探討肌肉激素與運動代謝物在肌肉骨骼系統中的作用

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姓名

江宇翔(Yu-Xiang Chiang) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討肌肉激素與運動代謝物在肌肉骨骼系統中的作用
(Investigating the effect of myokines and exercise metabolite on the musculoskeletal system)

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至系統瀏覽論文 (2026-9-30以後開放)

摘要(中)

越來越多的證據表明，運動可以刺激骨骼肌釋放蛋白質（稱為肌肉因子），這些蛋白質以類似荷爾蒙的方式發揮作用，並對遠端器官產生特定的內分泌影響。在我們的研究過程中發現MyoD促使 FNDC5（irisin）和纖維母細胞生長因子21 (FGF21)基因表現量上升。N-lactoyl-phenylalanine（N-LP）是一種新型運動代謝物，在劇烈運動後由多種細胞中的carnosine dipeptidase 2（CNDP2）合成，由乳酸和苯丙胺酸合成，N-LP對於脂肪細胞的氧化代謝等作用與irisin/FGF21相似。在本研究中，我們想要探討骨骼肌中鳶尾素/FGF21和N-LP之間的關係以及它們對肌肉骨骼系統的相互作用。首先我們發現 FGF21 啟動子的活性被 MyoD、N-LP 和神經傳導物質乙醯膽鹼 (Ach) 激活，但被細胞因子 TNFα 和 IL-1β 抑制，結果顯示FGF21啟動子透過分化和運動訊號活化。此外，雖然 Ach 或 N-LP 能夠在GM或DM環境中促使FGF21 啟動子活化，但它們共處理下會抑制 DM環境中的FGF21啟動子，這意味著拮抗途徑參與了它們的訊號傳導。使用N-LP 和Irisin/FGF21 處理骨源細胞（MC3T3），分析它們對於骨生成的作用。我們發現N-LP從早期就增強了骨生成作用，但irisin/FGF21的效果只能在晚期才能看到，特別是成骨相關基因Osteopontin(OPN)的活化。我們的研究結果提供了肌肉因子與N-LP之間的新關係，N-LP可能成為骨質疏鬆症或相關疾病的新療法。

摘要(英)

A growing body of evidence has shown that exercise can stimulate the release of proteins from skeletal muscle, termed myokines, that work in a hormone-like fashion and exert specific endocrine effects on distant organs. In our study, we have found that FNDC5 (irisin) and fibroblast growth factor 21 (FGF21) were up-regulated by MyoD. N-lactoyl-phenylalanine (N-LP) is a newly identified metabolite synthesized from lactate and phenylalanine by the enzyme carnosine dipeptidase 2（CNDP2）in various cells after strenuous exercise, and it share similar effect with irisin/FGF21 on adipocytes, such as oxidative metabolism enhancement. In this study, we aim to investigate the relationship between irisin/FGF21 and N-LP in skeletal muscle and their synergistic effect on the musculoskeletal system. We found that the promoter activity of FGF21 was activated by MyoD, N-LP, and the neurotransmitter acetylcholine (Ach) but repressed by cytokines TNFα and IL-1β, suggesting its activation by differentiation and exercise signals. Additionally, although either Ach or N-LP can enhance FGF21 promoter activity in GM or DM, together they repressed FGF21 promoter in DM, implying antagonistic pathways are involved in their signaling. MC3T3 osteoblast cells were treated with N-LP and irisin/FGF21 to analyze their distinct and synergistic effect on osteogenesis. We found that N-LP enhanced osteogenesis starting from early stage but the effect of irisin/FGF21 could only be seen at late stage, especially the activation of the osteogenic gene Osteopontin. Our results provide new relationship between myokines and the exercise metabolite N-LP, and N-LP might become a new therapy for osteoporosis or related diseases.

關鍵字(中)

★ 肌肉激素
★ 運動代謝物
★ 骨生成

關鍵字(英)

★ myokines
★ exercise metabolite
★ bone formation

論文目次

中文摘要 VI
英文摘要ABSTRACT VII
誌謝 VIII
目錄 IX
一、緒論 1
1-1　肌肉骨骼系統 1
1-2　肌肉激素 1
1-3　N-lactoyl-phenylalanine (N-LP) 3
1-4　肌肉生成（Myogenesis） 3
1-5　骨骼生成（Osteogenesis） 4
1-6　肌肉激素與骨骼間關係 5
1-7　生物反應裝置 7
1-8　研究動機 8
二、研究方法 9
2-1　實驗材料 9
2-1-1 小鼠肌纖維母細胞：Mouse myoblasts cells（C2C12） 9
2-1-2 人胚胎腎細胞：Human embryonic kidney epithelial cells（HEK293T） 9
2-1-3 小鼠纖維母細胞：Fibroblasts cells（C3H10T1/2） 9
2-1-4 小鼠前骨源細胞：Mouse preosteoblast cells（MC3T3） 9
2-1-5 小鼠纖維母細胞：Fibroblasts cells（3T3-L1） 9
2-1-6 小鼠結腸癌細胞：Mouse colon adenocarcinoma cells（C26） 10
2-1-7 穩定細胞株：C2C12-pStable 3K FGF21 promoter 10
2-1-8 穩定細胞株：293T- PyCAG-IP vector 10
2-1-9 穩定細胞株：293T- PyCAG-IP -Irisin 10
2-1-10 穩定細胞株：293T-PyCAG-IP-FGF21 10
2-1-11 穩定細胞株：C2C12-tTA-Irisin 10
2-1-12 穩定細胞株：C2C12-tTA-FGF21 10
2-2　質體建構 10
2-3　細胞轉染（Transfection） 12
2-5-1 細胞培養（Cell culture） 12
2-5-2 細胞轉染作用 13
2-4　冷光素?報導檢測（Luciferase reporter assay） 13
2-5　重組蛋白質純化 13
2-5-1 誘導重組蛋白表現 13
2-5-2 谷胱甘?瓊脂膠純化(Purification of glutathione S-transferase) 14
2-5-3 置換培養基 14
2-6　RNA萃取（RNA extraction） 15
2-7　反轉錄聚合?連鎖反應（Reverse-transcription PCR, PT-PCR） 15
2-8即時定量聚合?連鎖反應（Quantitative real-time PCR, qRT-PCR） 15
2-9茜素紅染色（Alizarin Red Staining） 16
2-10 Calcium Assay（O-Cresolphthalein Chromogenic Method） 16
2-11 生物反應裝置的組裝 17
三、研究結果 19
3-1　在肌肉生成過程中篩選及檢測Myokines之表現量 19
3-2　GST-Irisin及GST-FGF21是具有功能性之重組蛋白 19
3-3　FGF21 promoter受到MyoD及N-LP調控並且活性受到分化狀態影響 20
3-4　循環拉伸及電刺激對於FGF21 promoter活性的影響 21
3-5　檢測運動代謝物及Myokines對於不同細胞的增殖作用 22
3-6　檢測運動代謝物及Myokines對於不同細胞的遷移作用 22
3-7　檢測Myokines（Irisin及FGF21）和運動代謝物對於骨細胞礦化的影響 23
3-8　Myokines（Irisin及FGF21）和運動代謝物增強硬骨相關基因及軟骨相關基因表現 23
四、討論 25
4-1　分析N-LP對於FGF21 promoter之作用 25
4-2　運動代謝物與肌肉激素對於不同型態細胞的增殖與遷移作用 25
4-3　運動代謝物與肌肉激素對於骨生成過程中的影響 26
4-4　GST重組蛋白與真核細胞產生之蛋白對於細胞功能是否一致 27
4-5　結論與未來展望 28
五、圖表 29
圖5-1　肌肉生成過程中篩選及檢測Myokines之表現量 29
圖5-2　GST-Irisin及GST-FGF21是具有功能性之重組蛋白 31
圖5-3 FGF21 promoter受到MyoD及N-LP調控並且活性受到分化狀態影響 34
圖5-4 循環拉伸及電刺激對於FGF21 promoter活性的影響 35
圖5-5 檢測運動代謝物及Myokines對於不同細胞的增殖作用 36
圖5-6 檢測運動代謝物及Myokines對於不同細胞的遷移作用 39
圖5-7 檢測Myokines（Irisin及FGF21）和運動代謝物對於骨細胞礦化的影響 41
圖5-8 檢測Myokines（Irisin及FGF21）和運動代謝物增強硬骨相關基因及軟骨相關基因表現 45
六、參考資料 46
七、附錄 52
圖7-1 檢測癌症惡病質環境下與運動代謝物（N-LP）對於肌肉分化的情形 53
圖7-2 檢測pPyCAG-IP-Irisin及pPyCAG-IP-FGF21是否建立成功 54
7-3 引子條目（primer list） 55
7-4 溶液及溶劑配法 58

參考文獻

1. Buckingham, M. and P.W. Rigby, Gene regulatory networks and transcriptional mechanisms that control myogenesis. Dev Cell, 2014. 28(3): p. 225-38.
2. Quarto, N., et al., Origin matters: differences in embryonic tissue origin and Wnt signaling determine the osteogenic potential and healing capacity of frontal and parietal calvarial bones. J Bone Miner Res, 2010. 25(7): p. 1680-94.
3. Berendsen, A.D. and B.R. Olsen, Bone development. Bone, 2015. 80: p. 14-18.
4. Chan, W.C.W., et al., Regulation and Role of Transcription Factors in Osteogenesis. Int J Mol Sci, 2021. 22(11).
5. Iizuka, K., T. Machida, and M. Hirafuji, Skeletal muscle is an endocrine organ. J Pharmacol Sci, 2014. 125(2): p. 125-31.
6. Hoffmann, C. and C. Weigert, Skeletal Muscle as an Endocrine Organ: The Role of Myokines in Exercise Adaptations. Cold Spring Harb Perspect Med, 2017. 7(11).
7. McCarthy, J.J. and K.A. Esser, Anabolic and catabolic pathways regulating skeletal muscle mass. Curr Opin Clin Nutr Metab Care, 2010. 13(3): p. 230-5.
8. McPherron, A.C., A.M. Lawler, and S.J. Lee, Regulation of skeletal muscle mass in mice by a new TGF-beta superfamily member. Nature, 1997. 387(6628): p. 83-90.
9. Weigert, C., et al., The secretome of the working human skeletal muscle--a promising opportunity to combat the metabolic disaster? Proteomics Clin Appl, 2014. 8(1-2): p. 5-18.
10. Henningsen, J., et al., Dynamics of the skeletal muscle secretome during myoblast differentiation. Mol Cell Proteomics, 2010. 9(11): p. 2482-96.
11. Jodeiri Farshbaf, M. and K. Alvina, Multiple Roles in Neuroprotection for the Exercise Derived Myokine Irisin. Front Aging Neurosci, 2021. 13: p. 649929.
12. Peng, H., et al., Myokine mediated muscle-kidney crosstalk suppresses metabolic reprogramming and fibrosis in damaged kidneys. Nature Communications, 2017. 8.
13. Severinsen, M.C.K. and B.K. Pedersen, Muscle-Organ Crosstalk: The Emerging Roles of Myokines. Endocr Rev, 2020. 41(4): p. 594-609.
14. Lee, P., et al., Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Cell Metab, 2014. 19(2): p. 302-9.
15. Ribas, F., et al., FGF21 expression and release in muscle cells: involvement of MyoD and regulation by mitochondria-driven signalling. Biochem J, 2014. 463(2): p. 191-9.
16. Bostrom, P., et al., A PGC1-alpha-dependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature, 2012. 481(7382): p. 463-8.
17. LeBleu, V.S., et al., PGC-1alpha mediates mitochondrial biogenesis and oxidative phosphorylation in cancer cells to promote metastasis. Nat Cell Biol, 2014. 16(10): p. 992-1003, 1-15.
18. Loffler, D., et al., Serum irisin levels are regulated by acute strenuous exercise. J Clin Endocrinol Metab, 2015. 100(4): p. 1289-99.
19. Moon, H.S. and C.S. Mantzoros, Regulation of cell proliferation and malignant potential by irisin in endometrial, colon, thyroid and esophageal cancer cell lines. Metabolism, 2014. 63(2): p. 188-93.
20. Liu, J., et al., Irisin inhibits pancreatic cancer cell growth via the AMPK-mTOR pathway. Sci Rep, 2018. 8(1): p. 15247.
21. Provatopoulou, X., et al., Serum irisin levels are lower in patients with breast cancer: association with disease diagnosis and tumor characteristics. BMC Cancer, 2015. 15: p. 898.
22. Itoh, N., FGF21 as a Hepatokine, Adipokine, and Myokine in Metabolism and Diseases. Front Endocrinol (Lausanne), 2014. 5: p. 107.
23. Fisher, F.M., et al., Integrated regulation of hepatic metabolism by fibroblast growth factor 21 (FGF21) in vivo. Endocrinology, 2011. 152(8): p. 2996-3004.
24. Fisher, F.M., et al., FGF21 regulates PGC-1alpha and browning of white adipose tissues in adaptive thermogenesis. Genes Dev, 2012. 26(3): p. 271-81.
25. Izumiya, Y., et al., FGF21 is an Akt-regulated myokine. FEBS Lett, 2008. 582(27): p. 3805-10.
26. Yan, B., et al., FGF21-FGFR1 controls mitochondrial homeostasis in cardiomyocytes by modulating the degradation of OPA1. Cell Death Dis, 2023. 14(5): p. 311.
27. Lee, J.H. and H.S. Jun, Role of Myokines in Regulating Skeletal Muscle Mass and Function. Front Physiol, 2019. 10: p. 42.
28. Schranner, D., et al., Metabolite Concentration Changes in Humans After a Bout of Exercise: a Systematic Review of Exercise Metabolomics Studies. Sports Med Open, 2020. 6(1): p. 11.
29. Li, V.L., et al., An exercise-inducible metabolite that suppresses feeding and obesity. Nature, 2022. 606(7915): p. 785-790.
30. Tajbakhsh, S., Skeletal muscle stem cells in developmental versus regenerative myogenesis. J Intern Med, 2009. 266(4): p. 372-89.
31. Bentzinger, C.F., J. von Maltzahn, and M.A. Rudnicki, Extrinsic regulation of satellite cell specification. Stem Cell Res Ther, 2010. 1(3): p. 27.
32. Rudnicki, M.A., et al., The molecular regulation of muscle stem cell function. Cold Spring Harb Symp Quant Biol, 2008. 73: p. 323-31.
33. Bentzinger, C.F., Y.X. Wang, and M.A. Rudnicki, Building muscle: molecular regulation of myogenesis. Cold Spring Harb Perspect Biol, 2012. 4(2).
34. Kuang, S., et al., Distinct roles for Pax7 and Pax3 in adult regenerative myogenesis. J Cell Biol, 2006. 172(1): p. 103-13.
35. Collins, C.A., et al., Stem cell function, self-renewal, and behavioral heterogeneity of cells from the adult muscle satellite cell niche. Cell, 2005. 122(2): p. 289-301.
36. Hojo, H., S. Ohba, and U.I. Chung, Signaling pathways regulating the specification and differentiation of the osteoblast lineage. Regen Ther, 2015. 1: p. 57-62.
37. Karsenty, G. and E.F. Wagner, Reaching a genetic and molecular understanding of skeletal development. Dev Cell, 2002. 2(4): p. 389-406.
38. Calvi, L.M., et al., Osteoblastic cells regulate the haematopoietic stem cell niche. Nature, 2003. 425(6960): p. 841-6.
39. Zhang, J., et al., Identification of the haematopoietic stem cell niche and control of the niche size. Nature, 2003. 425(6960): p. 836-41.
40. Cancedda, R., F. Descalzi Cancedda, and P. Castagnola, Chondrocyte differentiation. Int Rev Cytol, 1995. 159: p. 265-358.
41. Roughley, P.J. and J.S. Mort, The role of aggrecan in normal and osteoarthritic cartilage. J Exp Orthop, 2014. 1(1): p. 8.
42. Gao, Y., et al., The ECM-cell interaction of cartilage extracellular matrix on chondrocytes. Biomed Res Int, 2014. 2014: p. 648459.
43. Kirk, B., et al., Muscle, Bone, and Fat Crosstalk: the Biological Role of Myokines, Osteokines, and Adipokines. Curr Osteoporos Rep, 2020. 18(4): p. 388-400.
44. Colaianni, G., et al., The myokine irisin increases cortical bone mass. Proc Natl Acad Sci U S A, 2015. 112(39): p. 12157-62.
45. Kim, H., et al., Irisin Mediates Effects on Bone and Fat via alphaV Integrin Receptors. Cell, 2018. 175(7): p. 1756-1768 e17.
46. Wei, W., et al., Fibroblast growth factor 21 promotes bone loss by potentiating the effects of peroxisome proliferator-activated receptor gamma. Proc Natl Acad Sci U S A, 2012. 109(8): p. 3143-8.
47. Wang, X., et al., A Liver-Bone Endocrine Relay by IGFBP1 Promotes Osteoclastogenesis and Mediates FGF21-Induced Bone Resorption. Cell Metab, 2015. 22(5): p. 811-24.
48. Ishida, K. and D.R. Haudenschild, Interactions between FGF21 and BMP-2 in osteogenesis. Biochem Biophys Res Commun, 2013. 432(4): p. 677-82.
49. Yang, S., et al., Effect of FGF-21 on implant bone defects through hepatocyte growth factor (HGF)-mediated PI3K/AKT signaling pathway. Biomed Pharmacother, 2019. 109: p. 1259-1267.
50. Engler, A.J., et al., Myotubes differentiate optimally on substrates with tissue-like stiffness: pathological implications for soft or stiff microenvironments. J Cell Biol, 2004. 166(6): p. 877-87.
51. Genchi, G.G., et al., Bio/non-bio interfaces: a straightforward method for obtaining long term PDMS/muscle cell biohybrid constructs. Colloids Surf B Biointerfaces, 2013. 105: p. 144-51.
52. Kaji, H., et al., Electrically induced contraction of C2C12 myotubes cultured on a porous membrane-based substrate with muscle tissue-like stiffness. Biomaterials, 2010. 31(27): p. 6981-6.
53. Kamal, K.Y., et al., Bioreactor development for skeletal muscle hypertrophy and atrophy by manipulating uniaxial cyclic strain: proof of concept. NPJ Microgravity, 2024. 10(1): p. 62.
54. Zhang, Y., et al., Applying exercise-mimetic engineered skeletal muscle model to interrogate the adaptive response of irisin to mechanical force. iScience, 2022. 25(4): p. 104135.
55. Adams, A.C., et al., Thyroid hormone regulates hepatic expression of fibroblast growth factor 21 in a PPARalpha-dependent manner. J Biol Chem, 2010. 285(19): p. 14078-82.
56. Dai, H., et al., FGF21 facilitates autophagy in prostate cancer cells by inhibiting the PI3K-Akt-mTOR signaling pathway. Cell Death Dis, 2021. 12(4): p. 303.
57. Zhang, T., et al., FGF21 increases the sensitivity of hepatocellular carcinoma to sorafenib under hypoxia. Malignancy Spectrum, 2024. 1(2): p. 99-112.
58. Tian, J., et al., OPN Deficiency Increases the Severity of Osteoarthritis Associated with Aberrant Chondrocyte Senescence and Apoptosis and Upregulates the Expression of Osteoarthritis-Associated Genes. Pain Res Manag, 2020. 2020: p. 3428587.
59. Zhu, H., et al., CRISPRa-based activation of Fgf21 and Fndc5 ameliorates obesity by promoting adipocytes browning. Clin Transl Med, 2023. 13(7): p. e1326.

指導教授

陳盛良(Shen-Liang Chen)

審核日期

2024-10-15

推文