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研究生:謝偉鋒
研究生(外文):Hsieh Wei-Feng
論文名稱:RIG-1基因啟動區負調節子之鑑定及TCDD對其基因表現之作用
論文名稱(外文):Identification of the Negative Regulatory Region in RIG-1 Gene Promoter and the Effect of TCDD on RIG-1 Gene Expression
指導教授:張自忠
學位類別:碩士
校院名稱:國防醫學院
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
中文關鍵詞:rig
外文關鍵詞:rig
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RIG-1 (Retinoid-Induced Gene 1) 被認為屬於第二型腫瘤抑制基因。其表現受全反式維甲酸 (ATRA) 以及 p53 的作用而增加。RIG-1表現量的增加會抑制細胞的生長並且促進分化甚至造成細胞凋亡或壞死,這些現象暗示著 RIG-1 為一個細胞生長的調節者,且其可能具有抑制癌細胞生長的功能。先前實驗室的研究找出了受 RIG-1 基因啟動子區上 ATRA 和 p53 所調控的區域,在研究過程中,發現當 rig(-442/-110) 片段存在時,報告載體的基本螢光酶活性降低且失去了被全反式維甲酸所活化的現象,因此說明了在 rig(-442/-110) 的片段中可能存在了一段負調節的區域,因此我們將此片段做刪除片段載體分析,結果發現此負調節區存在於 rig(-342/-270) 片段中,接著做電泳凝膠位移分析的實驗,發現有特定蛋白與 rig(-342/-301) (簡稱 nf-A) 片段結合,另外我們在 rig0705 片段上發現了一個 IR 6 的單位元,許多研究顯示此單位元與細胞對 xenobiotics 之反應有關,因此我們利用 2,3,7,8-tetrachlorodibenzo- -dioxin (TCDD) 來檢測其與 RIG-1 基因的關係,結果我們發現 TCDD 可以促進 RIG-1 的蛋白質及 mRNA 的表現量增加,另外我們也發現 TCDD 促進 RIG-1 基因表現的區域是位在 rig0705 片段上
目錄

目 錄…………………………………………….………………. Ⅰ
縮 寫 表……………………………………………………………. Ⅲ
圖表目錄…………………………………………….……………… Ⅳ
中文摘要……………………………………………………………. Ⅵ
英文摘要……………………………………………………………. Ⅶ
緒 論……………………………………………………………. 1
材料與方法…………………………………………………………. 8
一、主要儀器及藥品試劑………………………………..……… 8
二、小量質體的製備…….………………………….…………… 9
三、核酸限制酶反應 …………………………………………… 11
四、DNA片段的回收…………………………………………… 11
五、DNA連結反應………………………………………………. 12
六、勝任細胞的製備………………….…… …..……………….. 12
七、轉形作用………………………….………….……………… 14
八、大量質體的備製…………………………………..………… 14
九、細胞培養………………………….…………………………. 18
十、細胞轉染…………………………………….………………. 22
十一、相對螢光酶活性分析…………………….………………. 22
十二、蛋白質濃度的測定…………………………………........... 24
十三、西方墨漬轉移法………………………………………… 25
十四、抽取細胞核蛋白………………………….……………….. 32
十五、Electrophoretic Mobility Shift Assay………………………. 34
結 果…….……………………………………………………… 39
一、rig(-442/-110) 刪除片段的構築…………………………….. 39
二、RA誘導 rig(-442/-110) 刪除片段載體之轉錄活性分析….. 39
三、在 Hep3B 細胞中,rig(-393/-343) 與 rig(-342/-270) 區域之電泳凝膠位移分析……………………………………….. 42
四、rig(-342/-270) 片段之電泳凝膠位移分析…………………. 43
五、rig(-342/-270) 片段的次片段 nf-A 及 nf-B 之電泳凝膠位
移分析………………………………………………………… 44
六、TCDD 對肝癌細胞 HepG2 與 Hep3B 中 RIG-1 蛋白含量之影響…………………………………………………….. 45
七、TCDD 對肝癌細胞 HepG2 與 Hep3B 中 RIG-1 mRNA 表現量之影響……………………………………………….. 47
八、TCDD誘導 RIG-1 基因啟動區刪除片段報告載體之轉錄
活性分析…………………………………………………….. 47
討 論………….………………………………………………… 49
結 論………………………………………………………….… 52
圖表………………………………………………….……………… 53
參考文獻……………………………………………………………. 81
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