探討FoxO1在肌肉生成細胞中的表現位置變化及抑制肌肉細胞分化的機制

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研究生：	吳怡儒 Yi-Ju Wu
論文名稱：	探討FoxO1在肌肉生成細胞中的表現位置變化及抑制肌肉細胞分化的機制 Defining the intra-cellular localization of FoxO1 and its implication in the inhibition of myogenesis
指導教授：	陳盛良 Shen-Liang Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
畢業學年度：	99
語文別：	中文
論文頁數：	65
中文關鍵詞：	肌肉分化
外文關鍵詞：	myogenesis, differentiation, FoxO1, MRF family
相關次數：	點閱：8 下載：0
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Forkhead box protein 1 (簡稱FoxO1) 又稱為FoxO1，屬於Forkhead family蛋白質成員中的〝O〞subclass。FoxO1為轉錄因子(transcription factor) 在結構上具有一段高保留性的forkhead DNA binding domain (DBD)，此DNA binding domain包含三個α-helix及三個β-strand及兩個wings；其中α-helix負責辨識並接觸到DNA的major groove。FoxO1可調控細胞的生長、繁殖、肌肉細胞的分化及壽命。FoxO1會與Pax3及Pax7發生染色體異位，而產生Pax3-FoxO1和Pax7- FoxO1融合蛋白；此功能類似致癌蛋白的融合蛋白會誘使正常的細胞轉型成癌細胞、抑制肌肉細胞分化及抑制細胞凋亡。為了要探討FoxO1與肌肉細胞分化的關係，我們觀察大量表現FoxO1的穩定細胞株C2C12- FoxO1，發現FoxO1有抑制細胞分化的能力；並且在肌肉分化過程中會表現的調控因子MyoD、Myogenin及Myf5表現也被抑制。在本篇研究利用GFP-FoxO1觀察到在尚未分化的肌肉細胞中，FoxO1表現累積在細胞核；但在開始表現MHC的單顆細胞中發現FoxO1移動到細胞質，並且在已分化的肌管中FoxO1也表現在細胞質。表示當細胞分化前FoxO1在細胞核內進行轉錄功能；直到細胞開始分化時，FoxO1移動到細胞質，因此推測已失去轉錄能力。接著透過免疫螢光染色持續觀察肌肉細胞分化過程中FoxO1的移動方式，發現在更換分化培養液或加入insulin及LiCl刺激細胞分化的藥劑時，FoxO1表現量會先減少並且轉移到細胞質中；但在8小時後FoxO1便漸漸的聚集回細胞核，直到細胞分化成肌管才又送出至細胞質中。由實驗室之前的研究指出，當FoxO1大量表現時會抑制細胞中MyoD 的表現，因此我們更近一步利用過渡性轉染實驗得知，FoxO1 可能作用於MyoD promoter而抑制其表現。比較持續表現FoxO1的C2C12 FoxO1-AAA和C2C12的葡萄糖攝取，得知FoxO1會減少細胞因insulin的刺激而影響葡萄糖攝取，且葡萄糖輸送蛋白GLUT4的表現量也會受到抑制。未來將著重於FoxO1對於MyoD下游基因的影響分析。

FoxO1 belongs to the forkhead family that bind to their target sites by their forkhead DNA-binding domain. FoxO1 has been shown to play important roles in the regulation of cell growth, proliferation, differentiation, and longevity. Its chimeric fusion with Pax3 or Pax7, generating either Pax7-or Pax3- FoxO1, has been demonstrated as strong inhibitors of muscle cell differentiation and apoptosis. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which led to a consistently inhibitory effect on myogenic differentiation. The expression of myogenic regulatory factors, including MyoD, Myogenin and Myf5, was significantly reduced in C2C12-FoxO1 cells. In this study, we observed constitutive nuclear accumulation of FoxO1 in differentiating myoblasts. However, simultaneous nuclear exclusion of FoxO1 and MHC expression was observed in mononucleated cells ready for fusion into multinucleated myotubes. FoxO1 remains cytoplasmic in multinucleated myotubes and GPF-FoxO1 introduced into myotubes also stays in the cytoplasm, implying that nuclear exclusion of FoxO1 is prerequisite for terminal differentiation. The nuclear exclusion process of FoxO1 can be mimicked by insulin treatment, but they return to the nucleus 8 hr after the treatment. To further elucidate the roles of FoxO1 in myogenesis and its possible implication in tumorigenesis, FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which strongly inhibited myogenic differentiation. Both insulin treatment and over-expression of MyoD partially rescued myogenic differentiation of C2C12-FoxO1 cells, implying that direct targeting of FoxO1 on myogenic genes, including MyoD, is required for its inhibitory effect. In silico analysis and transient transfection promoter assays have identified putative FoxO1-binding sites in the 24 kb upstream regulatory region of MyoD. These putative FoxO1-binding sites will be confirmed by EMSA and chromatin immunoprecipitation assay. Our results suggest that FoxO1 inhibits myogenesis by direct targeting the regulatory elements of myogenic genes.

目 錄
中文摘要-------------------------------------------------------------------------------------------------------------- I
ABSTRACT --------------------------------------------------------------------------------------------------------- II
聲明(DECLARATION) ----------------------------------------------------------------------------------------- III
誌謝------------------------------------------------------------------------------------------------------------------IV
目錄-------------------------------------------------------------------------------------------------------------------V
縮寫與全名對照表----------------------------------------------------------------------------------------------VIII
藥品及材料...................................................................................................................................VIII
第一章、緒論------------------------------------------------------------------------------------------------------- 1
1-1 . FOXO1 (FORKHEAD BOX PROTEIN 1)：........................................................................................1
1-2. 肌肉的起源與生成(MYOGENESIS):............................................................................................5
1-3.MYOD (MYOGENIC DIFFERENTIATION FACTOR 1):........................................................................6
1-4.葡萄糖輸送蛋白(GLUCOSE TRANSPORTER):................................................................................6
1-5.泛素連接酶(UBIQUITION LIGASE):...............................................................................................7
1-6. 研究動機與目的: ........................................................................................................................8
第二章、實驗材料與方法---------------------------------------------------------------------------------------- 9
Ⅰ. 實驗材料......................................................................................................................................9
1. 細胞株----------------------------------------------------------------------------------------------------- 9
2. 菌株-------------------------------------------------------------------------------------------------------- 9
II. 質體建構.......................................................................................................................................9
1. 質體建構方式-------------------------------------------------------------------------------------------- 9
2. 構築載體的修飾----------------------------------------------------------------------------------------10
3. p-Stable-MyoD601514 ----------------------------------------------------------------------------------10
4. p-Stable-MyoD60-EM----------------------------------------------------------------------------------- 11
5. p-Stable-MyoD60-enhancer (human)------------------------------------------------------------------ 11
6. p-Stable-MyoD601514-enhancer (human)------------------------------------------------------------ 11
7. p-Stable-human Atrogin-1 promoter------------------------------------------------------------------- 11
8. p-Stable-mouse MuRF1 promoter --------------------------------------------------------------------- 11
9. pPyCAGIP-GFP- FOXO1-wt -------------------------------------------------------------------------- 11
10. p-Stable-mouse Atrogin-1 promoter------------------------------------------------------------------ 11
11. p-Stable-mouse Glut4 promoter ----------------------------------------------------------------------12
12.聚合酶連鎖反應(Polymerase Chain Reaction, PCR) --------------------------------------------12
III. 轉染作用 (TRANSFECTION) ........................................................................................................12
1. 穩定細胞株的製備-------------------------------------------------------------------------------------12
VI
2. 過渡性轉染實驗----------------------------------------------------------------------------------------12
Ⅳ. 螢火蟲冷光活性方法( LUCIFERASE ACTIVITY ASSAY ) ............................................................13
Ⅴ. 葡萄糖攝取實驗(GLUCOSE UPTAKE) ........................................................................................13
Ⅵ. 反轉錄酶反應(REVERSE TRANSCRIPTASE, RT).........................................................................14
1. Total RNA 製備-----------------------------------------------------------------------------------------14
2. 反轉錄酶反應(Reverse Transcription) -------------------------------------------------------------14
3. Real-time PCR--------------------------------------------------------------------------------------------15
Ⅶ螢光免疫染色(IMMUNOFLUORESCENCE) .....................................................................................15
Ⅷ 細胞質與細胞核蛋白的抽取......................................................................................................16
1. 質核蛋白的抽取----------------------------------------------------------------------------------------16
2. 蛋白質透析----------------------------------------------------------------------------------------------16
3. 蛋白質定量----------------------------------------------------------------------------------------------17
Ⅸ 西方墨點轉漬法(WESTERN BLOT) .............................................................................................17
第三章、實驗結果------------------------------------------------------------------------------------------------18
Ⅰ FOXO1可降低細胞受到INSULIN刺激的葡萄糖攝取反應。......................................................18
Ⅱ 比較C2C12 CONTROL及C2C12 FOXO1-AAAMONOCLONE 5 在CMB與MT時期，葡萄糖輸送蛋白表現量差異。...............................................................................................................................18
Ⅲ FOXO1可抑制MYOD PROMOTER的轉錄活性。..........................................................................19
Ⅳ 探討長時間細胞分化過程中，FOXO1的分布位置。.............................................................20
Ⅴ 觀察處理 INSULIN 及LICL 細胞中，FOXO1隨著時間的變化其存在位置的改..................20
變20
Ⅵ FOXO1的轉錄活性對其他PROMOTER的影響。.........................................................................22
第四章、討論------------------------------------------------------------------------------------------------------23
Ⅰ FOXO1對肌肉細胞葡萄糖攝取的影響。..................................................................................23
Ⅱ FOXO1抑制細胞分化與MYOGENIC FACTORS的關係。.............................................................23
Ⅲ 持續時間觀察FOXO1在細胞分化各階段表現位置。.............................................................24
Ⅳ 探討FOXO1的轉錄活性對其他PROMOTER的影響。................................................................25
Ⅴ 結論.............................................................................................................................................26
第五章、圖表 ----------------------------------------------------------------------------------------------------27
圖一、大量表現FOXO1的穩定細胞株C2C12 FOXO1-AAA較後期代數分別處理INSULIN及LICL， 分化四天後測量葡萄糖攝取的差異。..............................................................................28
圖二、C2C12 CONTROL及大量表現FOXO1的穩定細胞株C2C12 FOXO1-AAAMONOCLONE 5較前期代數分別處理INSULIN及LICL，分化四天後測量葡萄糖攝取的差異。...............................31
圖三、將C2C12 CONTROL及C2C12 FOXO1-AAAMONOCLONE 5以2% HS處理刺激分化四天後，比較GLUT1與GLUT4 MRNA表現量。.............................................................................................32
圖四、構築P-STABLE-MYOD601514-ENHANCER (HUMAN) 質體。.................................................34
VII
圖五、FOXO1-WT與MSX1轉錄活性對MYOD PROMOTER的影響。.............................................35
圖六、大量表現FOXO1-WT的穩定細胞株C2C12 FOXO1-WT在2% HS中加入INSULIN及LICL刺激分化四天後，以西方墨點法與C2C12 CONTROL比較細胞核中FOXO1表現量。........................36
圖七、C2C12 CONTROL細胞培養至CMB後，更換2% HS並處理INSULIN及LICL刺激分化，利用免疫螢光染色觀察兩天及四天細胞中FOXO1的表現位置。.......................................................38
圖八、C2C12 FOXO1-WT細胞培養至CMB後， 更換2% HS並處理INSULIN及LICL刺激分化， 利用免疫螢光染色觀察兩天及四天細胞中FOXO1的表現位置。...................................................40
圖九、利用免疫螢光染色觀察C2C12 FOXO1-WT在PMB及CMB時期，FOXO1在細胞中表現位置。..................................................................................................................................................41
圖十、利用免疫螢光染色觀察C2C12 FOXO1-WT，在2% HS中隨著時間FOXO1的表現位置。43
圖十一、利用免疫螢光染色觀察C2C12 FOXO1-WT，在2% HS加入LICL 10 MM後，FOXO1隨著時間表現位置的變化。...............................................................................................................45
圖十二、利用免疫螢光染色觀察C2C12 FOXO1-WT，在2% HS加入INSULIN 50 MM後，FOXO1隨著時間表現位置的變化。...........................................................................................................47
圖十三、C2C12 FOXO1-WT細胞經2% HS (加入INSULIN 50 NM) 刺激分化三天後，再加入INSULIN 50 NM。利用免疫螢光染色觀察，FOXO1隨著時間表現位置的變化。.......................49
圖十四、將PPYCAGIP-GFP-FOXO1-WT轉染到已分化的C2C12 MYOTUBE。............................51
圖十五、老鼠小腿肌(GASTROCNEMIUS MUSCLE) 切片FOXO1表現位置。................................52
圖十六、質體P-STABLE-HATROGIN-1 PROMOTER與P-STABLE-MMURF1 PROMOTER的構築。.......53
圖十七、FOXO1-WT轉錄活性對ATROGIN-1及MURF1 PROMOTER的影響。...............................54
第六章、參考文獻 ----------------------------------------------------------------------------------------------55
附錄一 -------------------------------------------------------------------------------------------------------------59
附錄二 -------------------------------------------------------------------------------------------------------------60
附錄三 -------------------------------------------------------------------------------------------------------------61
附錄四 -------------------------------------------------------------------------------------------------------------62
Ⅰ. 溶液及試劑配方.........................................................................................................................62
Ⅱ. 藥品試劑....................................................................................................................................64
Ⅲ. 酵素和限制酶.............................................................................................................................64
Ⅳ. 抗體............................................................................................................................................64
附錄五 -------------------------------------------------------------------------------------------------------------65
建構質體PRIMER 對照表................................................................................................................65
REAL-TIME PCR PRIMER 對照表......................................................................................................65
BAC CLONE對照表 ............................................................................................................................65

                                

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