探討大量表現 FoxO6 對肌肉終極分化的影響以及尋找 FoxO6 蛋白質在 PGC-1 alpha 啟動子上的結合位

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李冠緯(Kuan-Wei Lee) 查詢紙本館藏

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生命科學系

論文名稱

探討大量表現 FoxO6 對肌肉終極分化的影響以及尋找 FoxO6 蛋白質在 PGC-1 alpha 啟動子上的結合位
(Effects of FoxO6 over-expression on terminal myogenic differentiation and defining the FoxO6- binding site in the PGC-1 alpha promoter)

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摘要(中)

Forkhead box O (FoxO)轉錄因子能夠調節細胞中各種生理功能，包含細胞週期、葡萄糖代謝、壓力反應、細胞分化以及細胞的死亡與存活機制等。在2003年發現了一個新的Forkhead家族成員-FoxO6。由於它缺少了一些重要的胺基酸使得進出細胞核的能力受到影響，其中最重要的是缺少了C端保守的PKB可磷酸化胺基酸，這個位置是FoxO成員主要移出核的調控區域，也因此FoxO6會比其它FoxO成員更容易滯留在細胞核中。雖然目前研究發現，FoxO6主要表現在腦部組織，但在肌肉中也有發現FoxO6的表現，而且FoxO6對於骨骼肌的功能目前尚未清楚，因此FoxO6對骨骼肌的影響成為我們主要的研究目標。肌肉中另一個重要因子-PGC-1 alpha，它是一個PPAR-gamma?的輔助轉錄因子能夠與 PPAR-gamma 相互作用，在粒線體的代謝中更扮演一個非常重要的角色，若是在肌肉中大量表現PGC-1 alpha?會促使肌肉的型態從TypeII肌纖維變成TypeI肌纖維。從我們的研究中發現大量表現FoxO6的穩定細胞株C2C12-mFoxO6比起控制組更能降低肌管的數量及大小，這表示FoxO6能夠抑制肌肉的分化。於是我們推測也許會干擾細胞週期來影響肌肉細胞的分化。然而在流式細胞儀分析實驗中，confluent時期的C2C12-mFoxO6也能與控制組一樣停留在G0/G1期。此外，我們發現了過量表現FoxO6會抑制PGC-1 alpha?基因的表現。於是我們推測FoxO6可能可以直接調控PGC-1 alpha?啟動子的轉錄活性。在啟動子活性試驗中，發現PGC-1 alpha 啟動子的活性會受到FoxO6的調控，而利用EMSA以及Footprinting也證實了FoxO6能夠直接結合在PGC-1?alpha 啟動子上，而在未來，我們希望能利用chip試驗來加以印證FoxO6與PGC-1 alpha?啟動子的關係。

摘要(英)

Forkhead box O (FoxO) transcription factors could regulate many cellular functions, including cell cycle, glucose metabolism, stress response, cell differentiation, cell death and survival. FoxO6, one of the forkhead family, was discovered in 2003. It was different from other FoxO factor because FoxO6 remained mostly nuclear due to its impaired shuttling ability . It lacked the conserved C-terminal PKB motif, which was the cause of the shuttling impairment. Although FoxO6 is majorly expressed in developing brain, it is also expressed in muscle and its function in skeletal muscle was unknown. PGC-1 alpha??was a transtcription coactivator that interacted with PPAR-gamma?and?played a key role in mitochondrial metabolism. When PGC-1 alpha?was?overexpressed in muscle, it transformed type2 myofiber into type1 myofiber. Our study had discovered that C2C12-mFoxO6 cell line could decrease the number and size of myotube compared with C2C12-control. We supposed that FoxO6 may be interfere with cell cycle to inhibit myogenic differentiation. However in flow cytometer assay, confluent C2C12-mFoxO6 myoblast also could majorly stay at G0/G1 phase during cell cycle, it meaned that C2C12-mFoxO6 myoblast also had normal cell cycle exit. We also found that overexpression of FoxO6 inhibited expression of PGC-1 alpha gene in confluent C2C12 myoblast. Thus, we supposed that FoxO6 may regulate PGC-1 alpha promoter activity. By using reporter assay we demonstrated that FoxO6 could inhibit the PGC-1?alpha?promoter activity. Then, we supposed FoxO6 may bind directly to PGC-1?alpha promoter. By EMSA and footprinting we demonstrated that FoxO6 really could bind to PGC-1?alpha?promoter. In the future, we hope to confirm the binding site by chromatin immunoprecipitation assy.

關鍵字(中)

★ 肌肉終極分化

關鍵字(英)

★ PGC-1 alpha
★ FoxO6

論文目次

致謝 I
中文摘要 II
ABSTRACT III
目錄 IV
第一章緒論 1
一肌肉的起源 1
二FoxO (Forkhead box O) 2
三PGC-1 (Peroxisome proliferator-activated receptor?? co-activator 1) 5
四研究動機與目的 7
第二章實驗方法與材料 9
2-1細胞株與細胞培養 9
2-2質體建構 9
2-3 聚合酶連鎖反應(Polymerase Chain Reaction, PCR) 10
2-3-1 PCR反應條件: 10
2-3-2 載體的製備 11
2-3-3 插入(insert)DNA純化 11
2-3-4 接合反應(ligation) 12
2-3-5 大腸桿菌的轉型作用(transformation) 12
2-4 pPyCAGIP-FoxO6-flag穩定細胞株製備 12
2-4-1 質體pPyCAGIP-FoxO6-flag製備 12
2-4-2 細胞培養 13
2-4-3 轉染作用 (Transfection) 13
2-4-4 篩選穩定細胞株 13
2-5 RT-PCR 13
2-5-1 Total RNA 製備 13
2-5-2 反轉錄酶反應(Reverse Transcriptase, RT) 14
2-6 免疫染色 (Immunohistochemistry) 14
2-7 流式細胞儀分析 15
2-8 啟動子活性分析 (promoter assays) 16
2-8-1 轉染作用(trasnsfection) 16
2-8-2 螢火蟲冷光活性方法 ( Luciferase Activity Assay ) 16
2-9 EMSA (Electrophoresis Mobility Shift Assay) 16
2-10 Footprinting 18
第三章實驗結果 20
3-1 探討 Insulin 對 C2C12-FoxO6s 穩定細胞株分化的影響 20
3-2 觀察過量表現FoxO6s的肌纖維母細胞對細胞週期的影響 21
3-3 大量表現FoxO6的穩定細胞株對PGC-1α表現量的影響 21
3-4 FoxO6轉錄因子在肌纖維母細胞對於PGC-1?啟動子的影響 22
3-5 尋找PGC-1? 啟動子上是否有FoxO6的結合位 22
3-6 利用Footprinting來定序PGC-1? 啟動子上FoxO6的結合位 23
3-7 C2C12 PyCAGIP-FoxO6-flag穩定細胞株分化的情形 23
第四章討論 25
4-1 在C2C12中大量表現FoxO6對肌肉分化的影響 25
4-2 FoxO6與PGC-1? 之間的關係對於肌肉分化可能的影響 26
第五章参考文獻 29
第六章圖表 33
圖一、將大量表現 FoxO6 的穩定細胞株：C2C12-FoxO6 wt、C2C12-FoxO6 S184A、C2C12-FoxO6 T26A 做肌肉分化實驗，並處理 insulin，探討 FoxO6 是否影響分化。 36
圖二、將大量表達 FoxO6 的穩定細胞株：C2C12-FoxO6 wt、C2C12-FoxO6 S184A、C2C12-FoxO6 T26A 分別收取 PMB、CMB 時期，利用流式細胞儀來觀察 FoxO6 對細胞週期的影響。 38
圖三、定量 RT-PCR 觀察大量表現 FoxO6 的穩定細胞株中 PGC-1?的表現量 41
圖四、在 C2C12 中轉染 mPGC-1 ?啟動子以及 pMSCV-FoxO6，觀察 FoxO6 是否調控 mPGC-1 ???啟動子。 43
圖五、利用 EMSA 來尋找 mPGC-1?? 啟動子上是否有 FoxO6 的結合位 47
圖六、利用 Footprinting 分析 mPGC-1? 啟動子上 FoxO6 的結合位 49
圖七、將 C2C12 PyCAGIP-Vector 與 C2C12 PyCAGIP-FoxO6-flag 穩定細胞株進行肌肉分化實驗，並處理 insulin 觀察。 51
附錄一 52
圖一、利用免疫染色觀察 C2C12 control 在處理 insulin 後的分化形態 (2009年方彥心研究資料) 53
圖二、2008年黃暐捷研究資料 54
附錄二 55
Primer對照表 55
附錄三 57
縮寫與全名對照表 57
附錄四 59
I 溶液及試劑配方 59
II 藥品試劑 62
III 酵素和限制酶 62

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2010-7-29

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