博碩士論文 992204028 詳細資訊




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姓名 王筱雯(Xiao-wen Wang)  查詢紙本館藏   畢業系所 生命科學系
論文名稱 尋找MyoD啟動子上Wnt3a訊號路徑的目標區域並探討FoxO1在肌肉分化過程中所扮演的角色
(Identifying the Wnt3a signaling pathway targeted regions in MyoD promoter and the role of FoxO1 in Myogenesis.)
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摘要(中) 在骨骼肌發育過程中,MyoD是決定肌肉品系形成的重要轉錄因子。MyoD只會表現在骨骼肌肉細胞以及肌肉前驅細胞中。它屬於myogenic basic helix-loop-helix transcription factors (MRFs)家族的一員,此家族是由體節鄰近的組織,像是神經索及神經管所分泌的訊號分子所誘導表現的。Wnt訊號分子即是其中的一個誘導分子,可以在肌纖維母細胞發現它。Wnt家族是會被分泌到細胞外的醣蛋白,它的訊號傳遞路徑在多細胞生物的發育過程中扮演相當重要的角色。關於Wnt訊號分子,特別是Wnt3a 分子,在胚胎發育過程中,會誘導MRFs的表現已經被清楚的知道,但是Wnt的訊號傳遞路徑如何去調控MRFs的轉錄活性目前還沒有被研究。本研究中,我們發現Wnt3a可以調控MyoD的表現以及MyoD promoter的活性,因此為了去找尋MyoD promoter上游Wnt3a所誘發相關因子的目標結合位,我們分別將MyoD promoter上游的片段,與帶有MyoD 6k promoter及遠端core enhancer(-25k~-20k)的載體進行接合,利用這些建構載體去檢視由293T所釋放出來的Wnt3a是否可以調控MyoD promoter的活性。結果發現在有Wnt3a的情況下,MyoD core enhancer會促進MyoD 6k promoter的活性,並且MyoD上游約-9Kb到-8Kb區域也會促進MyoD 6k promoter的活性,其餘的片段則發現會抑制
MyoD 6k promoter的活性。未來,由Wnt訊號分子誘發參與調控MyoD表現的因子也將會做進一步研究。
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可調控細胞的生長、繁殖、肌肉細胞的分化及壽命。在大量表現FoxO1的穩定細胞株C2C12-FoxO1,發現FoxO1有抑制細胞分化的能力。為了要探討FoxO1與肌肉細胞分化的關係,透過免疫螢光染色並處理Leptomycin B這個出核運輸抑制劑,觀察肌肉細胞分化過程中FoxO1的移動方式。結果發現在PMB時期,FoxO1是從核內不斷運送至核外,CMB時期FoxO1則是持續停留在核內。在尚未分化的肌肉細胞中,FoxO1表現累積在細胞核;但在單顆細胞中發現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,推測已失去轉錄能力。認為FoxO1肌肉分化過程中所扮演的角色和其存在位置有著相當重要的關聯性。
摘要(英) In skeletal muscle, the terminal differentiation is initiated by the muscle-specific transcription factor MyoD. MyoD belongs to the family of myogenic basic helix-loop-helix transcription factors (MRFs) that are expressed in response to inductive signals coming from tissues adjacent to somites, such as notochord and the neural tube. Wnt signals are among these inductive signals and they are also released by myoblasts. Although it is clear that Wnt, especially Wnt3a, signaling events result in the expression of MRFs during embryogenesis, the mechanistic role for Wnt signaling in the regulation of MRFs expression has not yet been established. In this study we have found that Wnt3a can regulate MyoD expression and its promoter activity. To identify the Wnt3a-targeted region in the MyoD control region, I have inserted different upstream regions of MyoD gene into a vector that contains the MyoD 6k promoter and a distal enhancer (-20k~-25k) to test their response to 293T cells secreted Wnt3a. We found that MyoD 6k promoter is responsive to Wnt3a signal when the distal enhancer exsit. The -9k~-8k elements of upstream regions of MyoD gene have been found that activate the activity of MyoD promoter The other elements of upstream regions of MyoD gene have been found that inhibit the activity of MyoD promoter. In the future, factors mediating the activation effect of Wnt3a signaling on MyoD expression will also be screened.
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. FoxO1 was over-expressed in C2C12 myoblasts (C2C12-FoxO1) and which led to a consistently inhibitory effect on myogenic differentiation. To further elucidate the roles of FoxO1 in myogenesis, Immunofluorescence was used to observed nuclear accumulation of FoxO1 in differentiating myoblasts. Besides, we use the Leptomycin B that inhibitor of nuclear export to observed the localization of FoxO1.We found that FoxO1 was transported from nucleus to cytoplasm in PMB stage, stayed in the nucleus in CMB stage and the expression of FoxO1 was observed in mononucleated cells ready for fusion into multinucleated myotubes. FoxO1 remains cytoplasmic in multinucleated myotubes, implying nuclear exclusion of FoxO1 is prerequisite for terminal differentiation.
關鍵字(中) ★ MyoD啟動子
★ Wnt3a訊號路徑
★ FoxO1
關鍵字(英) ★ FoxO1
★ Wnt3a signaling pathway
★ MyoD promoter
論文目次 聲明-------------------------------------------------------------------------------------------------------------------I
中文摘要 ------------------------------------------------------------------------------------------------------------VI
ABSTRACT --------------------------------------------------------------------------------------------------------VII
誌謝 ----------------------------------------------------------------------------------------------------------------VIII
目錄 ------------------------------------------------------------------------------------------------------------------IX
縮寫與全名對照表 ----------------------------------------------------------------------------------------------XIII
第一章、緒論 ------------------------------------------------------------------------------------------------------- 1
一、肌肉的起源------------------------------------------------------------------------------------------------- 1
二、肌肉的形成--------------------------------------------------------------------------------------------------2
三、MRF (muscle regulatory factors) 與肌肉細胞的發育與分化---------------------------------3
四、MyoD (Myogenic Determination Factor)------------------------------------------------------------ 5
五、調控MRFs的上游機制------------------------------------------------------------------------------------7
六、The Wnt family:------------------------------------------------------------------------------------------ 8
七、FoxO1 (Forkhead box protein 1)-------------------------------------------------------------------- 10
八、研究動機與目的-------------------------------------------------------------------------------------------11
第二章、實驗材料與方法 ----------------------------------------------------------------------------------------13
Ⅰ. 實驗材料----------------------------------------------------------------------------------------------------13
1. 細胞株 ------------------------------------------------------------------------------------------------ 13
2. 菌株 --------------------------------------------------------------------------------------------------- 13
II. 質體建構 -------------------------------------------------------------------------------------------------- 14
1.質體建構流程圖-------------------------------------------------------------------------------------- 14
1.1載體DNA製備-------------------------------------------------------------------------------------- .14
1.2載體DNA限制酶剪切------------------------------------------------------------------------------- 15
1.3載體DNA的5’端修飾----------------------------------------------------------------------------- 15
1.4載體DNA的純化------------------------------------------------------------------------------------- 16
1.5聚合酶連鎖反應 (Polymerase Chain Reaction ,PCR)------------------------------------- 17
1.6聚合酶反應產物修飾------------------------------------------------------------------------------ 18
1.7插入 (Insert) DNA的純化----------------------------------------------------------------------- 18
1.8接合反應 (Ligation)---------------------------------------------------------------------------- 19
1.9大腸桿菌的轉型作用 (Transformation)---------------------------------------------------- 19
1.9-1大腸桿菌勝任細胞之製備 (Preparation of E.coli competent cells)---------- 19
1.10質體DNA的少量製備 (Mini-preparation)------------------------------------------------ 20
1.11 篩選 (Screening)----------------------------------------------------------------------------------.20
III.轉染作用 (Transfection)-------------------------------------------------- 20
1.穩定細胞株的製備-------------------------------------------------------20
2. 過渡性轉染實驗-------------------------------------------------------.21
Ⅳ. 螢火蟲冷光活性方法 ( Luciferase Activity Assay )--------------------------21
V. 反轉錄酶反應 (Reverse Transcriptase, RT)---------------------------------..22
1. Total RNA 製備-------------------------------------------------------..22
2. 反轉錄酶反應 (Reverse Transcription)-----------------------------------22
3. Real-time PCR----------------------------------------------------------23
VI. 西方墨點轉漬法 (western blot)-------------------------------------------- 23
1.Total lysate的製備-----------------------------------------------------23
2.SDS-polyacrylamide Gel Electropheresis----------------------------------24
3.Transfer---------------------------------------------------------------24
4.Blocking 以及 Antibody 辨識--------------------------------------------24
5.Striping---------------------------------------------------------------25
Ⅶ 螢光免疫染色 (immunofluorescence) ----------------------------------------25
第三章 實驗結果-----------------------------------------------------------------27
I.收集含有Wnt3a protein 的medium 並測試Wnt3a的功能-------------------------27
II.293T-pyWnt3a medium在C2C12 CMB時期會增加MyoD的表現量,另外同時處理Wnt3a和RA 並不會增加MyoD表現量---------------------------------------------------28
III. 293T-pyWnt3a medium 對mouse MyoD 6k promoter及MyoD 4.5k core enhancer的影響----------------------------------------------------------------------------29
IV.293T-pyWnt3a medium 對MyoD promoter上游序列的影響-------------------------30
V.探討C2C12-FoxO1-wt在不同時期時,FoxO1存在位置的變化------------------------31
第四章 討論----------------------------------------------------------------------33
I. 不同medium培養293T-pPyCAGIP-Wnt3a,其培養液中Wnt3a含量差異---------------33
II.Wnt3a和RA對於MyoD表現量的影響-------------------------------------------33
III MyoD轉錄起始點上游序列的質體建構----------------------------------------34
IV.Wnt3a調控MyoD表現---------------------------------------------------------35
V. C2C12-FoxO1-AAA在PMB、CMB時期及MT時期,FoxO1在細胞中存在位置------------36
VI.結論-----------------------------------------------------------------------38
第五章 圖表----------------------------------------------------------------------39
圖一、收取含有Wnt3a 蛋白質的medium並證實其功能性-----------------------------41
圖二、293T-pyWnt3a medium會增加MyoD的表現量,而同時處理Wnt3a和RA 並不會增加MyoD表現量------------------------------------------------------------------------44
圖三、構築p-Stable-MyoD6.0-enhancer (mouse) 質體------------------------------47
圖四、293T-pyWnt3a medium會影響MyoD promoter及core enhancer的活性-----------49
圖五、構築C2C12-pStable-MyoD6.0-Enhancer-promoter upstream fragments質體-----52
圖六、構築C2C12-pStable-MyoD6.0-Enhancer-promoter upstream fragments -10k~-9k質體----------------------------------------------------------------------------54
圖七、構築C2C12-pStable-MyoD6.0-Enhancer-promoter upstream fragments -10k~-9k質體----------------------------------------------------------------------------56
圖八、FoxO1-wt轉錄活性對MyoD 4.2 k promoter的影響----------------------------57
圖九、利用免疫螢光染色觀察C2C12-FoxO1-wt在PMB、CMB時期及MT時期,FoxO1在細胞中存在位置----------------------------------------------------------------------59
圖十、利用免疫螢光染色觀察C2C12-FoxO1-AAA在PMB、CMB時期及MT時期,FoxO1在細胞中存在位置--------------------------------------------------------------------61
圖十一、利用免疫螢光染色觀察C2C12-FoxO1-wt處理 Leptomycin B 24小時後, 在PMB、CMB時期及MT時期,FoxO1在細胞中存在位置----------------------------------------63
第六章 參考文獻------------------------------------------------------------------64
附錄一---------------------------------------------------------------------------68
附圖一、帶有EcoR I及 Mlu I兩個切位的adaptor ----------------------------------68
附錄二---------------------------------------------------------------------------69
建構質體 primer 對照表--------------------------------------------------------69
BAC clone對照表---------------------------------------------------------------69
附錄三---------------------------------------------------------------------------70
附圖三、MyoD core enhancer上預測的factor binding site整理表格----------------70
附錄四---------------------------------------------------------------------------72
附圖四、以RT-PCR觀察在五個不同的FOXOs 穩定細胞株Sol-8中,PMB、CMB及MT時期的MyoD表現量-------------------------------------------------------------------72
附錄五---------------------------------------------------------------------------73
附圖五、FoxO1-wt與Msx1轉錄活性對MyoD promoter的影響------------------------73
附錄六---------------------------------------------------------------------------74
附圖六、C2C12 FoxO1-AAA monoclones的FoxO1 mRNA表現量及對分化反應的影響-------74
附錄七---------------------------------------------------------------------------75
附圖七、MyoD 在 C2C12、 Sol8 的表現量-----------------------------------------75
附錄八---------------------------------------------------------------------------76
Ⅰ. 溶液及試劑配方------------------------------------------------------------76
Ⅱ. 藥品試劑------------------------------------------------------------------78
Ⅲ. 酵素和限制酶--------------------------------------------------------------78
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指導教授 陳盛良(Shen-Liang Chen) 審核日期 2012-7-27
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