博碩士論文 952204009 詳細資訊




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姓名 錢葉盛(Yeh-Sheng Chien)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 FoxO1 調節抗胰島素激素基因的表現
(FoxO1 regulates resistin gene expression)
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摘要(中) 抗胰島素激素(Resistin)為一種已知的脂肪細胞所分泌的細胞激素, 它會造成胰島素抗性作用,並扮演著調節能量平衡的角色。之前的文獻中已經找出許多會去調節resistin基因表現的轉錄因子以及其結合位﹐但是其調控機制仍然不清楚。FoxO1 (Forkhead/winged helix box gene,group O-1) 轉錄因子會受到胰島素調控將其磷酸化﹐去決定是否能在細胞核內作用。之前的研究中指出FoxO1與resistin隨著脂肪細胞分化的程度越好,兩者基因在脂肪細胞內皆會有改變﹐而FoxO1參與調控一些胰島素所調控的基因表現。胰島素又會抑制抗胰島素機速基因的表現。因此本論文假設FoxO1轉錄因子會參與脂肪細胞內抗胰島素激素基因表現的調控作用﹐實驗結果證明﹕(1) FoxO1,3,4與resistin 基因之表現確實在脂肪細胞分化的過程中,逐步升高。(2)轉染FoxO1-AAA與human resistin promoter(-819~+24 bp)在HEK 293T細胞株中。發現FoxO1隨著劑量增加而有負調控該啟動子的活性。 (3) 轉染FoxO1-AAA與human resistin promoter(-819~+24 bp)在NIH 3T3細胞株中。發現FoxO1隨著劑量增加而有負調控該啟動子的活性。由以上結果顯示,Foxo1調節human resistin promoter(-819~+24 bp)活性,不會因轉染細胞株不同而有差異性。 (4)FoxO1-AAA與以架構的三種(-3880~-55 bp,-1330~-55 bp,-990~-55 bp)共同轉染到HEK 293T細胞內,發現FoxO1-AAA均負調節三種mouse resistin promoter的活性。(5)野生型FoxO1,突變型FoxO1-AAA,以及FoxO1-H215R,分別與Human resistin promoter(-819~+24 bp)共同轉染到HEK 293,HEK 293T,或3T3細胞株中。皆發現是負調控該promoter活性。所以顯示FoxO1是間接性調控resistin promoter的活性。綜合以上的結果得知,FoxO1是負調節Human resistin promoter的活性。因為FoxO1-AAA與mouse resistin promoter共同轉染到 3T3細胞株中,FoxO1-AAA正調節該promoter 的活性(採自於學妹楊淑雅的實驗數據),所以FoxO1調節mouse resistin promoter的活性會因轉染不同細胞株而有所差異。
摘要(英) Resistin is also known as adipocyte secreted factor (ADSF) that plays potential role in insulin resistiance and homeostasis. Although resistin can be regulated by transcriptional factors, the action mechanism is still not clear. FoxO1 (Forkhead/winged helix box gene,group O-1) is a member of a family of nuclear transcriptional factors that can be regulated by insulin. It has been reported that FoxO1 and resistin genes express during adipocyte differentiation, and are negatively regulated by insulin. In this thesis,the hypothesis that FoxO1 regulates resistin gene expression was examined. The results were as follows. First, expression of FoxO1, FoxO3, FoxO4, and resistin genes were increased as 3T3-L1 adipocyte differentiation proceeded. Second, cotransfection of FoxO1-AAA with human resistin promoter (-819~+24) to HEK 293 or 293T cells indicated that FoxO1-AAA dose-dependently inhibited activity of human resistin promoter. Third, cotransfection of FoxO1-AAA with human resistin promoter (-819~+24) to NIH 3T3 cells indicated that FoxO1-AAA does-dependently inhibited activity of human resistin promoter. These results suggest the cell type-independent effect of FoxO1-AAA on human resistin promoter. Fourth, cotransfection of FoxO1-AAA with mouse resistin promoter (-3546~+165 bp, -1145~+165 bp, -805~+165 bp) to HEK 293 or 293T cells showed a downregulatory effect of FoxO1-AAA on mouse resistin promoter. Finally, both the wild type of FoxO1 and the mutant types of FoxO1-AAA and FoxO1-H215R downregulated human resistin promoter activity, indicating an indirect effect of FoxO1 on resistin gene.
Taken together, FoxO1 downregulates human resistin promoter activity. Because FoxO1-AAA upregulated mouse resistin promoter activity in 3T3 cells, FoxO1 may regulate mouse resistin promoter activity in the cell type-dependent manner (adapted from susu).
關鍵字(中) ★ 抗胰島素激素 關鍵字(英) ★ resistin
★ FoxO1
論文目次 中文摘要 ................................................ i
英文摘要 ................................................ ii
謝誌..................................................... iii
縮寫與全名對照表......................................... iv
目錄..................................................... v
圖目錄................................................... ix
一、前言.................................................. 1
1. Resistin ....................................................1
2. FoxO family .................................................3
二、研究動機與目的........................................ 6
三、材料與方法............................................ 8
壹、 實驗材料.....................................................8
1-1. 細胞株..................................................8
1-1-1. 細胞培養..............................................8
1-2. 菌株....................................................8
1-2-1 菌株培養...............................................9
1-2-2 菌株的保存.............................................9
貳、 質體建構.....................................................9
2-1. 質體建構之基本流程......................................9
2-1-1 大腸桿菌勝任細胞之製備.................................9
2-1-2 大腸桿菌的轉型作用.....................................10
2-1-3 質體DNA 的少量製備.....................................10
2-1-4 聚合酶鏈反應...........................................10
2-1-5 聚合酶鏈反應產物的修飾.................................11
2-1-6 插入 (Insert) DNA的純化................................11
2-1-7 載體DNA的製備..........................................11
2-1-8 載體DNA限制酶的剪切....................................12
2-1-9 載體DNA的5'端去磷酸根反應.............................12
2-1-10 載體DNA的純化.........................................12
2-1-11 接合反應 ( Ligation ) ................................13
2-1-12篩選 (Screening) ......................................13
2-1-12-1 核酸限制酶的剪切.....................................13
參、 Human rstn promoter-Luc報告質體之構築.........................14
3-1. Human rstn promoter 0.8kb的製備..........................14
3-1-1 Human rstn p 引子的設計.................................14
3-1-2 聚合酶鏈反應............................................14
3-1-3 載體的製備..............................................14
3-1-4 接合作用與轉型作用......................................14
3-1-5 篩選 (Screening) .......................................14
3-1-5-1 質體DNA 的少量製備....................................15
3-1-5-2 核酸限制酶剪切........................................15
3-1-5-3 純化插入子(insert) ...................................15
3-2. 載體DNA (pGL3 basic)的製備...............................15
3-2-1 pGL3 basic的剪切......................... ...............15
3-2-2 載體DNA (pGL3 basic)的5'端去磷酸根反應..................15
3-2-3 篩選 (Screening) .......................................16
3-2-3-1 質體DNA 的少量製備....................................16
3-2-3-2 核酸限制酶剪切........................................16
3-2-3-3定序(DNA sequencing) ..................................16
肆、 Human rstn promoter於細胞表現之活性...........................16
4-1. 質體pGL3- Human Rstn promoter的轉染......................16
4-1-1 細胞培養................................................17
4-1-2 轉染作用於人類胚胎腎臟細胞株............................17
4-1-3 螢火蟲冷光活性方法......................................17
伍、 利用反轉錄病毒將FOXOs基因表現於哺乳類細胞中..................18
5-1. 反轉錄病毒的製備........................................18
5-1-1 細胞培養................................................18
5-1-2 轉染作用................................................18
5-2. 利用反轉錄聚合酶鏈反應篩選FOXOs反轉錄病毒製備............18
5-2-1 Total RNA的抽取.........................................18
5-2-2 反轉錄酶反應............................................19
5-2-3 聚合酶鏈反應............................................19
5-3. 質體pMSCV-neo-FOXOs的感染................................19
5-3-1 細胞培養................................................19
5-3-2 感染作用................................................19
5-4. 利用反轉錄聚合酶鏈反應篩選FOXOs穩定表達細胞株............20
5-4-1 Total RNA的抽取.........................................20
5-4-2 反轉錄酶反應............................................20
5-4-3 聚合酶鏈反應............................................20
陸、 DAPI將細胞核染色.............................................20
四、實驗結果與討論........................................ 21
1. 在不同分化程度的3T3-L1細胞株中﹐觀察Resistin、FoxO1、FoxO3、FoxO4的mRNA表現量.................................................21
2. 確認FoxO1-WT、FoxO1-AAA、FoxO1-H215R在HEK 293中會大量表現與其位置...........................................................22
3. 在HEK 293 cell中FoxO1-AAA對於Human resistin promoter活性的影響...........................................................22
4. 在HEK 293 cell中FoxO1-AAA 的抑制作用與劑量相關. .............24
5. FoxO1-AAA抑制Human rstn promoter的作用並沒有隨著不同的轉染細胞株而產生差異性.................................................25
6. FoxO1在HEK 293T轉染細胞株內可能藉與轉錄因子交互作用﹐間接影響human resistin promoter的活性.................................26
7. FoxO1-AAA對於Mouse rstn promoter活性的影響.....................27
五、討論....................................................29
六、結論................................................... 32
七、參考文獻 .............................................. 49
圖一、不同分化程度的3T3-L1脂肪細胞中﹐Resistin、FoxO1、FoxO3、FoxO4的mRNA
表現..................................................................33
圖二、轉染之pcDNA-FoxO1-WT、pcDNA-FoxO1-AAA、pcDNA-FoxO1-H215R在HEK 293 cell中表現之相對位置 ................................................34
圖二、Human rstn promoter的架構.......................................35
圖三、C/EBPα與pMSCV-FoxO1-AAA對於Human rstn promoter活性的影響在HEK 293 與 293T細胞中.........................................................36
圖四、FoxO1-AAA抑制 human resistin 活性有劑量相關的影響﹐在 HEK 293 cell 中
......................................................................37
圖五、FoxO1-AAA抑制 human resistin 活性有劑量相關的影響﹐在 NIH 3T3 cell 中 ...................................................................38
圖六、DNA binding deficiency FoxO1-H215R 對於FoxO1抑制 human resistin 活性的作用並無影響﹐在 NIH 3T3 cell 中....................................39
圖七、不同長度Human resistin promoter的構築............................40
圖八、FoxO1-WT,FoxO1-AAA ,FoxO1-H215R對不同長度Human rstn promoter片段活性的影響 ...........................................................41
圖九、Mouse resistin promoter 序列以及FoxO1結合位在此序列中的可能結合位IRS ..................................................................42
圖十、不同長度Mouse resistin promoter的構築............................43
圖十一、FoxO1-AAA抑制Mouse rstn promoter ( 3.8 KB )活性的影響有劑量相關性,在HEK 293 cell 中..................................................44
圖十二、FoxO1-WT,FoxO1-AAA,以及FoxO1-H215R皆抑制 Human and Mouse resistin promoter 的活性﹐在HEK 293T cell 中....................................45
圖十三、Human and Mouse resistin promoter 在不同細胞株中的活性與受FoxO1的調節作用............................................................46
圖十四、在HEK 293T中 ﹐各種長度片段的 human and Mouse resistin promoter 。活性..................................................................47
圖十五、human resistin promoter 上有著許多可調節其表現的轉錄因子結合位
.....................................................................48
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指導教授 高永旭(Yung-Hsi Kao) 審核日期 2008-7-24
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