FOXO1 轉錄因子刺激小鼠抗胰島素激素基因啟動子的活性

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姓名

楊淑雅(Shu-ya yang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

FOXO1 轉錄因子刺激小鼠抗胰島素激素基因啟動子的活性
(The forkhead transcription factor FOXO1 stimulates activity of mouse resistin gene promoter)

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摘要(中)

抗胰島素激素是一種由脂肪細胞所分泌的多胜肽荷爾蒙，會阻抗胰島素作用及減低脂肪細胞的分化過程，並且會被轉錄因子所調節，但轉錄因子中，FOXO1是否會調節抗胰島素激素基因的表現尚未被證實，所以本論文利用NIH3T3纖維母細胞、HEK293T人類腎胚細胞，及C3H10T1/2前脂肪細胞進行實驗，發現未被磷酸化且持續活化型FOXO1AAA在細胞中短暫大量表達時，會隨劑量升高時而逐漸增強活化抗胰島素激素驅動子所架構的冷光蛋白的表現，然而野生型FOXO1或是DNA結合位變異型FOXO1H215R在細胞中短暫大量表達時，幾乎無法促進冷光蛋白的表現；另外，將顯性抑制型FOXO1△256在細胞中短暫大量表達時，會降低抗胰島素激素驅動子的活性。FOXO1轉錄蛋白在抗胰島素激素驅動子上結合位大約是在從5’端算來第-3546到-788鹼基位置上，此外，持續活化型FOXO1AAA和持續活化型FOXO4AAA轉錄因子會比FOXO3AAA和FOXO6T26A或FOXO6S184A轉錄因子有較強的活化效果。經由凝膠遷移滯後實驗發現，glutathione-S-transferase-FOXO1融合蛋白會直接與抗胰島素激素驅動子的-1436到-1387及-936到-697核苷酸區域的位置相結合。另外，在細胞生理狀態之下，隨著C3H10T1/2及3T3-L1前脂肪細胞分化轉變為脂肪細胞的階段，抗胰島素激素及FOXO1的核醣核酸表現量都會有增加的趨勢。綜合以上所述，顯示FOXO1轉錄因子似乎會透過抗胰島素激素的驅動子而正調節脂肪細胞中抗胰島素激素的基因表現。

摘要(英)

Resistin is known as an adipocyte-specific secretory hormone that can cause insulin resistance and decrease adipocyte differentiation. It can be regulated by transcriptional factors, but the possible role of the forkhead transcription factor FOXO1 in regulating resistin gene expression is still unknown. Using NIH3T3 fibroblast, HEK293T, and C3H10T1/2 preadipocytes, we found that transient overexpression of a non-phosphorylatable, constitutively active FOXO1AAA activated resistin promoter-directed luciferase expression in a dose-dependent manner. Whereas, transient overexpression of either the wild type of FOXO1 or a DNA binding-deficient FOXO1H215R mutant was much less to increase luciferase expression. However, transient overexpression of a dominant-negative FOXO1△256 tended to inactivate resistin promoter activity. The FOXO1 protein target sites on resistin promoter were localized to the proximal -3546 to -788 bp of 5’-flanking region of the resistin promoter.In addition, FOXO1AAA and FOXO4AAA were more effective than FOXO3AAA and FOXO6T26A or FOXO6S184A in stimulating the resistin promoter-directed luciferase expression. Electrophoretic mobility shift assay indicated that glutathione-S-transferase-FOXO1 protein directly bound the nucleotide regions of resistin promoter at -1436 to -1387 and at -936 to -697. In vivo, levels of resistin mRNA and FOXO1 mRNA increased during differentiation of 3T3-L1 preadipocytes to adipocytes. These data suggest that FOXO1 transcription factor likely upregulates expression of preadipocyte resistin gene via activation of the upstream resistin promoter.

關鍵字(中)

★ 小鼠抗胰島素激素
★ 轉錄因子

關鍵字(英)

★ mouse resistin gene promoter
★ FOXO1

論文目次

壹、中文摘要 i
貳、英文摘要 ii
参、致謝 iii
肆、縮寫與全名對照表 iv
伍、目錄 v
陸、表目錄 viii
柒、圖目錄 ix
捌、正文 1
一、前言 1
1. 抗胰島素激素(Resistin) 1
2. FOXO轉錄因子(FOXO transcription factor) 2
3. 研究動機與目的 4
二、材料與方法 4
1. 細胞株 6
2. 細胞培養 6
3. pGL3 mRSTN promoter質體建構 6
3-1 菌株的培養 6
3-2 聚合酶鏈鎖反應 6
3-3 嵌入DNA的純化 7
3-4 載體DNA限制酶的剪切 7
3-5 接合反應 8
3-6 大腸桿菌的轉型作用 8
3-7 質體DNA 的少量製備 8
3-8 質體DNA的大量製備 9
4. Mouse resistin promoter於細胞表現之活性 10
4-1 轉染作用於老鼠纖維母細胞 10
4-2 甲蟲及水母冷光活性方法 11
5. 凝膠遷移滯後實驗 11
5-1 聚合酶鏈鎖反應 11
5-2 探針的標定 11
5-3 探針的純化 12
5-4 Binding assay 12
6.共軛焦雷射顯微鏡(Laser Confocal) 13
6-1 細胞培養 13
6-2 DAPI細胞核染色封片 13
7. 核內蛋白質萃取(Nuclear extract) 14
三、實驗結果 15
1. 建構不同片段的mRSTN promoter於含有Luciferase reporter基因質體上 15
2. C/EBPα會活化mRSTN promoter的活性，FOXO1會活化IGFBP-1 promoter
的活性 15
3. 持續活化型FOXO1AAA會活化mRSTN promoter的活性 15
4. 證明FOXO1AAA在細胞核內持續表達 16
5. 不同型FOXO轉錄蛋白差異性調節mRSTN promoter之活性 17
6. FOXO1AAA調節Mouse resistin promoter或human resistin promoter會因轉染細胞的種類而有差異 18
7. 利用EMSA確認FOXO1的蛋白質是否會與Resistin啟動子直接結合 18
四、討論 20
五、結論與未來展望 24
六、圖表 25
七、參考文獻 54
捌、附錄 57
附錄一 57
附錄二 58
附錄三 60
附錄四 62
附錄五 63

參考文獻

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指導教授

高永旭(Yung-hsi Kao)

審核日期

2010-1-5

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