臺灣博碩士論文加值系統

中文摘要
免疫反應為抵擋外來物侵入時會活化內皮細胞、巨噬細胞(macrophage)及白血球細胞等，釋放出大量生物標記分子，如巨噬細胞移行抑制因子(macrophage migration inhibitory factor, MIF)及發炎相關細胞激素等，如TNF-、IL-1、IL- 6、IL-8等，直接或間接造成器官衰竭。其中，促發炎反應細胞激素 MIF基因之啟動子調控，在過去的文獻中，在細菌感染模式下的表現調控以及MIF 啟動子中轉錄因子之功能及其重要性，並沒有太多探討，因此本篇研究MIF基因之啟動子活性，與在感染模式下，不同的轉錄因子在MIF基因啟動子上是否扮演重要角色。我們建構不同長度的啟動子-報導基因表現載體來探討MIF基因啟動子活性，並使用定點突變技術將可能的不同轉錄因子結合部位突變，來探討轉錄因子對MIF基因表現的影響。我們發現MIF-157啟動子片段具有基礎啟動子活性。除MIF+44片段啟動子以外，在所有的啟動子長度下，處理LPS都可以誘導MIF基因啟動子活性，但同時給予dexamethasone則會抑制這個作用。MIF-157啟動子片段上，突變兩個Sp1位點及CRE和HIF-1位點，可以顯著降低啟動子活性。突變HIF-1位點也抑制了LPS和dexamethasone的作用。MIF-508啟動子片段上突變可能的GR位點，會降低啟動子活性，包括LPS和dexamethasone的作用，但突變可能的F-ACT1位點，則會增加MIF基因啟動子活性。根據以上結果，一些轉錄因子在MIF基因啟動子活性上扮演重要角色，同時也會影響LPS和dexamethasone對MIF基因表現的作用。

Abstract
Immune response to the invasion of foreign objects will activate endothelial cells, macrophages and other white blood cells to release a large number of biomarkers, such as macrophage migration inhibitory factor (MIF) and inflammation-related cytokines, such as TNF-α, IL-1 IL-6, IL-8 and so on, which directly or indirectly cause organ failure. Among them, few literatures reported the promoter studies of the pro-inflammatory cytokine MIF gene in the bacterial infection models, as well as the functions and importance of transcription factors in regulation of the MIF promoter activity. Therefore, in this study, we investigated the role of transcription factors in the regulation of MIF gene promoter and immune functions in an infection model. We constructed several promoter-reporter constructs with different length of MIF promoter fragments to study the promoter activities and used sited-directed mutagenesis to mutate the putative transcription factor sites to elucidate the role of these sites in regulating MIF gene expression. We found that MIF-157 fragment contains a basal promoter activity. Treatment of LPS induced MIF promoter activities but dexamethasone reduced these effects in all fragments except for MIF+44. Mutation of two Sp1 sites, CRE and HIF-1 on MIF-157 fragment decreased the MIF promoter activity. Mutation of HIF-1 on MIF-157 fragment also decreased the effects of LPS and dexamethasone. Mutation of putative GR site on MIF-508 fragment also decreased the promoter activity. However, mutation of putative F-ACT1 site increased the promoter activity. According to these results, we found that several transcription factors may play important roles in regulating basal promoter activity of MIF as well as the effects of LPS and dexamethasone on MIF gene expression.

目錄
論文口試委員審定書 i
誌謝 ii
中文摘要 iv
Abstract vi
縮寫表 viii
目錄 ix
圖表目錄 xii
第一章 前言 1
第一節 免疫系統的種類 1
第二節 MIF的角色 2
第三節 MIF在免疫反應中的角色 2
第四節 MIF拮抗醣皮質醇抑制免疫反應 4
第二章 研究動機與目標 6
第三章 實驗材料與方法 7
第一節 建構質體和定點突變(Site-directed mutagenesis) 7
第二節 Raw264.7細胞培養 7
第三節 抽取質體DNA (Plasmid DNA extraction)和去除內毒素(Endotoxin remove) 8
第四節 短暫性轉染(Transient transfection) 10
第五節 冷光酵素分析(Daul-luciferase assay)-啟動子分析 10
第六節 統計方法 11
第四章 實驗結果 12
第一節 MIF基因啟動子序列比對 12
第二節 MIF基因啟動子的表現 13
第三節 藥物調控MIF基因啟動子活性 13
第四節 不同轉錄因子結合部位在MIF基因表現所扮演的角色 14
第五章 討論與結論 16
參考文獻 18
附表與圖 22
作者簡歷 37

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