大量表現幹細胞專有轉錄因子抑制肌肉細胞走向分化

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姓名

張毓容(Yu-Jung Chang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

大量表現幹細胞專有轉錄因子抑制肌肉細胞走向分化
(Over-expressing ES cell-specific transcription factors suppresses the differentiation of myoblasts)

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摘要(中)

利用幹細胞發展培養再生組織或器官來進行醫療研究，為醫療發展的一大趨勢，但是由於免疫系統對外來幹細胞的排斥，以及自體幹細胞的取得與培養相對的並不容易，因此就目前為止，取得不會引起免疫排斥的幹細胞是目前幹細胞醫療的瓶頸，但是如果能利用病人本身的體細胞做為材料，進行胞外實驗使其回復到前驅細胞的時期，再依所需而誘導其轉分化，對於幹細胞的醫療研究將會是一大突破。在我們的研究中，選擇兩個幹細胞的轉錄因子Nanog與Oct3為標的，利用已建立好的Retrovirus系統，來建立大量表現標的基因的Sol8穩定細胞，進而利用此穩定細胞株來分析幹細胞因子對於肌肉細胞分化過程的影響。在我們的實驗中發現，由形態來看，當同時表現兩個幹細胞因子Nanog與Oct3的Sol8穩定細胞株時，會抑制肌肉細胞走向終末分化，且在利用RT-PCR及Western blot分析中得到肌肉細胞中的肌肉特化因子MyoD、Myogenin同時會受到抑制，另外我們在同時表現Nanog與Oct3的穩定細胞株中處理β-mercaptoethanol，發現當細胞中添加的量越高時，myf5的表現量相對的受到抑制的現象。在我們的實驗中，我們發現了當肌肉細胞中同時表現幹細胞因子Nanog與Oct3時，會促使肌肉細胞不會走向終末分化，至於要使肌肉細胞回復到前驅細胞且帶有pluripotency的能力，將需
要再進一步的研究來達成這遠程的目標。

摘要(英)

The potential of cell mediated tissue regeneration is hampered by both immune
rejections of engrafted allogenic ES cells and the difficulties of isolating and
propagating autologous stem cells. However, if the somatic cells can be induced to
transdifferentiate into precursors of target cell types in vitro, cell mediated tissue
regeneration will be more applicable. In this study, we introduced two ES cell-specific
transcription factors, Nanog and Oct3, into sol8 myoblast using retrovirus system and
examined the effects on differentiation stage of the stable clones. Over expressing
Nanog and Oct3 simultaneously, but not independently, impaired the terminal
differentiation of Sol8 myoblasts. Furthermore, the expression of myogenic regulator
factors (MRFs), MyoD and Myogenin, is abolished in Sol8-Nanog/Oct3 cells. In addition, the slightly repression of Myf5 expression could be further diminished after β-mercaptoethanol treatment. Our data suggest that although over expressing Nanog and Oct3 in myoblasts can surpress myogenic differentiation, whether those cells acquired pluripotency still needs to be further investigated.

關鍵字(中)

★ 幹細胞因子
★ 肌肉細胞分化

關鍵字(英)

★ Myogenisis
★ Oct3
★ Nanog

論文目次

目錄..............................................................................................................................i
圖目錄..................................................................................................................... iii
序論.............................................................................................................................1
I. 肌肉由來與發育...................................................................................1
II. MRFs 家族與肌肉發育的關係............................................................2
III. 幹細胞簡介...........................................................................................3
IV. Oct3 與Nanog 的角色.........................................................................6
V. 研究動機與目的...................................................................................8
材料與方法...........................................................................................................9
I. 細胞培養...............................................................................................9
II. 穩定細胞株的製備...............................................................................9
III. 質體建構.............................................................................................10
IV. Promoter 分析....................................................................................11
V. 單株細胞株的製備.............................................................................12
VI. RT-PCR...............................................................................................12
VII. 西方墨點實驗(Western blot).............................................................14
VIII. 篩選Sol8-reserve cell 細胞株............................................................16
IX. In vitro transcription and translation...............................................16
結果...........................................................................................................................18
I. 建立並檢測Nanog 與Oct3 以及兩者同時表現的穩定細胞株......18
II. pMSCV-Oct3 與pMSCV-Nanog-IRES-Oct3 序列確認.................18
III. 觀察穩定細胞株之Myogenic differentiation ..................................19
IV. 利用RT-PCR 的方式偵測不同穩定細胞株中Myogenic related
factors 的表現量.................................................................................................19
ii
V. 利用Western blot 的方式偵測MRFs 在不同穩定細胞株中表現.21
VI. 觀察Nanog 與Oc3 對Pax3 Promoter 的調控................................22
VII. 篩選同時表現Nanog 與Oct3 穩定細胞株的單株細胞株..............22
VIII. 比較Sol8-Nanog/Oct3 與Sol8-reserve cell 之間的關係.................23
IX. 利用35S 標定方式在胞外做出OCT3 蛋白質..................................24
X. Oct3-N terminal 融合蛋白降低轉譯效率........................................25
XI. β-ME 抑制myf5 的表現量................................................................25
XII. β-ME 與LIF 對於myf5 基因表現量的影響....................................26
XIII. Nanog 與Oct3 對於MyoD promoter 活性的調控.........................26
討論...........................................................................................................................28
I. 幹細胞因子Nanog、Oct3 與肌肉細胞分化之間的關係................28
II. 回復肌肉細胞至幹細胞階段.............................................................29
III. 促使同時表現Nanog 與Oct3 的穩定細胞株重新走向終末分化..30
IV. 探討ΔN-Oct3 與Oct3 之間的差異..................................................31
參考文獻...............................................................................................................32
附圖一.....................................................................................................................52
附圖二.....................................................................................................................53
附錄...........................................................................................................................54
I. Primer 對照表....................................................................................54
II. 縮寫與全名對照表.............................................................................57
III. 溶劑及試劑配方.................................................................................58

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2006-7-21

推文