Nanog和Oct4表現對肌肉分化之影響

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姓名

黃苡甄(I-chen Huang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

Nanog和Oct4表現對肌肉分化之影響
(Effects of Nanog and Oct4 overexpression on myogenic differentiation)

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摘要(中)

胚胎幹細胞可分化發育成生物個體的所有組織，究竟為何幹細胞能具有如此的特性而有別於體細胞？近來的研究，發現幹細胞當中存在著獨特的因子能維持其多能性，這些獨特的調控因子可藉由刺激或抑制其標的基因的表現來調節胚胎發育分化的過程。Nanog是一個近年來才被確定的基因，其在維持幹細胞獨特的特性扮演很重要的角色。而Oct4，亦命名為Oct3，是屬於POU家族中的其中一員，藉由和序列ATGCAAAT相互作用來調節其標的基因的表現。在這個實驗當中，我們要來探討，當Nanog或是Oct4大量表現在C2C12肌肉細胞時，是否能使得肌肉細胞重新具有多能性？利用反轉錄病毒來感染C2C12肌肉細胞，使其能夠穩定並且大量的表現Nanog或Oct4，以及同時表現兩者基因。我們發現，在Nanog的穩定表達細胞株於分化的狀態其形成的肌管數目僅僅為控制組的五分之一，其形成的肌管數目分別是59條/單位面積以及236條/單位面積。在形態上來說，Nanog的穩定表達細胞株的分化狀態較控制組來得成熟，所以，Nanog不僅僅影響了肌管形成的數目，也影響了肌肉分化的程度。另外，當我們將Oct4大量表現時，MyoD以及Pax7的表現量會下降。利用轉染的方式，我們進一步的證明瞭在C2C12細胞當中，Nanog所扮演的是刺激者的角色，而Oct4所扮演的則是抑制者的角色，Nanog會形成同質聚合體，Nanog和Oct4之間具有微弱的交互作用。依據Nanog中央的homeodomain的部分，將Nanog分為三個domain，並且將Nanog的N terminal domain利用Gal-4回報系統來測試其是否具有transactivation的能力，我們發現Nanog N terminal domain的transactivation能力會依據細胞株以及promoter的不同而有所不同。另外，利用GST-pull down的方式來證明了在in vivo 的情況下Nanog、Oct4以及SMRT三者之間具有交互作用。

摘要(英)

Embryonic stem cells are pluripotent progenitors for virtually all cell types in our body. Recent studies have shown that some transcription factors can sustain pluripotency of the stem cells. These regulators activate or repress patterns of gene expression that mediate phenotypic changes during stem cell differentiation. Nanog has recently been proposed to play a key role in maintaining stem cell pluripotency. Oct4 (Oct3), a member of the POU family of homeodomain proteins, can regulate the expression of their target genes through binding to an octameric sequence ATGCAAAT. Here, we try to explore whether over-expression of Nanog or Oct4 (Oct3) will re-establish the pluripotency of determined myoblasts and thus prevent its terminal differentiation? Using retrovirus carrying Nanog or Oct4 cDNA, we have engineered cell lines that can stably express Nanog or Oct4 or both of them. We further demonstrated that the number of the myotubes in Nanog over-expressed cell is about one fifth to that of retrovirus control infected cell. In the meantime, the number of myotubes is 59 in Nanog over-expressed cell and is 236 in retrovirus control infected cell. Morphologically, the Nanog overexpressed myotube is more matured than the retrovirus control infected cell. Our preliminary results show that Nanog actually influence the degree of differentiation not only in the number of myotube formation but also in the state of myotube maturation. Furthermore, we also found that expression of MyoD and Pax7 is downregulated in Oct4 overexpressed cells. In transient transfection reporter assays, Nanog functions as an activator, but Oct4 functions as a repressor in C2C12 cell line. We also provide evidence showing that Nanog can form homodimer complex and also interacts with Oct4 weakly. Based on the homeodomain, Nanog can be divided into three regions. In order to investigate the transactivation activity of Nanog N terminal domain, we used the well-established Gal-4 reporter system. We report here that N terminal domain of Nanog could perform different transactivation activity in different cell type under different promoter context. By using GST pull down experiment, we further demonstrated the relationship among Nanog, Oct4 and SMRT.

關鍵字(中)

關鍵字(英)

★ Nanog
★ Oct4

論文目次

中文摘要………………………………………………………………Ⅰ
英文摘要………………………………………………………………Ⅱ
目錄……………………………………………………………………Ⅲ
圖目錄…………………………………………………………………Ⅷ
縮寫與全名對照表……………………………………………………Ⅹ
第一章、緒論
I. 概論幹細胞……………………..……….……...…………….. ..1
II. 肌肉的起源………..………………..………………..................6
III. MRF falimy在肌肉細胞的功能………...…………………….8
IV. 研究動機與目的…………..………………………………......10
第二章、材料與方法
І.實驗材料
1. 細胞株
1.1細胞培養…………………………………………….………..11
2. 菌株
2.1菌株培養……………………………………………………….11
2.2菌株的保存…………………………………………………….12
Ⅱ. 質體建構
1. 質體建構之基本流程
1.1 大腸桿菌勝任細胞之製備（Preparation of E. coli
competent cells)……………………………………….......12
1.2 大腸桿菌的轉型作用 (Transformation)…………………....12
1.3 質體DNA 的少量製備 (Mini-preparation) – 用鹼處理法 (Alkaline lysis method) …………………………………………13
1.4 聚合酶鏈反應(Polymerase chain reaction)……………13
1.5 聚合酶鏈反應產物的修飾………………………………….14
1.6插入(Insert)DNA的純化………….…………………………14
1.7 載體DNA的製備……………………………………………14
1.8 載體DNA限制酶的剪切……………………………………...15
1.9載體DNA的5＇去磷酸根反應……………………………….15
1.10 載體DNA的純化…………………………………………..15
1.11接合反應(ligation)…………………………………………16
1.12篩選(screening)…………………………………………….16
1.12.1核酸限制酶的剪切……………………………………16
Ⅲ. 特殊質體之建構
1.1插入DNA(Nanog)的製備……………………………………17
1.2插入DNA的製備(Oct4、Oct4△N)………………………….17
1.3特殊載體DNA的製備…………………………..………….19
1.4接合反應……………………………………………,,…….19
1.5篩選……………………………………………………..….19
1.6核酸定序………………………………………………..….20
Ⅳ. 哺乳細胞細胞中之Two hybrid……………………………….…20
1. 質體轉染
1.1細胞培養…………………………………………..………20
1.2轉染作用…………………………………………...………20
1.3螢火蟲冷光活性方法…………………………………...…21
Ⅴ. 蛋白質純化、Antibody的製備、GST pull down assay………..21
1.1轉型作用…………………………………………….……..21
1.2蛋白質的表現………………………………………….…..21
1.3蛋白質的純化…………………………………………..….21
1.4Antibody的製備……………………………………..……..23
1.5GST pull down assay………………………………...……..23
Ⅵ.pMSCV-neo-Nanog、pMSCV-neo-Oct4、pMSCV-neoOct4△N以及pMSCV-neo-Nanog-IRES-Oct4表現載體之構築…………………………………………………………………24
Ⅶ. 利用反轉錄病毒( Retrovirus )將Nanog、Oct4、Oct4△N以及Nanog-IRES-Oct4基因表現於哺乳類細胞中…………………………………………………….……………24
1. 反轉錄病毒的製備：
細胞培養……………………………………………..…..…25
轉染作用……………………………………………………25
2. 利用反轉錄聚合酶鏈反應( RT-PCR )篩選反轉錄病毒製備成功
2.1 Total RNA的抽取………………………………..…………..25
2.2 反轉錄酶反應(Reverse Transcriptase, RT)………..……......25
2.3 聚合酶鏈反應( Polymerase Chain Reaction, PCR )………25
3. 質體pMSCV-neo-Nanog、pMSCV-neo-Oct4、pMSCV-neo-Oct4△N以及pMSCV-neo-Nanog-IRES-Oct4的感染……………………..26
3.1 細胞培養……………………………………………………...26
3.2 感染作用………………………………………………….…..26
4. 利用反轉錄聚合酶鏈反應篩選穩定表達細胞株
4.1 Total RNA的抽取…………………………………………..26
4.2 反轉錄酶反應(Reverse Transcriptase, RT)…………..…....26
4.3 聚合酶鏈反應( Polymerase Chain Reaction, PCR )………27
5. 利用西方點墨法(Immunoblotting)偵測Nanog的表現………..27
Ⅷ.利用反轉錄聚合酶鏈反應判別穩定表達株之基因表現…………………………………………………………………..… 28
Ⅸ.利用DAPI染細胞染色體………………………………..………..28
第三章、結果……………………………………………………...29
第四章、討論………………………………………………...…….41
第五章、參考文獻…………………………………………...……67
反轉錄病毒的製備流程…………………………………..…70
附錄一……………………………………………..…………………71
附錄二……………………………………………..…………………75
附錄三……………………………………………..…………………77

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2005-12-20

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