhang),李明學(Ming-Xiao Li) | |
dc.subject.keyword | 發炎反應,脂多醣, | zh_TW |
dc.subject.keyword | LPS,iNOS,trichostatin A, | en |
dc.relation.page | 83 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2007-07-10 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 生物化學暨分子生物學研究所 | zh_TW |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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