觀察肌肉生成轉錄因子如何調控 Ｍ- 和Ｎ- cadherin 表現

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姓名

蕭勝斌(Sheng-Pin Hsiao) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

觀察肌肉生成轉錄因子如何調控Ｍ- 和Ｎ- cadherin 表現
(The regulation of M- and N-cadherin expression by myogenic factors)

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摘要(中)

M-和N-cadherin 都是屬於鈣離子依賴型細胞與細胞間黏著分子家族的一員，M-cadherin主要表現在正在發育生成中的骨骼肌中，且在之前已被證實其在肌肉的終極分化階段扮演著非常重要的腳色尤其是在肌纖維母細胞進行融合的時期特別的明顯。藉由N-cadherin的細胞與細胞間的黏著作用同樣的也在骨骼肌的生成過程中有著顯要的地位。ＭyoD、Myogenine、Myf-5和 MRF4 這四個轉錄因子同時都具有Basic-loop-helix motif 且同屬於肌肉調節因子(Muscle Regulatory Factor , MRF)。ＭyoD和Myf-5在整個胚胎發育中整個肌肉生成的全部步驟中都是不可缺少的，但是Myogenin 和MRF4則不然，他們是在肌肉生成的後期也就是終極分化時才有顯著的影響。之前的研究指出在MyoD-/- 肌原性細胞中，並無法行最後的終極分化且M-cadherin的表現量也有很明顯的下降。我們也知道當細胞缺乏M-cadherin時 N-cadherin 會互補的表現出來而維持細胞正常的功能。在我們的研究中我們要了解究竟這些肌肉生成調節因子是如何去調控M-和N-cadherin promoter的表現。首先我製備了M-cadherin promoter質體並且發現MyoD會藉由結合到M-cadherin promoter上而去活化M-cadherin的表現。接下來我們突變了M-cadherin core promoter上面的 E-box更正確的定義出MyoD及Myogenin 的結合位置。同時我們也觀察及比較MRFs對於N-cadherin的調控是否有不同於M-cadherin調控的部份，發現 MRFs 以及 MEF-2C 對於N-和M-cadherin 活性的調控並不相同，所以之後我們要找出為什麼會不同以及這種不同在生理上代表了什麼意義，未來我們會用ChIP assay (chromatin-immunoprecipitation ) 分析MRF調控M-cadherin和N-cadherin 的生理意義。

摘要(英)

M- and N-cadherin are members of the Ca2+ dependent cell-cell adhesion molecule family. M-cadherin is predominantly expressed in developing skeletal muscles and has been implicated in terminal myogenic differentiation, particularly in myoblast fusion. N-cadherin-mediated cell-cell adhesion also plays an important role in skeletal myogenesis. MyoD, Myf-5, Myogenine and MRF-4 are basic helix-loop-helix (bHLH) transctiption factors are that form the Myogenic regulatory factors (MRFs) family. MyoD and Myf-5 are required for proper myogenesis during embryogenesis. In contrast, myogenine and MRF-4 are important only for terminal myogenic differentiation. In MyoD-/- myOblast terminal myogenic differentiation is impaired and the expression of M-cadherin is also reduced. We also known that N-cadherin can largely compensate for lack of M-cadherin. In our study we will want to know how myogenic factors regulate M- and N-cadherin expression. Firstly we cloned M-cadherin promoter and found that MyoD could activate M-cadherin by binding to it’s promoter. Then we mutated the putative E-boxs of M-cadherin promoter to define the MRF binding sites. At the same time we cloned and analysed N-cadherin promoter and compare it’s regulation by MRFs with that of M-cadherin. We found that the regulation of M- and N-Cadherin by MRFs and MEF-2C were different. In the future we will use CHIP assay to analyze the physiology meaning in vivo of this regulation by MRFs.

關鍵字(中)

★ 肌肉調節因子
★ 聚合酶鏈鎖反應
★ 肌纖維母細胞

關鍵字(英)

★ N-cadherin
★ M-cadherin
★ MyoD
★ promoter

論文目次

中文摘要............................................................I
Abstract...........................................................II
誌謝..............................................................III
目錄.............................................................. VI
圖目錄...........................................................VIII
縮寫與全名對照表....................................................X
一、序論............................................................1
I. 肌肉起源....................................................1
II. 肌肉調節因子(Muscle Regulatory Factor,MRF) .................2
III. MEF2 family.................................................3
IV. Cadherin family.............................................4
V. 實驗動機與目的..............................................8
二、材料與方法.....................................................10
I. 實驗材料...................................................10
1. 細胞株........................................... .....10
1.1細胞培養...........................................10
2. 菌株................................................... 10
2.1菌株培養........................................... 11
II. 質體構築............................................... .. 11
1. M-cadherin promoter–pGL3-basic 報告質體(Reproter plasmid) 建構.......................................................11
1.1 插入DNA(M-cadherin promoter)的製備.................11
1.1.1聚合酶鏈鎖反應Polymerase Chain Reaction (PCR) .11
1.1.2聚合酶鏈反應產物的修飾.........................12
1.1.3插入 (Insert) DNA的純化........................12
1.2載體 DNA 的製備..................................... 13
1.2.1 載體 DNA 限制酶的剪切...........................14
1.2.2 載體 DNA 的 5＇端去磷酸根反應................. .14
1.2.3載體DNA的純化........... ......................15
1.2.4 接合反應 ( Ligation ) ......................... 15
1.2.5篩選 (Screening) ............................... 15
2. N-cadherin promoter–pStable 報告質體(Reproter plasmid) 建構.................................................................15
2.1插入DNA(N-cadherin promoter)的製備.................15
2.1.1聚合酶鏈鎖反應Polymerase Chain Reaction (PCR) .16
2.1.2插入 (Insert) DNA的純化....................... 16
2.1.3聚合酶鏈反應產物的修飾..........................16
2.2載體DNA 製備........................................16
2.2.1載體 DNA 限制酶的剪切..........................16
2.2.2載體 DNA 的 5＇端去磷酸根反應..................17
2.2.3載體DNA的純化..................................17
2.2.4接合反應 ( Ligation ) ......................... 17
2.2.5篩選 (Screening) .............................. 17
3. M-cadherin promoter 較短片段的製備...................... 17
3.1限制酶的剪切....................................... 17
3.1.1 pGL 3-M-cadherin(-2535~+200) promoter-Lu........ 17
3.1.2 pGL 3-M-cadherin(-1773~+200) promoter-L........18
3.2聚合鏈鎖反應製備...................................18
3.2.1 pGL3-M-cadherin(-508~+200) promoter-Luc...... 18
3.2.2 pGL3-M-cadherin(-252~+200) promoter-Luc...... 18
3.2.3 pGL3-M-cadherin(-91~+200) promoter-Luc...... 18
3.2.4 pGL3-M-cadherin(-2 ~ +200) promoter-Luc...... 19
3.2.5 pGL3-M-cadherin(+20 ~ +143) promoter-Luc......19
III.質體pGL3-M-cadherin promoter 及pGL3-M-cadherin promoter較短片段的轉染(Transfection) ..................................19
1. 細胞培養............................................... 19
2. 轉染作用 (Transfaction) ............................... 19
3. 螢火蟲冷光活性方法(Luciferase Activity Assay) .......... 19
IV. RT-PCR.................................................... 20
1. Total RNA 的抽取....................................... 20
2. 反轉錄酶反應 (Reverse Transcriptase, RT) ............... 20
3. 聚合酶鏈反應 ( Polymerase Chain Reaction, PCR ) .........21
V. 凝膠遷移滯後實驗electrophoretic mobility shift assays，EMSA.23
1. 聚合鏈鎖反應...........................................21
2. 探針的標定(labelling) .................................21
3. 探針的純化............................................ 21
4. Binding assay......................................... 22
VI. Mutagenesis...............................................22
1. M-cadherin -2~+200 promoter 各突變型質體製備...........22
1.1 M-cadherin -2~+200 promoter MT4 製備................23
1.2 M-cadherin -2~+200 promoter MT5 製備................23
1.3 M-cadherin -2~+200 promoter DB MT 製備..............24
2. M-cadherin -2~+143 各突變型片段製備..................24
2.1 M-cadherin -2 ~ +143 MT4 製備.......................24
2.2 M-cadherin -2 ~ +143 MT5 製備.......................24
2.3 M-cadherin -2 ~ +143 DB MT 製備.....................24
三、實驗結果.................................................... 25
四、實驗討論.....................................................33
五、圖表....................................................... .38
六、參考文獻.....................................................63
七、附錄一.......................................................66
I. PCR primer.............................................. 66
II. RT-PCR primer........................................... 68
八、附錄二...................................................69
I. 溶液及試劑配方...........................................69
II. 藥品試劑.................................................72
III. 酵素和限制酶.............................................72
九、 <表一>...................................................73
<表二>...................................................74
<表三>...................................................75

參考文獻

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2008-7-22

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