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研究生:武氏錦仙
研究生(外文):VO, THI CAM TIEN
論文名稱:槲皮素促進骨骼肌細胞遷移和肌肉分化的分子機制
論文名稱(外文):Molecular Mechanisms of Quercetin Promotes the Migration and Myogenic Differentiation in Skeletal Muscle Cells
指導教授:侯自銓
指導教授(外文):HOUR, TZYH-CHUYAN
口試委員:潘敏雄張基隆
口試委員(外文):PAN, MIN-HSIUNGCHANG, KEE-LUNG
口試日期:2022-07-22
學位類別:碩士
校院名稱:高雄醫學大學
系所名稱:醫學研究所碩士班
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2022
畢業學年度:110
語文別:英文
論文頁數:95
中文關鍵詞:肌少症
外文關鍵詞:Sarcopenia
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肌肉減少症是一種與年齡相關的疾病,被定義為老年人族群中肌肉質量和功能的喪失。最近的研究表明,台灣南部約有五分之一的社區老年人受到肌肉減少症的威脅.老化肌肉的肌肉再生能力下降,這是肌肉減少症的一個重要的影響。骨骼肌再生是一個複雜的生物學過程,涉及成肌細胞增生、細胞週期退出、細胞遷移和分化。最近研究顯示,一些類黃酮可刺激骨骼肌再生和生長。槲皮素是一種天然類黃酮,已有研究顯示可以防止肥胖引起的骨骼肌萎縮作用。因此,研究槲皮素的有益功能可刺激遷移和肌原性分化,這與肌肉再生的改善有關.在本研究中結果顯示,12.5 µM 的槲皮素對調節C2C12成肌細胞遷移和肌肉分化的影響最大。然而,槲皮素不會通過調節 G0/G1 細胞週期退出以影響肌肉分化的早期階段。此外,C2C12處理槲皮素在第3天和第4天時會使IGF-1R活化,但在第7天活性下降,結果顯示槲皮素可以通過活化IGF-1R在早期誘導肌肉分化。槲皮素促進肌肉分化的分子機制是通過活化轉錄因子 STAT3 和 AKT訊息傳遞路徑進而調控目標的肌肉特異性基因結合。此外,我們證明槲皮素促進肌肉分化需要 AKT 活化。此外,槲皮素通過調節 ITGB1 訊息傳遞路徑和活化 FAK 和paxillin的磷酸化促進成肌細胞遷移。總而言之,槲皮素在刺激遷移和分化中具有潛在作用,有助於改善骨骼肌的再生。
Sarcopenia is an age-related disease that is defined as the loss of muscle mass and function in the elderly population. Recent studies show that approximately one-fifth of the community elderly is intimidation by sarcopenia in southern Taiwan. Aged muscles have declined the ability of muscle regeneration, which is an important aspect of sarcopenia. Skeletal muscle regeneration is a complex biological process involving myoblast proliferation, cell cycle exit, migration, and differentiation. Several studies have proven that some flavonoids stimulate skeletal muscle regeneration and growth. Quercetin is a natural flavonoid and has shown an effect protecting against obesity-induced skeletal muscle atrophy. Therefore, investigating the beneficial function of quercetin stimulates migration and myogenic differentiation, which is correlated to muscle regeneration improvement. In this study, our results showed that quercetin (12.5 µM) was the highest effect on regulated C2C12 myoblast migration and myogenic differentiation. However, quercetin did not influence the early stage of myogenic differentiation by regulating cell cycle exit at G0/G1 phase. Moreover, quercetin showed early up-regulation of IGF-1R activity on day 3 and day 4 but decreased on day 7. These results indicated quercetin could induce myogenic differentiation at the early stage through activated IGF-1R. The molecular mechanisms of quercetin promote myogenic differentiation investigated via activated transcription factors STAT3 and AKT signaling pathway. In addition, we demonstrated that AKT activation is required for myogenic differentiation by quercetin. Besides, quercetin promotes myoblast migration by regulating the ITGB1 signaling pathway and activating phosphorylation of FAK and paxillin. In conclusion, quercetin has a potential role in inducing migration and differentiation, which contributes to improving muscle regeneration.



Chinese abstract 3
Abstract 5
Introduction 6
1. Skeletal muscle cell structure and function 7
2. Sarcopenia 8
3. Skeletal muscle regeneration 12
4. Molecular mechanisms of migration and differentiation 16
5. Quercetin 23
Research purpose 26
Materials and methods 27
A. Materials 28
1. Cell line 28
2. Antibodies 28
3. Reagents 31
4. Buffers 32
5. Equipment 33
B. Methods. 34
1. Cell culture 34
2. Subculture 35
3. Cell counting with a hemocytometer 36
4. Freezing cells 37
5. Thawing cells 37
6. Dissolving Quercetin 38
7. Cell differentiation 38
8. Cell viability assay (MTT assay) 39
9. Immunofluorescence staining and morphology analysis 39
10. Western blot 40
11. Wound-healing assay 42
12. Transwell migration 43
13. Myoblast cell analysis and flow cytometry 43
14. AKT inhibitor treatments 44
Results 45
1. Cytotoxicity of quercetin in C2C12 cells. 46
2. Quercetin significantly promoted myoblast fusion and myogenic differentiation. 46
3. Quercetin did not affect the exit of myoblasts cell cycle at the early stage of differentiation. 46
4. Quercetin significantly enhanced myoblast migration. 47
5. Quercetin induced the phosphorylation of AMPK. 47
6. Quercetin inhibited the protein expression of phosphorylation PDGFR-β 47
7. Quercetin induced early up-regulation of IGF-1R and promoted myogenic differentiation, and protein synthesis via downstream of the IGF-1R signaling pathway. 48
8. AKT activation is required for induction of myogenic differentiation by quercetin. 49
9. Quercetin regulated myoblast migration through ITBG1 signaling pathway. 49
Figures 59
Figure 1. Cytotoxicity of quercetin in C2C12 cells. 60
Figure 2. Quercetin significantly promoted myoblast fusion and myogenic differentiation. 62
Figure 3. Quercetin did not affect the exit of myoblasts cell cycle at the early stage of differentiation. 65
Figure 4. Quercetin significantly enhanced myoblast migration. 66
Figure 5. Quercetin induced the phosphorylation of AMPK. 67
Figure 6. Quercetin inhibited the protein expression of phosphorylation PDGFR-β 68
Figure 7. Quercetin induced early up-regulation of IGF-1R and promoted myogenic differentiation, and protein synthesis via downstream of the IGF-1R signaling pathway. 71
Figure 8. AKT activation is required for the induction of myogenic differentiation by quercetin. 74
Figure 9. Quercetin regulated myoblast migration through ITGB1 signaling pathway. 75
Figure 10. Schematic diagram of quercetin promotes myoblast migration and myogenic differentiation 76
Reference 77
Appendixes 91


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