p53和M3-p53過度表現對細胞週期、代謝 和肌肉細胞分化的影響

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姓名

普蒂(Lulus Putri Aninda) 查詢紙本館藏

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生命科學系

論文名稱

p53和M3-p53過度表現對細胞週期、代謝和肌肉細胞分化的影響
(The effect of p53 and M3-p53 overexpression on cell cycle, metabolism, and myogenesis)

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摘要(中)

腫瘤抑制蛋白p53 (Tumor protein 53)為最早被發現的腫瘤抑制基因，目前已知當DNA受到外界影響而受損時，細胞便會誘導p53表現，透過影響細胞週期或細胞凋亡，避免使不正常的細胞繼續生長；然而p53在肌肉細胞中所扮演的角色目前並不清楚。因此本研究透過肌肉細胞C2C12，探討p53在肌肉發育過程中所造成的影響。本實驗使用p53或M3-p53 (p53基因與MyoD的轉錄激活區域），觀察如何影響肌肉細胞的分化。結果顯示，大量表現p53使細胞週期停滯於G1 / G0、降低細胞生長速度、提高細胞的氧化壓力及誘導p21和Rb1的表現量。雖然p53造成細胞週期停滯，但並沒有降低細胞週期蛋白Cyclin D的表現量，並且在M3-p53大量表現時也呈現相同的結果；但M3-p53會稍微的減少Cyclin D表現。此外，我們還探討了p53及M3-p53對肌肉細胞分化的影響，結果顯示兩者皆會減少肌肉細胞分化，並且抑制分化相關基因Mef2c、Myogenin及MRF4的表現；提高Myf5的表現量。實驗結果表示，p53和M3-p53會延遲肌肉細胞分化過程，但並不會抑制肌肉細胞的分化程度。本研究接下來可探討，大量表現被p53和M3-p53抑制的Mef2c或Myogenin，是否可回復肌肉細胞的分化比例。將來可透過此方式提高肌肉細胞的分化，用於治療肌肉相關疾病。

摘要(英)

The overexpression of p53 has been known widely to be stimulated by cellular stress; however, its aberrant towards muscle development that leads to muscle disease is not apparent. Therefore, we are investigating how p53 or M3-p53, a chimeric gene of p53 and the activation domain of MyoD, affects cell cycle, metabolism, and muscle differentiation in myoblast. Up-regulation of both genes exhibited interchangeable results. The over-activation of p53 caused cell cycle progression to arrest at G1/G0, increased oxidative stress, decreased cell viability, and up-regulated p21 and Rb1. Intriguingly, Cyclin D was not repressed. Meanwhile, M3-p53 induction also exhibited similar results. It arrested cell cycle at G0/G1, reduced cell number, and up-regulated oxidative stress, p21, and Rb1 but slightly induced cyclin D. We also explored the effects at myotube stage in which the activation of p53 showed lesser fusion index value than uninduced cells as well as M3-p53. Reduction of late differentiation marker, Myosin, was confirmed at the protein level. At this stage, p53 and M3-p53 activation shared a similar result in repressing Mef2c, Myogenin, MRF4, and inducing Myf5. These results suggest that p53 or M3-p53 activation leads to delay but not to terminate the differentiation program. Further research would be performing the restoration of Mef2c or Myogenin in excessive activation of p53 or M3-p53 at the myotube formation stage. Expectedly, this strategy could be developed to enhance the differentiation that may have the prospect to cure muscle-related disease.

關鍵字(中)

★ Myogenesis
★ p53
★ M3-p53
★ Cell cycle

關鍵字(英)

★ Myogenesis
★ p53
★ M3-p53
★ Cell cycle

論文目次

摘要 i
Abstract ii
Declaration iii
Acknowledgment iv
Table of Contents v
Chapter I: Introduction 1
1.1. Introduction of Tumor protein 53 (tp53) 1
1.2. p53 involvement towards cell cycle progression 2
1.3. Introduction of M3-p53 4
1.4. Tp53 and myogenesis 4
1.5. The importance of Myogenic Regulatory Factors and Mef2c 5
Chapter II: Materials and Methods 8
2.1. Cell lines and maintenance 8
2.2. Plasmid construction 9
2.2.1. pMSCV-neo-Eb-Mef2c 9
2.2.2. pMSCV-neo-Eb-Myogenin 9
2.3. Antibiotic killing dose test 10
2.3.1. Puromycin killing dose test 10
2.3.2. G418 killing dose test 10
2.4. Transfection, retroviral transduction, and electroporation 10
2.4.1. Transfection 10
2.4.2. Retroviral transduction 11
2.4.3 Electroporation 11
2.5. Real-Time PCR 12
2.5.1. Total RNA extraction 12
2.5.2. Reverse transcription 12
2.5.3. Quantitative Real-Time PCR 13
2.6. Western Blot 13
2.6.1. Protein extraction and quantification 13
2.6.2. Western Blotting 13
2.7. Immunofluorescence 14
2.8. Flow Cytometer 14
2.9. Cell Viability 15
2.10. Reactive Oxygen Species (ROS) Assay 15
Chapter III: Results 17
3.1. Overexpression of p53 inhibits cell cycle progression and induces oxidative stress 17
3.2. Overexpression of p53 delays myoblast differentiation through suppressing Mef2c, Mrf4, and Myogenin 20
3.3 Overexpression of M3-p53 arrests the cell cycle and increases oxidative stress 21
3.4. Overexpression of M3-p53 terminates myoblast differentiation through repressing MRFs and its co-regulator 23
3.5. The construction of Mef2c expression vector 25
3.6. The construction of Myogenin expression vector 25
3.7. Mef2c or Myogenin Restoration in C2C12-tTA-p53 cells 26
3.8. Overexpression of p53 and M3-p53 in K562 cells 26
Chapter IV: Discussion 29
Chapter V: References 33
Chapter VI: Figures 38
Appendix A 61
Appendix B 68

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2019-7-25

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