探討FoxO1抑制肌肉生成的機制｜國立中央大學博碩士論文系統

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研究生：	方彥心 Yen-Hsin Fang
論文名稱：	探討FoxO1抑制肌肉生成的機制 Defining the mechanisms of FoxO1-mediated inhibition of myogenesis
指導教授：	陳盛良 Shen-Liang Chen
口試委員:
學位類別：	碩士 Master
系所名稱：	生醫理工學院 - 生命科學系 Department of Life Science
畢業學年度：	97
語文別：	中文
論文頁數：	122
中文關鍵詞：	肌肉分化
外文關鍵詞：	myogenesis, differentiation, FoxO1, MRF family
相關次數：	點閱：17 下載：0
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肺泡型橫紋肌肉瘤為一好發於孩童時期的惡性腫瘤，其致病原因為在第二對染色體上的PAX3與第十三對染色體上的FoxO1形成一個基因異位的融合蛋白質PAX3-FoxO1，或較少發生的情形為: 第一對染色體上的PAX7與第十三對染色體上的FoxO1形成一個基因異位的融合蛋白質Pax7-FoxO1，此融合蛋白質有使正常細胞轉型成癌細胞的能力，並且會抑制肌肉的分化作用 ; 所以我們設想FoxO1對肌肉生成應扮演著重要的角色。近期的研究指出，當大量表現FoxO1會導致肌肉萎縮，且類型 I 的肌纖維數量會減少。為了更深入了解FoxO1於肌肉生成中的定位，我們探討當大量表現FoxO1時，對於C2C12肌肉細胞末期分化的影響，由我們的研究指出，FoxO1對於肌肉細胞末期分化為抑制效果。接下來，我們想找出是何條訊息傳遞路徑影響了這樣的抑制情形，哪一條訊息傳遞路徑可以回復由FoxO1所抑制的分化情形，很幸運的，我們找出insulin和LiCl可回復FoxO1抑制肌細胞分化的情形，並且這兩者的影響為一加成反應。除了這個發現，我們還找出了p38 這條訊息傳遞路徑可回復C2C12-FoxO1-H215R 肌纖維母細胞對 insulin導致肌肉細胞末期分化的效果。未來我們會著重於LiCl和Wnt-3a 對FoxO1在細胞的細胞核與細胞質轉移情形去研究。

Alveolar rhabdomyosarcoma is the most frequently detected malignant solid tumor in children and is associated with the translocated chimeric genes PAX3-, and Pax7- FoxO1. These chimeric proteins have potent transforming effects and are inhibitors of myogenic differentiation; thus it suggests that FoxO1 should play important roles in myogenesis. Recent studies have shown that over-expression of FoxO1 caused muscular atrophy and reduced type I fibers. To further elucidate the roles of FoxO1 in myogenesis, we examined the effects of FoxO1 over-expression on myogenic terminal differentiation, and we found that the effect was inhibitory. Subsequently, we like to identify the pathways mediating this effect to test whether they can rescue FoxO1 mediating inhibition of myogenesis. Luckly, we found that both insulin and LiCl could rescue FoxO1 inhibited myogenesis and their effects were synergistic. Another surprise was that we found p38 pathway can restore C2C12-FoxO1-H215R myoblasts insulin induced terminal differentiation. In the future, we will investigate whether LiCl and Wnt-3a treatment trigger the translocation of FoxO1 from nucleus.

中文摘要………………………………………………………………………………i
英文摘要………………………………………………………………………………ii
誌謝 ………………………………………………………………………………… iii
目錄 ………………………………………………………………………………… iv
圖目錄……………………………………………………………………………… viii
縮寫與全名對照表 ………………………………………………………………… xi
藥品及材料 ………………………………………………………………………… xi
第一章、序論………………………………………………………………………… 1
1-1  橫紋肌肉瘤(Rhabdomyosarcoma) …………………………………………… 1
1-2  肌肉的起源與生成(myogenesis) ………………………………………………2
1-3  肌肉衛星細胞(satellite cell) ……………………………………………………4
1-4  FoxO轉錄因子(FoxO transcription factor) …………………………………… 6
1-5  研究動機與目的……………………………………………………………… 10
第二章、實驗材料與方法……………………………………………………………11
2-1  細胞株………………………………………………………………………… 11
2-2  穩定細胞株的製備……………………………………………………………11
2-3  細胞培養……………………………………………………………………… 12
2-4  以Insulin處理C2C12肌纖維母細胞…………………………………………14
2-4-1 細胞培養 …………………………………………………………………… 14
2-4-2 DAPI染色…………………………………………………………………… 14
2-5  RT-PCR ……………………………………………………………………… 15
2-5-1 Total RNA的抽取…………………………………………………………… 15
2-5-2 反轉錄酶反應(Reverse Transcription, RT)………………………………… 15
2-5-3 聚合酶鏈反應( Polymerase Chain Reaction, PCR )………………………… 15
2-5-4 Semi-quantitative-PCR 反應在肌肉特異性基因中的各個條件…………… 16
2-6  西方墨點轉漬法(Western blot) ……………………………………………… 17
2-6-1 總蛋白質(Total protein)的製備 …………………………………………… 17
2-6-2 SDS-polyacryamide Gel Electropheresis …………………………………… 17
2-6-3 Blocking 以及 Antibody 辨識………………………………………………18
2-6-4 抗體脫附(Stripping)………………………………………………………… 18
2-7  Immunohistochemistry ……………………………………………………… 19
2-8  RNA穩定度測試………………………………………………………………19
2-9  基因選殖(Gene Cloning)………………………………………………………19
2-9-1 菌株 ………………………………………………………………………… 19
2-9-2 菌株培養 …………………………………………………………………… 20
2-9-3 pPyCAGIP-Pax7的質體構築…………………………………………………20
1.insert DNA的製備 ………………………………………………………… 20
2.載體DNA的製備…………………………………………………………… 21
3.載體 DNA 限制酶的剪切………………………………………………… 22
4.載體 DNA 的 5’端去磷酸根反應………………………………………… 22
5.載體DNA的純化…………………………………………………………… 22
6.接合反應( Ligation ) …………………………………………………………22
7.篩選(Screening)………………………………………………………………23
2-9-4 pStable-Myostatin promoter的質體構築…………………………………… 24
1.insert DNA的製備……………………………………………………………24
2.聚合酶鏈鎖反應產物的修飾 ……………………………………………… 24
3.Insert DNA的純化 ………………………………………………………… 25
4.載體 DNA 的製備 …………………………………………………………25
5.載體DNA限制酶的剪切…………………………………………………… 25
6.載體 DNA 的 5’端去磷酸根反應………………………………………… 25
7.載體DNA的純化…………………………………………………………… 25
8.接合反應(Ligation) ………………………………………………………… 25
9.篩選(Screening)………………………………………………………………25
2-10  流式細胞儀分析 …………………………………………………………… 26
2-10-1 細胞培養…………………………………………………………………… 26
2-10-2 細胞前置處理……………………………………………………………… 26
1.酒精固定 …………………………………………………………………… 26
2.PI染色 ……………………………………………………………………… 26
3.流式細胞儀數據分析 ……………………………………………………… 27
2-11  轉染(Transfection)……………………………………………………………27
2-11-1 細胞培養…………………………………………………………………… 27
2-11-2 轉染作用(Transfection) …………………………………………………… 27
2-11-3 螢火蟲冷光活性方法(Luciferase Activity Assay) …………………………27
第三章、實驗結果……………………………………………………………………28
3-1  FoxO1可抑制C2C12 肌纖維母細胞的分化 ……………………………… 28
3-2  在大量表達FoxOs的C2C12細胞株中，對於肌肉細胞分化特異性相關基因表現的影響………………………………………………………………………… 29
3-3  在大量表現FoxO1的C2C12 細胞中探討細胞週期對於分化的影響…… 30
3-3-1 大量表現FoxO1s的C2C12細胞中PMB時期對於細胞週期的影響……30
3-3-2 大量表現FoxO1的C2C12細胞中CMB時期對於細胞週期的影響………31
3-4  探討Insulin對C2C12、Sol8肌纖維母細胞分化的影響…………………… 31
3-5  在C2C12-FoxO1s細胞株中處理Insulin來觀察其對細胞分化型態的影響…………………………………………………………………………………… 32
3-6  試圖找出C2C12-FoxO1-AAA 抑制細胞分化的機制是牽涉何條路徑…… 33
3-7  Insulin 和LiCl可協同回復由FoxO1所抑制的分化情形 ………………… 34
3-8  FoxO1-AAA轉錄因子可輕微抑制Myogenin promoter …………………… 35
3-9  活化p38可回復C2C12-FoxO1-H215R由Insulin所導致的分化……………36
第四章、討論…………………………………………………………………………37
4-1  大量表現FoxO1s細胞株對於肌肉分化的影響 …………………………… 37
4-2  探討由Insulin和LiCl引起的細胞分化與Myogenic factors的關係 ……… 37
4-3  p38對於肌纖維母細胞分化的影響………………………………………… 38
4-4  MyoD、FoxO1-AAA、β-catenin轉錄因子對Myogenin promoter的調控影響…………………………………………………………………………………… 39
4-5  Wnt與β-catenin轉錄因子對肌纖維母細胞分化可能的調控影響………… 40
第五章、圖表…………………………………………………………………………42
第六章、參考文獻……………………………………………………………………93
附錄一……………………………………………………………………………… 96
附錄二……………………………………………………………………………… 97
附錄三……………………………………………………………………………… 98
I. 溶液及試劑配方………………………………………………………………… 98
II. 藥品試劑……………………………………………………………………… 101
III. 酵素和限制酶………………………………………………………………… 101
IV. 抗體…………………………………………………………………………… 101
附錄四………………………………………………………………………………103
與肌肉分化相關基因的primer對照表……………………………………………103
附錄五………………………………………………………………………………105
PCR primer 對照表 ……………………………………………………………… 105
附錄六………………………………………………………………………………106
中原大學暨中央大學生科系聯合學術研討會佳作獎……………………………106
圖  目  錄
圖一、以RT-PCR、Western blot偵測C2C12 control、C2C12-FoxO1-wt、C2C12-FoxO1-AAA、C2C12-FoxO1-H215R穩定細胞株的mRNA、蛋白質表現量
……………………………………………………………………………………… 42
圖二、將C2C12 control以及大量表現FoxO1的穩定細胞株: C2C12-FoxO1-wt、C2C12-FoxO1-AAA、C2C12-FoxO1-H215R做分化實驗，試探討FoxO1對分化的影響……………………………………………………………………………… 44
圖三、試探討C2C12 control，C2C12-FoxO1-wt，C2C12- FoxO1-AAA，C2C12-FoxO1-H215R Total protein中mTOR，β-catenin，FoxO1， MyoD，MEF-2，Myogenin，P-p38，Pax7蛋白質表現情形………………………………………… 46
圖四、探討C2C12 control，C2C12-FoxO1-AAA中MyoD，Myogenin的RNA穩定性………………………………………………………………………………… 47
圖五、將C2C12-FoxO1-wt-Py(pPyCAGIP)、C2C12-FoxO1- wt-Py-MyoD、C2C12-FoxO1-wt-Py –Mef-2C、C2C12-FoxO1- wt-Py-M-cadherin、C2C12-FoxO1- wt-Py-Pax7穩定細胞株使之分化4天，觀察其細胞型態的改變………………… 49
圖六、在大量表達FoxO1s的C2C12細胞株，分別收取PMB、CMB時期利用流式細胞儀來觀察FoxO1對細胞週期的調控影響………………………………… 50
圖七、Insulin對C2C12細胞株分化型態及其肌肉分化特異基因mRNA表現量變化…………………………………………………………………………………… 52
圖八、Insulin對Sol8細胞株分化型態及其肌肉分化特異基因mRNA表現量變化…………………………………………………………………………………… 56
圖九、在early passage C2C12-FoxO1-AAA 細胞株處理Insulin、IGF-1以檢視其對細胞分化型態的影響…………………………………………………………… 60
圖十、處理Insulin對C2C12 control 細胞分化型態的影響……………………… 61
圖十一、處理Insulin對C2C12-FoxO1-wt 細胞分化型態的影響…………………62
圖十二、處理Insulin對C2C12-FoxO1-AAA P9 細胞分化型態的影響………… 63
圖十三、處理Insulin對C2C12-FoxO1-AAA P16 細胞分化型態的影響………… 64
圖十四、試探討C2C12-FoxO1-AAA Monoclones對分化反應的影響…………… 65
圖十五、處理Insulin對C2C12-FoxO1-H215R 細胞分化型態的影響…………… 67
圖十六、Insulin，PI3K inhibitor (LY294002)對C2C12 control 細胞分化型態之影響…………………………………………………………………………………… 68
圖十七、Insulin，PI3K inhibitor (LY294002)對C2C12-FoxO1-AAA P9細胞分化型態之影響…………………………………………………………………………… 69
圖十八、Insulin，MAP kinase inhibitor對C2C12-FoxO1-AAA P15細胞分化型態之影響……………………………………………………………………………… 70
圖十九、處理Insulin，ROCK inhibitor (Y27632)，SIRT1 inhibitor (NAM) 對C2C12 control 細胞分化型態的影響……………………………………………………… 71
圖二十、處理Insulin， ROCK inhibitor (Y27632)，SIRT1 inhibitor (NAM) 對C2C12-FoxO1-AAA P10 細胞分化型態的影響………………………………… 72
圖二十一、處理Insulin，mTOR inhibitor (Rapamycin)對C2C12- FoxO1-AAA P17 細胞分化型態的影響……………………………………………………………… 73
圖二十二、探討Insulin和LiCl對C2C12 control 細胞分化型態的影響………… 75
圖二十三、探討Insulin和LiCl對C2C12-FoxO1-wt 細胞分化型態的影響………77
圖二十四、探討Insulin和LiCl對C2C12- FoxO1-AAA 細胞分化型態的影響… 79
圖二十五、探討Insulin和LiCl對C2C12-FoxO1- H215R 細胞分化型態的影響…………………………………………………………………………………… 81
圖二十六、試探討C2C12-FoxO1-AAA處理Insulin、LiCl分化4天，核蛋白(nuclear protein)中β-catenin，Myogenin，MyoD，MEF-2，PAX7，FoxO1蛋白質表現情形…………………………………………………………………………………… 83
圖二十七、模擬細胞分化狀況下，觀察MyoD、FoxO1AAA、β-catenin S37A之轉錄活性對於Myogenin promoter活性的影響…………………………………… 84
圖二十八、以Western blot 偵測C2C12 control，C2C12-FoxO1-H215R，C2C12-FoxO1-H215R-Py (C2C12-FoxO1-H215R-pPyCAGIP)，
C2C12-FoxO1-H215R-Py-MKK6EE (C2C12-FoxO1-H215R- pPyCAGIP -MKK6EE)中β-catenin，PAX7和P-p38蛋白質表現情形…………………………………… 85
圖二十九、探討Insulin和LiCl對C2C12-FoxO1- H215R-pPyCAGIP 細胞分化型態的影響…………………………………………………………………………… 87
圖三十、探討Insulin和LiCl對C2C12-FoxO1- H215R-pPyCAGIP -MKK6EE細胞分化型態的影響…………………………………………………………………… 89
圖三十一、將C2C12-FoxO1-wt-Py (pPyCAGIP)、C2C12-FoxO1- wt-Py-MKK6EE、C2C12-FoxO1-wt-Py –Wnt-3a、C2C12-FoxO1- wt-Py-β-catenin-S37A穩定細胞株使之分化4天，觀察其細胞型態的改變………………………………………… 91

                                

Andres, V. and K. Walsh (1996). "Myogenin expression, cell cycle withdrawal, and phenotypic differentiation are temporally separable events that precede cell fusion upon myogenesis." J. Cell Biol. 132(4): 657-666.
Asakura, A., G. E. Lyons, et al. (1995). "The Regulation of MyoD Gene Expression: Conserved Elements Mediate Expression in Embryonic Axial Muscle." Developmental Biology 171(2): 386-398.
Borello, U., M. Coletta, et al. (1999). "Transplacental delivery of the Wnt antagonist Frzb1 inhibits development of caudal paraxial mesoderm and skeletal myogenesis in mouse embryos." Development 126(19): 4247-4255.
Brunet, A., L. B. Sweeney, et al. (2004). "Stress-Dependent Regulation of FOXO Transcription Factors by the SIRT1 Deacetylase." Science 303(5666): 2011-2015.
Buckingham, M. and F. Relaix (2007). "The Role of Pax Genes in the Development of Tissues and Organs: Pax3 and Pax7 Regulate Muscle Progenitor Cell Functions." Annual Review of Cell and Developmental Biology 23(1): 645-673.
Carter, M. E. and A. Brunet (2007). "FOXO transcription factors." Current Biology 17(4): R113-R114.
Chi hsiang-chung (2006). “The roles of FOXO transcriptional factors in skeletal muscle terminal differentiation”
Chun, Y. K., J. Kim, et al. (2000). "Phosphatidylinositol 3-Kinase Stimulates Muscle Differentiation by Activating p38 Mitogen-Activated Protein Kinase." Biochemical and Biophysical Research Communications 276(2): 502-507.
Crabtree, G. R. and E. N. Olson (2002). "NFAT Signaling: Choreographing the Social Lives of Cells." 109(2): S67-S79.
Eric, N. O. and R. S. Williams (2000). "Remodeling muscles with calcineurin." BioEssays 22(6): 510-519.
Fredericks, W. J., N. Galili, et al. (1995). "The PAX3-FKHR fusion protein created by the t(2;13) translocation in alveolar rhabdomyosarcomas is a more potent transcriptional activator than PAX3." Mol. Cell. Biol. 15(3): 1522-1535.
Gallego Melcón, S. and J. Sánchez de Toledo Codina (2007). "Molecular biology of rhabdomyosarcoma." Clinical and Translational Oncology 9(7): 415-419.
Graf Finckenstein, F., V. Shahbazian, et al. (2007). "PAX-FKHR function as pangenes by simultaneously inducing and inhibiting myogenesis." Oncogene 27(14): 2004-2014.
Greer, E. L. and A. Brunet "FOXO transcription factors at the interface between longevity and tumor suppression." Oncogene 24(50): 7410-7425.
Halevy, O., B. G. Novitch, et al. (1995). "Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD." Science 267(5200): 1018-1021.
Hennebry, A., C. Berry, et al. (2009). "Myostatin regulates fiber-type composition of skeletal muscle by regulating MEF2 and MyoD gene expression." Am J Physiol Cell Physiol 296(3): C525-534.
Hribal, M. L., J. Nakae, et al. (2003). "Regulation of insulin-like growth factor-dependent myoblast differentiation by Foxo forkhead transcription factors." J. Cell Biol. 162(4): 535-541.
Huang, H. and D. J. Tindall (2007). "Dynamic FoxO transcription factors." J Cell Sci 120(15): 2479-2487.
Jacobs, F. M. J., L. P. van der Heide, et al. (2003). "FoxO6, a Novel Member of the FoxO Class of Transcription Factors with Distinct Shuttling Dynamics." J. Biol. Chem. 278(38): 35959-35967.
Keren, A., Y. Tamir, et al. (2006). "The p38 MAPK signaling pathway: A major regulator of skeletal muscle development." Molecular and Cellular Endocrinology 252(1-2): 224-230.
Kim, C.-H., H. Neiswender, et al. (2008). "{beta}-Catenin Interacts with MyoD and Regulates Its Transcription Activity." Mol. Cell. Biol. 28(9): 2941-2951.
Ludolph, D. C. and S. F. Konieczny (1995). "Transcription factor families: muscling in on the myogenic program." FASEB J. 9(15): 1595-1604.
McEwen, D. G. and M. Peifer (2000). "Wnt signaling: Moving in a new direction." 10(15): R562-R564.
McEwen, D. G. and M. Peifer (2001). "Wnt signaling: The Naked truth?" 11(13): R524-R526.
Molkentin, J. D., B. L. Black, et al. (1995). "Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins." Cell 83(7): 1125-1136.
Nishiyama, T., I. Kii, et al. (2004). "Inactivation of Rho/ROCK Signaling Is Crucial for the Nuclear Accumulation of FKHR and Myoblast Fusion." J. Biol. Chem. 279(45): 47311-47319.
Nuno, C., T. Lurdes, et al. (2005). "Multimodal genetic diagnosis of solid variant alveolar rhabdomyosarcoma." Cancer genetics and cytogenetics 163(2): 138-143.
Perrot, V. and M. M. Rechler (2005). "The Coactivator p300 Directly Acetylates the Forkhead Transcription Factor Foxo1 and Stimulates Foxo1-Induced Transcription." Mol Endocrinol 19(9): 2283-2298.
Rochat, A., A. Fernandez, et al. (2004). "Insulin and Wnt1 Pathways Cooperate to Induce Reserve Cell Activation in Differentiation and Myotube Hypertrophy." Mol. Biol. Cell 15(10): 4544-4555.
Ruben Conejo, A. M. V. M. B. M. L. (2001). "Insulin produces myogenesis in C2C12 myoblasts by induction of NF-kappaB and downregulation of AP-1 activities." Journal of Cellular Physiology 186(1): 82-94.
Schmidt, M., S. Fernandez de Mattos, et al. (2002). "Cell Cycle Inhibition by FoxO Forkhead Transcription Factors Involves Downregulation of Cyclin D." Mol. Cell. Biol. 22(22): 7842-7852.
van der Velden, J. L. J., R. C. J. Langen, et al. (2006). "Inhibition of glycogen synthase kinase-3{beta} activity is sufficient to stimulate myogenic differentiation." Am J Physiol Cell Physiol 290(2): C453-462.
Wagers, A. J. and I. M. Conboy (2005). "Cellular and Molecular Signatures of Muscle Regeneration: Current Concepts and Controversies in Adult Myogenesis." Cell 122(5): 659-667.
Wang, M. C., D. Bohmann, et al. (2005). "JNK Extends Life Span and Limits Growth by Antagonizing Cellular and Organism-Wide Responses to Insulin Signaling." 121(1): 115-125.
Wei Chieh Huang (2008) .“The roles of FoxO6 in myoblast metabolism and differentiaion”
Wu, A.-L., J.-H. Kim, et al. (2008). "Forkhead Box Protein O1 Negatively Regulates Skeletal Myocyte Differentiation through Degradation of Mammalian Target of Rapamycin Pathway Components." Endocrinology 149(3): 1407-1414.
Wu, Z., P. J. Woodring, et al. (2000). "p38 and Extracellular Signal-Regulated Kinases Regulate the Myogenic Program at Multiple Steps." Mol. Cell. Biol. 20(11): 3951-3964.

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