14-3-3蛋白目前被證實參與許多生理功能，如細胞存活、細胞凋亡間的訊號平衡、細胞分化，及腫瘤生成。做為一個橋接蛋白，14-3-3也參與許多訊號傳遞，包含phosphoinositide(PI3K)/protein kinase B (PKB/Akt)、mitogen-activated protein kinase (MAPK)…等。14-3-3在許多組織均有表現，但是在肌肉中存在何種異構體(isoform)目前還沒有明確的研究結果。在接收到分化的訊號後，肌纖維母細胞(myoblast)會終止細胞週期並活化肌肉特化基因，而在肌肉分化的過程中，肌纖維母細胞會移動並與相鄰的細胞發生融合的現象，形成多核的肌小管(myotube)。C2C12細胞是一種用來研究肌肉分化非常好的模式材料，因為可以藉由在低血清的環境下，誘發它們形成肌小管。在這篇研究中，我們經由即時定量聚合酶連鎖反應(qPCR)發現肌肉細胞中存在四種異構體，beta、epsilon、gamma、theta且他們在肌肉分化過程的基因表現各有趨勢上的不同，其中14-3-3 beta、epsilon、gamma在分化後是有表現量上升的現象，theta則是下降。而在過量表現14-3-3 epsilon下，可以發現肌肉分化的指標- Myosin heavy chain(MyHC) 上升，並且過量表現beta、epsilon異構體也會促進肌小管形成。最後，14-3-3 epsilon與gamma的基因敲落(knockdown)也會使得肌小管的形成能力下降。因此我們認為14-3-3 beta、epsilon、gamma對於肌肉分化扮演正向調控的角色。

14-3-3 participates in regulating many cellular functions including signaling between cell survival and apoptosis, cell differentiation, and tumor progression. As an adaptor protein, 14-3-3 involved in many signaling pathways, such as phosphoinositide-3-kinase (PI3K)/protein kinase B (PKB/AKT) pathway and mitogen-activated protein kinase (MAPK) pathway. Although 14-3-3 is found in many tissues, the main isoform in muscle remains unknown. Myogenesis is the process that myoblasts exit cell cycle, activate muscle-related genes, and finally fuse with other myoblasts to form multi-nucleated myotubes. C2C12 cells are a well-established model system for myogenesis based on their ability to differentiate into myotubes in a low-serum condition. In this study, we determined the expression profiles of different isoforms of 14-3-3 using semi-quantitative real-time polymerase chain reaction (qPCR). Our results showed that 14-3-3 beta, epsilon, gamma, and theta expressed differently during muscle differentiation. Beta, epsilon, gamma are up-regulated, and theta is down-regulated. Overexpression of 14-3-3 epsilon up-regulated the myogenesis-determining gene --- myosin heavy chain (MyHC), and overexpression of 14-3-3 beta and 14-3-3 epsilon promotes myotube formation. Knockdown of 14-3-3 epsilon and gamma also limited the formation of myotube. These data suggest that 14-3-3 beta, epsilon, and gamma promote myogenesis.

中文摘要 I
英文摘要 II
致謝 III
目錄 VI
引言 1
肌肉分化 1
14-3-3 蛋白 3
實驗材料與方法 7
試劑 7
細胞培養 7
質體與轉染 8
半定量之即時定量聚合酶連鎖反應(qPCR) 8
西方點墨法 8
免疫螢光染色及fusion index 9
由RNA interference進行14-3-3異構體之基因敲落 9
統計分析 9
結果 10
14-3-3的各個異構體在肌肉分化過程中表現量有所差異 10
過量表現組織特定的14-3-3異構體對肌細胞分化的影響 11
抑制組織特定14-3-3異構體的基因表現影響肌細胞分化的情形 11
討論 14
圖 16
圖一、14-3-3的各個異構體在肌肉分化過程中基因表現量的變化 16
圖二、14-3-3異構體的總蛋白質含量在分化過程中沒有顯著差異 17
圖三、過量表現組織特定的14-3-3異構體影響肌肉分化的情形 18
圖四、過量表現組織特定的14-3-3異構體影響肌小管的形成能力 22
圖五、抑制特定14-3-3異構體的基因表現影響肌細胞分化的情形 26
表格 27
表1. qPCR及RT-PCR使用的引子序列 27
表2. 抗體的稀釋倍數及其應用 28
參考文獻 29

1. Braun, T. and M. Gautel, Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis. Nat Rev Mol Cell Biol, 2011. 12(6): p. 349-61.
2. Penn, B.H., et al., A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation. Genes Dev, 2004. 18(19): p. 2348-53.
3. Mal, A., et al., A role for histone deacetylase HDAC1 in modulating the transcriptional activity of MyoD: inhibition of the myogenic program. 2001. 20(7): p. 1739-1753.
4. Neuhold, L.A. and B.J.C. Wold, HLH forced dimers: tethering MyoD to E47 generates a dominant positive myogenic factor insulated from negative regulation by Id. 1993. 74(6): p. 1033-1042.
5. Spicer, D.B., et al., Inhibition of myogenic bHLH and MEF2 transcription factors by the bHLH protein Twist. 1996. 272(5267): p. 1476-1480.
6. Wei, Q. and B.M.J.F.l. Paterson, Regulation of MyoD function in the dividing myoblast. 2001. 490(3): p. 171-178.
7. Halevy, O., et al., Correlation of terminal cell cycle arrest of skeletal muscle with induction of p21 by MyoD. 1995. 267(5200): p. 1018-1021.
8. Martelli, F., et al., MyoD induces retinoblastoma gene expression during myogenic. 1994. 9: p. 3579-3590.
9. Guo, C.S., et al., Regulation of MyoD activity and muscle cell differentiation by MDM2, pRb, and Sp1. J Biol Chem, 2003. 278(25): p. 22615-22.
10. Molkentin, J.D., et al., Cooperative activation of muscle gene expression by MEF2 and myogenic bHLH proteins. 1995. 83(7): p. 1125-1136.
11. Edmondson, D.G., et al., Analysis of the myogenin promoter reveals an indirect pathway for positive autoregulation mediated by the muscle-specific enhancer factor MEF-2. 1992. 12(9): p. 3665-3677.
12. Hsiao, S.P. and S.L. Chen, Myogenic regulatory factors regulate M-cadherin expression by targeting its proximal promoter elements. Biochem J, 2010. 428(2): p. 223-33.
13. Thompson, W.R., B. Nadal-Ginard, and V.J.J.o.B.C. Mahdavi, A MyoD1-independent muscle-specific enhancer controls the expression of the beta-myosin heavy chain gene in skeletal and cardiac muscle cells. 1991. 266(33): p. 22678-22688.
14. Weintraub, H., et al., MyoD binds cooperatively to two sites in a target enhancer sequence: occupancy of two sites is required for activation. 1990. 87(15): p. 5623-5627.
15. Konermann, A., et al., Autoregulation of insulin-like growth factor 2 and insulin-like growth factor-binding protein 6 in periodontal ligament cells in vitro. Annals of Anatomy - Anatomischer Anzeiger, 2013. 195(6): p. 527-532.
16. Halevy, O. and L.C. Cantley, Differential regulation of the phosphoinositide 3-kinase and MAP kinase pathways by hepatocyte growth factor vs. insulin-like growth factor-I in myogenic cells. Exp Cell Res, 2004. 297(1): p. 224-34.
17. Porras, A., et al., p42/p44 mitogen-activated protein kinases activation is required for the insulin-like growth factor-I/insulin induced proliferation, but inhibits differentiation, in rat fetal brown adipocytes. 1998. 12(6): p. 825-834.
18. Chakravarthy, M.V., et al., Insulin-like growth factor-I extends in vitro replicative life span of skeletal muscle satellite cells by enhancing G1/S cell cycle progression via the activation of phosphatidylinositol 3'-kinase/Akt signaling pathway. J Biol Chem, 2000. 275(46): p. 35942-52.
19. Rommel, C., et al., Mediation of IGF-1-induced skeletal myotube hypertrophy by PI (3) K/Akt/mTOR and PI (3) K/Akt/GSK3 pathways. 2001. 3(11): p. 1009.
20. Hara, K., et al., Amino acid sufficiency and mTOR regulate p70 S6 kinase and eIF-4E BP1 through a common effector mechanism. 1998. 273(23): p. 14484-14494.
21. Vyas, D.R., et al., GSK-3β negatively regulates skeletal myotube hypertrophy. 2002. 283(2): p. C545-C551.
22. Otto, A. and K. Patel, Signalling and the control of skeletal muscle size. Exp Cell Res, 2010. 316(18): p. 3059-66.
23. Rochat, A., et al., Insulin and wnt1 pathways cooperate to induce reserve cell activation in differentiation and myotube hypertrophy. Mol Biol Cell, 2004. 15(10): p. 4544-55.
24. Wilker, E.W., et al., A structural basis for 14-3-3sigma functional specificity. J Biol Chem, 2005. 280(19): p. 18891-8.
25. Celis, J.E., et al., Comprehensive two‐dimensional gel protein databases offer a global approach to the analysis of human cells: The transformed amnion cells (AMA) master database and its link to genome DNA sequence data. 1990. 11(12): p. 989-1071.
26. Fanger, G.R., et al., 14-3-3 proteins interact with specific MEK kinases. 1998. 273(6): p. 3476-3483.
27. Freed, E., et al., Binding of 14-3-3 proteins to the protein kinase Raf and effects on its activation. 1994. 265(5179): p. 1713-1716.
28. Garcia-Guzman, M., et al., Cell adhesion regulates the interaction between the docking protein p130Cas and the 14-3-3 proteins. 1999. 274(9): p. 5762-5768.
29. Leffers, H., et al., Molecular cloning and expression of the transformation sensitive epithelial marker stratifin: a member of a protein family that has been involved in the protein kinase C signalling pathway. 1993. 231(4): p. 982-998.
30. Tang, S.J., et al., Association of the TLX-2 homeodomain and 14-3-3η signaling proteins. 1998. 273(39): p. 25356-25363.
31. TOKER, A., et al., Protein kinase C inhibitor proteins: Purification from sheep brain and sequence similarity to lipocortins and 14‐3‐3 protein. 1990. 191(2): p. 421-429.
32. Morgan, A. and R.D.J.N. Burgoyne, Exol and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaff in cells. 1992. 355(6363): p. 833.
33. Fu, H., J. Coburn, and R.J.J.P.o.t.N.A.o.S. Collier, The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. 1993. 90(6): p. 2320-2324.
34. Campbell, J.K., et al., Activation of the 43 kDa inositol polyphosphate 5-phosphatase by 14-3-3ζ. 1997. 36(49): p. 15363-15370.
35. Gelperin, D., et al., 14-3-3 proteins: potential roles in vesicular transport and Ras signaling in Saccharomyces cerevisiae. 1995. 92(25): p. 11539-11543.
36. Kuwana, T., P.A. Peterson, and L.J.P.o.t.N.A.o.S. Karlsson, Exit of major histocompatibility complex class II–invariant chain p35 complexes from the endoplasmic reticulum is modulated by phosphorylation. 1998. 95(3): p. 1056-1061.
37. Bertram, P.G., et al., The 14-3-3 proteins positively regulate rapamycin-sensitive signaling. 1998. 8(23): p. 1259-S1.
38. Rosiak, J. and J.B. Zawilska, [14-3-3 proteins--a role in the regulation of melatonin biosynthesis]. (0032-5422 (Print)).
39. Hsu, S.Y., et al., Interference of BAD (Bcl-xL/Bcl-2-associated death promoter)-induced apoptosis in mammalian cells by 14-3-3 isoforms and P11. (0888-8809 (Print)).
40. Braselmann, S. and F.J.T.E.J. McCormick, Bcr and Raf form a complex in vivo via 14‐3‐3 proteins. 1995. 14(19): p. 4839-4848.
41. Fu, H., R.R. Subramanian, and S.C. Masters, 14-3-3 Proteins: Structure, Function, and Regulation. Annual Review of Pharmacology and Toxicology, 2000. 40(1): p. 617-647.
42. Wan, P.T.C., et al., Mechanism of Activation of the RAF-ERK Signaling Pathway by Oncogenic Mutations of B-RAF. Cell, 2004. 116(6): p. 855-867.
43. Wellbrock, C., M. Karasarides, and R. Marais, The RAF proteins take centre stage. Nature Reviews Molecular Cell Biology, 2004. 5: p. 875.
44. Porter, G.W., F.R. Khuri, and H. Fu, Dynamic 14-3-3/client protein interactions integrate survival and apoptotic pathways. Seminars in Cancer Biology, 2006. 16(3): p. 193-202.
45. Cai, S.-L., et al., Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning. 2006. 173(2): p. 279-289.
46. DeYoung, M.P., et al., Hypoxia regulates TSC1/2-mTOR signaling and tumor suppression through REDD1-mediated 14-3-3 shuttling. Genes Dev, 2008. 22(2): p. 239-51.
47. Gwinn, D.M., et al., AMPK Phosphorylation of Raptor Mediates a Metabolic Checkpoint. Molecular Cell, 2008. 30(2): p. 214-226.
48. Wilker, E.W., et al., 14-3-3σ controls mitotic translation to facilitate cytokinesis. Nature, 2007. 446: p. 329.
49. Sabatini, D.D., et al., Mechanism of formation of post Golgi vesicles from TGN membranes: Arf-dependent coat assembly and PKC-regulated vesicle scission. Biocell : official journal of the Sociedades Latinoamericanas de Microscopia Electronica ... et. al, 1996. 20(3): p. 287-300.
50. Li, F.-Q., et al., Chibby cooperates with 14-3-3 to regulate β-catenin subcellular distribution and signaling activity. 2008. 181(7): p. 1141-1154.
51. Niemantsverdriet, M., et al., Cellular functions of 14-3-3ζ in apoptosis and cell adhesion emphasize its oncogenic character. Oncogene, 2007. 27: p. 1315.
52. McKinsey, T.A., C.L. Zhang, and E.N.J.P.o.t.N.A.o.S. Olson, Activation of the myocyte enhancer factor-2 transcription factor by calcium/calmodulin-dependent protein kinase-stimulated binding of 14-3-3 to histone deacetylase 5. 2000. 97(26): p. 14400-14405.
53. Strochlic, L., et al., 14-3-3 gamma associates with muscle specific kinase and regulates synaptic gene transcription at vertebrate neuromuscular synapse. (0027-8424 (Print)).
54. Choi, S.-J., S.-Y. Park, and T.-H.J.N.a.r. Han, 14-3-3τ associates with and activates the MEF2D transcription factor during muscle cell differentiation. 2001. 29(13): p. 2836-2842.
55. Yaffe, D. and O.R.A. Saxel, Serial passaging and differentiation of myogenic cells isolated from dystrophic mouse muscle. Nature, 1977. 270: p. 725.
56. Dowling, J.J., et al., Kindlin-2 is required for myocyte elongation and is essential for myogenesis. BMC Cell Biology, 2008. 9(1): p. 36.
57. Miyazaki, M., J.J. McCarthy, and K.A. Esser, IGF-1-induced phosphorylation and altered distribution of TSC1/TSC2 in C2C12 myotubes. The FEBS journal, 2010. 277(9): p. 2180-2191.