大量表現幹細胞專有轉錄因子抑制肌肉細胞走向分化

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：41

、訪客IP：18.188.107.47

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)

相關論文

★ Thirst control of water-seeking behavior in Drosophila	★ KLHL17在癲癇與自閉症中之角色
★ MyoD對於PGC-1α 基因表現之調控機制	★ 雄性素受體對於肌肉前驅細胞決定的功用
★ Nanog和Oct4表現對肌肉分化之影響	★ FOXOs 轉錄調控因子家族對肌肉細胞末期分化的影響
★ 大量表現 Oct4 與 Nanog 抑制肌纖維母細胞 C2C12 分化	★ 在終極肌肉分化時，肌肉性bHLH轉錄因子對PGC-1α的調控
★ FoxOs 大量表現對肌肉細胞末期分化的影響	★ 觀察肌肉生成轉錄因子如何調控Ｍ- 和Ｎ- cadherin 表現
★ Oc4和Nanog共同抑制末端肌肉分化	★ FoxO6在肌原母細胞中的代謝及分化中所扮演的角色
★ PGC-1α 與 Stra13 間之交互作用	★ 探討大量表現 FoxO6 對肌肉終極分化的影響以及尋找 FoxO6 蛋白質在 PGC-1 alpha 啟動子上的結合位
★ 探討丙戊酸 (Valporic acid) 於肌肉細胞中活化 Oct4 promoter 的機制	★ 探討小鼠骨骼肌中FoxO6的表現情形

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

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

參考文獻

1. Ordahl CP, Le Douarin NM 1992 Two myogenic lineages within the
developing somite. Development 114:339-53
2. Olson EN, Klein WH 1994 bHLH factors in muscle development: dead lines
and commitments, what to leave in and what to leave out. Genes Dev 8:1-8
3. Blackwell TK, Weintraub H 1990 Differences and similarities in
DNA-binding preferences of MyoD and E2A protein complexes revealed by
binding site selection. Science 250:1104-10
4. Lassar AB, Buskin JN, Lockshon D, et al. 1989 MyoD is a
sequence-specific DNA binding protein requiring a region of myc homology
to bind to the muscle creatine kinase enhancer. Cell 58:823-31
5. Buckingham M 2001 Skeletal muscle formation in vertebrates. Curr Opin
Genet Dev 11:440-8
6. Munsterberg AE, Kitajewski J, Bumcrot DA, McMahon AP, Lassar AB
1995 Combinatorial signaling by Sonic hedgehog and Wnt family members
induces myogenic bHLH gene expression in the somite. Genes Dev 9:2911-22
7. Cossu G, Borello U 1999 Wnt signaling and the activation of myogenesis in
mammals. Embo J 18:6867-72
8. Berkes CA, Tapscott SJ 2005 MyoD and the transcriptional control of
myogenesis. Semin Cell Dev Biol 16:585-95
9. Rudnicki MA, Schnegelsberg PN, Stead RH, Braun T, Arnold HH,
Jaenisch R 1993 MyoD or Myf-5 is required for the formation of skeletal
muscle. Cell 75:1351-9
10. Molkentin JD, Olson EN 1996 Defining the regulatory networks for muscle
development. Curr Opin Genet Dev 6:445-53
33
11. Lassar AB, Skapek SX, Novitch B 1994 Regulatory mechanisms that
coordinate skeletal muscle differentiation and cell cycle withdrawal. Curr
Opin Cell Biol 6:788-94
12. Weintraub H, Davis R, Tapscott S, et al. 1991 The myoD gene family: nodal
point during specification of the muscle cell lineage. Science 251:761-6
13. Spinner DS, Liu S, Wang SW, Schmidt J 2002 Interaction of the myogenic
determination factor myogenin with E12 and a DNA target: mechanism and
kinetics. J Mol Biol 317:431-45
14. Black BL, Olson EN 1998 Transcriptional control of muscle development by
myocyte enhancer factor-2 (MEF2) proteins. Annu Rev Cell Dev Biol
14:167-96
15. Molkentin JD, Black BL, Martin JF, Olson EN 1995 Cooperative activation
of muscle gene expression by MEF2 and myogenic bHLH proteins. Cell
83:1125-36
16. Wasserman WW, Fickett JW 1998 Identification of regulatory regions which
confer muscle-specific gene expression. J Mol Biol 278:167-81
17. Evans MJ, Kaufman MH 1981 Establishment in culture of pluripotential
cells from mouse embryos. Nature 292:154-6
18. Martin GR 1981 Isolation of a pluripotent cell line from early mouse
embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc
Natl Acad Sci U S A 78:7634-8
19. Thomson JA, Itskovitz-Eldor J, Shapiro SS, et al. 1998 Embryonic stem
cell lines derived from human blastocysts. Science 282:1145-7
20. Alison MR, Poulsom R, Forbes S, Wright NA 2002 An introduction to stem
cells. J Pathol 197:419-23
21. Boiani M, Scholer HR 2005 Regulatory networks in embryo-derived
34
pluripotent stem cells. Nat Rev Mol Cell Biol 6:872-84
22. Saijoh Y, Fujii H, Meno C, et al. 1996 Identification of putative downstream
genes of Oct-3, a pluripotent cell-specific transcription factor. Genes Cells
1:239-52
23. Ryan AK, Rosenfeld MG 1997 POU domain family values: flexibility,
partnerships, and developmental codes. Genes Dev 11:1207-25
24. Okamoto K, Okazawa H, Okuda A, Sakai M, Muramatsu M, Hamada H
1990 A novel octamer binding transcription factor is differentially expressed in
mouse embryonic cells. Cell 60:461-72
25. Imagawa M, Miyamoto A, Shirakawa M, Hamada H, Muramatsu M 1991
Stringent integrity requirements for both trans-activation and DNA-binding in
a trans-activator, Oct3. Nucleic Acids Res 19:4503-8
26. Vigano MA, Staudt LM 1996 Transcriptional activation by Oct-3: evidence
for a specific role of the POU-specific domain in mediating functional
interaction with Oct-1. Nucleic Acids Res 24:2112-8
27. Chambers I, Colby D, Robertson M, et al. 2003 Functional expression
cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.
Cell 113:643-55
28. Mitsui K, Tokuzawa Y, Itoh H, et al. 2003 The homeoprotein Nanog is
required for maintenance of pluripotency in mouse epiblast and ES cells. Cell
113:631-42
29. Pan G, Pei D 2005 The stem cell pluripotency factor NANOG activates
transcription with two unusually potent subdomains at its C terminus. J Biol
Chem 280:1401-7
30. Hatano SY, Tada M, Kimura H, et al. 2005 Pluripotential competence of
cells associated with Nanog activity. Mech Dev 122:67-79
35
31. Lin T, Chao C, Saito S, et al. 2005 p53 induces differentiation of mouse
embryonic stem cells by suppressing Nanog expression. Nat Cell Biol
7:165-71
32. Kuroda T, Tada M, Kubota H, et al. 2005 Octamer and Sox elements are
required for transcriptional cis regulation of Nanog gene expression. Mol Cell
Biol 25:2475-85
33. Boyer LA, Lee TI, Cole MF, et al. 2005 Core transcriptional regulatory
circuitry in human embryonic stem cells. Cell 122:947-56
34. Huang I-c 2005 Effects of Nanog and Oct4 overexpression on myogenic
differentiation. In: National Central University Dissertation, p 1
35. Ridgeway AG, Skerjanc IS 2001 Pax3 is essential for skeletal myogenesis
and the expression of Six1 and Eya2. J Biol Chem 276:19033-9
36. Yoshida N, Yoshida S, Koishi K, Masuda K, Nabeshima Y 1998 Cell
heterogeneity upon myogenic differentiation: down-regulation of MyoD and
Myf-5 generates 'reserve cells'. J Cell Sci 111 ( Pt 6):769-79
37. Nichols J, Evans EP, Smith AG 1990 Establishment of germ-line-competent
embryonic stem (ES) cells using differentiation inhibiting activity.
Development 110:1341-8
38. Drukker M, Katz G, Urbach A, et al. 2002 Characterization of the
expression of MHC proteins in human embryonic stem cells. Proc Natl Acad
Sci U S A 99:9864-9

指導教授

陳盛良(Shen-Liang Chen)

審核日期

2006-7-21

推文