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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 陳靜宜(Ching-Yi Chen) | |
| dc.contributor.author | Ting-Rui Zhang | en |
| dc.contributor.author | 張庭睿 | zh_TW |
| dc.date.accessioned | 2023-03-19T23:16:20Z | - |
| dc.date.copyright | 2022-07-22 | |
| dc.date.issued | 2022 | |
| dc.date.submitted | 2022-07-21 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/85416 | - |
| dc.description.abstract | 骨骼肌佔體組成40-50%，且負責身體能量代謝與運動收縮功能，其中粒線體品質 (mitochondrial quality control, MQC) 調控了骨骼肌功能正常運作。MQC包括生合成 (biogenesis)、融合 (fusion)、分裂 (fission) 與粒線體自噬 (mitophagy) 等動態平衡 (dynamics)，但是隨著肌肉老化，MQC將逐漸下降，如粒線體動態失衡，堆積過多破碎化 (fragmentation) 粒線體造成肌纖維結構異常以及粒線體能量代謝速率下降等，這些因素會造成骨骼肌收縮能力降低與萎縮 (antrophy)。過去限制飲食 (dietary restriction, DR) 之研究雖證實能增加MQC，但DR可否藉改善骨骼肌MQC以減緩老化卻付之闕如，因此本研究將探討DR對老化過程骨骼肌MQC之影響，研究分為三部分。 第一階段試驗目的為了解骨骼肌老化過程之變化，試驗針對7 (青年)、14 (中年) 及19 (老年) 月齡C57BL/6J小鼠之股四頭肌 (quadriceps) 進行比較。試驗首先發現老年組相較另兩組之血糖與骨骼肌纖維化皆顯著上升 (p< 0.05)，而在電子顯微鏡下觀察到肌纖維結構異常與肌膜內粒線體趨向破碎化的現象，因此，實驗進一步比較老年與中年在MQC之差異，老年組之粒線體動態分裂蛋白指標 (Drp1) 下降 (p< 0.05)，融合蛋白指標 (Mfn2, Opa1) 也降低 (p< 0.05)，而粒線體總量 (VDAC) 則為顯著上升 (p< 0.05)。推論老化過程發生粒線體動態失衡與破碎化粒線體累積，進而引發骨骼肌纖維化與肌纖維異常。 第二階段試驗目的為探討DR的長度對於延緩骨骼肌老化之影響。C57BL/6J小鼠於3月齡開始為期11或16個月40% DR或任食 (ad libtium, AL)，發現DR可有效降低中年及老年組血糖、骨骼肌纖維化 (p< 0.05)。在中年組中，DR藉促進粒線體自噬 (PINK1, Parkin, LC3I) (p< 0.05)。而老年組中，DR能顯著降低氧化壓力 (TBARS)，減少骨骼肌內粒線體總量 (VDAC) 並提高粒線體動態平衡蛋白指標 (Drp1, Opa1) 與粒線體體積 (p< 0.05)。推測DR在不同年齡階段所調控MQC的方式不同，在中年以提升粒線體自噬為主，而老年則由維持粒線體動態平衡，使兩個階段保留代謝功能較佳之粒線體以延緩骨骼肌纖維化。 第三階段建立體外骨骼肌老化模型，進一步探討DR對老化肌肉粒線體功能之可能機制。試驗首先將C2C12肌原母細胞分化為肌管後藉氧化壓力誘發成為老化體外模型，後續添加薑黃素 (curcumin) 作為DR模擬物，探討DR如何調控細胞以延緩老化之分子機制。目前已知薑黃素能達到減緩細胞凋亡蛋白 (p53, p21)，進而減緩骨骼肌受老化之影響。 綜上所述，經由活體試驗，推測DR能透過促進粒線體自噬與粒線體動態平衡，使骨骼肌減少老化萎縮之影響，而DR調控粒線體之詳細機制仍待未來體外試驗證實。 | zh_TW |
| dc.description.abstract | Skeletal muscle is responsible for energy metabolism of body and exercise contraction, is which mitochondrial functions and their quality control (MQC). MQC are highly associated with muscles functionality, refers to the balance of mitochondrial biogenesis, fusion, fission and mitophagy, namely mitochondrial dynamics. During aging, MQC imbalance, such as dysfuction of mitochodonrial dynamics and accumulation of fragmented mitochondria, are related to decreased contraction of muscle fiber and antrophy. Past studies showed that dietary restriction (DR) cloud increases MQC, but the underlying associtation between aging and MQC by DR in skeletal muscle is still unknown. Therefore, this study explored the effect of DR on MQC in skeletal muscle along the progression of aging. The study is divided into three parts. The first experiment explored the changes of skeletal muscle with aging process, by comparing quadriceps femoris muscle of 7 (young), 14 (middle-aged) and 19 (elderly)-month old C57BL/6J mice levels. The elderly group exhibited a significant increase of blood glucose, fibrosis of skeletal muscle, when compared with the other two groups (p< 0.05). Besides, a tendency to accumulate abnormal structures of myofibrils and fragmentation of mitochondria in the sarcolemma of elderly group were observed. Therefore, the differences in MQC between the middle-aged and elderly mice were further analyzed. The mitochondrial dynamic fission (Drp1) and fusion (Mfn2, Opa1) protein were decreased in the elderly group (p< 0.05), whereas the total mitochondrial volume (VDAC) was significantly increased (p<0.05), suggesting that an imbalance of mitochondrial dynamics and accumulation of fragmented mitochondria occurre with aging, leading to fibrosis of skeletal muscle and myofibril abnormalities. The second experiment investigated the effect of DR on attenuating aging of skeletal muscle. C57BL/6J mice at 3 months of age were treated with middle (11 months) or long (16 months) period of DR (60% of ad libtium (AL). DR effectively reduced blood glucose levels and fibrosis of skeletal muscle in both middle-aged and elderly groups (p< 0.05). DR promoted mitophagy (PINK1, Parkin, LC3I) in the middle-aged mice (p< 0.05). Otherwise, DR significantly reduced oxidative stress (TBARS) and total mitochondrial volume (VDAC), but increased mitochondrial dynamics (Drp1, Opa1) and mitochondrial area (p< 0.05) in the skeletal muscle of elderly mice. These results suggest that DR exhibits different actions on MQC during the progress of aging. DR mainly promots mitophagy in middle-aged whereas maintained mitochondrial dynamics in elderly-age, and therefore, DR sustains functional mitochondria to delay fibrosis of skeletal muscle. The third experiment aimed to establish in vitro aging model of skeletal muscle, and explore the mechanism of DR on MQC of the aging muscles. C2C12 myoblasts were differentiated into myotubes, and induced for aging by oxidative stress reduction. Curcumin was used as DR mimic to explore the molecular mechanism of DR on regulating aging. Results showed that curcumin decreased the expression of apoptotic proteins (p53, p21) to attenuate aging of skeletal muscle. In summary, in vivo experiments suggested that DR reduced the effects of aging and atrophy on skeletal muscle via promoting mitophagy and mitochondrial dynamics. The detailed mechanisms of DR in regulating mitochondrial functionality further studies. | en |
| dc.description.provenance | Made available in DSpace on 2023-03-19T23:16:20Z (GMT). No. of bitstreams: 1 U0001-1907202217585000.pdf: 5078554 bytes, checksum: 89e0e3b2b3fee96578a529cc54a41288 (MD5) Previous issue date: 2022 | en |
| dc.description.tableofcontents | 目錄 謝誌 i 中文摘要 ii ABSTRACT iv 目錄 vii 圖次 xi 表次 xiii 前言 1 壹、文獻回顧 2 一、 骨骼肌之生理與老化 2 (一) 骨骼肌之生理功能與組成 2 (二) 骨骼肌之老化 5 二、 粒線體與骨骼肌之關聯 8 (一) 粒線體功能 8 (二) 骨骼肌老化與粒線體之關聯 11 三、 限制飲食及其應用方式 12 (一) 任食與限制飲食的介紹 12 (二) 限制飲食之應用 13 (三) 限制飲食與代謝適應 17 (四) 限制飲食模擬物 18 四、 限制飲食對骨骼肌之影響 18 (一) DR影響骨骼肌的代謝機制 18 (二) DR透過粒線體對骨骼肌之影響 19 (三) DR於骨骼肌老化之應用 21 五、 試驗設計 21 貳、 材料與方法 23 一、 動物飼養模式 23 二、 細胞試驗 24 (一) 細胞培養 24 (二) 細胞分化 25 (三) 細胞活性測試 25 三、 動物血糖值 26 四、 組織內三酸甘油脂濃度 26 五、 硫代巴比妥酸反應產物 (Thiobarbituric Acid Reactive Substances, TBARS) 27 (一) 藥品配置 27 (二) 試劑反應 27 六、 抗氧化能力檢測 (Oxygen Radical Absorbance Capacity, ORAC) 28 (一) 藥品配製 28 (二) 試劑反應 28 七、 組織切片與膠原蛋白定量 29 (一) 馬森三色染色法 (Masson's trichrome) 29 (二) 膠原蛋白定量 30 八、 電子顯微鏡 (Transmission electron microscope, TEM) 30 九、 蛋白質萃取與濃度測定 32 (一) 細胞株之蛋白質萃取 32 (二) 組織蛋白質萃取 33 (三) 西方墨點法 33 十、 統計方法 35 參、 試驗結果 36 一、 老化對小鼠生理之影響 36 (一) 老化對體重、血液血糖值與三酸甘油脂影響 36 (二) 氧化壓力指標 38 (三) 組織切片與膠原蛋白定量 39 (四) 電子顯微鏡 41 (五) MQC之蛋白質表現 42 二、 DR對小鼠不同老化程度之影響 49 (一) 小鼠對體重、血液血糖值與三酸甘油脂影響 49 (二) 氧化壓力指標 51 (三) 組織切片與膠原蛋白定量 52 (四) 電子顯微鏡 53 (五) MQC之蛋白質表現 55 三、 體外試驗 65 (一) C2C12肌原母細胞分化 65 (二) C2C12肌管型態誘發成衰老模型 67 (三) DR模擬物減緩C2C12肌管衰老 68 肆、 討論 70 一、 年齡對骨骼肌之影響 70 (一) 生理指標之變化 70 (二) 老化骨骼肌中粒線體之變化 71 二、 DR延緩老化骨骼肌之影響 73 (一) 不同期間DR對於生理之影響 73 (二) 中年DR對於骨骼肌MQC之影響 73 (三) 老年DR對於骨骼肌MQC之影響 74 三、 結論 77 伍、 附錄 78 一、 骨骼肌之細胞凋亡指標 78 陸、 參考文獻 79 圖次 圖 1. 粒線體生合成路徑 9 圖 2. 粒線體融合分裂 10 圖 3. 粒線體自噬作用 11 圖 4. 試驗設計圖 24 圖 5. 粒線體測量示意圖 31 圖 6. 肌節長度測量示意圖 32 圖 7. 小鼠各階段之體重 36 圖 8. 小鼠各階段之血液血糖值 37 圖 9. 骨骼肌內三酸甘油酯量 37 圖 10. 骨骼肌氧化壓力指標 38 圖 11. 骨骼肌馬森三色染色法以及校正量化示意圖 39 圖 12. 骨骼肌纖維化之量化 40 圖 13. 小鼠青年組與老年組骨骼肌電子顯微鏡示意圖 41 圖 14. 骨骼肌粒線體總量 42 圖 15. 骨骼肌粒線體生合成相關蛋白 43 圖 16. 骨骼肌氧化磷酸化酶相關蛋白 44 圖 17. 骨骼肌檸檬酸合成酶活性 45 圖 18. 骨骼肌粒線體融合與分裂蛋白 46 圖 19. 骨骼肌粒線體自噬相關蛋白 48 圖 20. 小鼠DR與AL之體重 49 圖 21. 小鼠DR與AL之血液血糖值 50 圖 22. 小鼠DR與AL之骨骼肌三酸甘油脂 50 圖 23. 骨骼肌內硫代巴比妥酸反應產物與抗氧化能力檢測 51 圖 24. 骨骼肌馬森三色染色法與校正示意圖與纖維化量化 52 圖 25. 小鼠骨骼肌之肌小節縱切面測量 53 圖 26. 小鼠骨骼肌電子顯微鏡下粒線體測量示意圖 54 圖 27. 老年小鼠骨骼肌之粒線體面積、圓度與最大條帶直徑 55 圖 28. 骨骼肌粒線體總量 56 圖 29. 骨骼肌粒線體生合成相關蛋白 57 圖 30. 中年骨骼肌氧化磷酸化酶相關蛋白 58 圖 31. 老年骨骼肌氧化磷酸化酶相關蛋白 59 圖 32. 骨骼肌檸檬酸合成活性 60 圖 33. 中年之骨骼肌粒線體融合與分裂蛋白 61 圖 34. 老年之骨骼肌粒線體融合與分裂蛋白 61 圖 35. 中年骨骼肌粒線體自噬相關蛋白 63 圖 36. 老年骨骼肌粒線體自噬相關蛋白 64 圖 37. C2C12肌原母細胞分化 66 圖 38. 不同濃度H2O2 影響C2C12肌管細胞存活率 67 圖 39. C2C12肌管受氧化壓力後老化蛋白之表現 68 圖 40. 受氧化壓力C2C12肌管添加薑黃素與否之細胞活性 69 圖 41. 薑黃素添加與否對受氧化壓力之C2C12肌管影響 69 圖 42. 細胞凋亡路徑相關蛋白之比較 78 表次 表 1. 不同肌肉纖維之特性 4 表 2. 一級抗體 34 表 3. 二級抗體 35 表 4. 近年文獻探討DR對骨骼肌影響 76 | |
| dc.language.iso | zh-TW | |
| dc.subject | 薑黃素 | zh_TW |
| dc.subject | 老年 | zh_TW |
| dc.subject | 限制飲食 | zh_TW |
| dc.subject | 限制飲食模擬物 | zh_TW |
| dc.subject | 骨骼肌老化 | zh_TW |
| dc.subject | 粒線體品質 | zh_TW |
| dc.subject | mitochondrial quality control | en |
| dc.subject | curcumin | en |
| dc.subject | grontology | en |
| dc.subject | dietary restriction | en |
| dc.subject | dietary restriction mimic | en |
| dc.subject | skeletal muscle aging | en |
| dc.title | 限制飲食調控粒線體品質以預防骨骼肌老化 | zh_TW |
| dc.title | Dietary restriction attenuates skeletal muscle aging via manipulating mitochondrial quality control | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 110-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林原佑(Yuan-Yu Lin),陳洵一(Shuen-Ei Chen),江信毅(Hsin-I Chiang) | |
| dc.subject.keyword | 老年,限制飲食,限制飲食模擬物,骨骼肌老化,粒線體品質,薑黃素, | zh_TW |
| dc.subject.keyword | grontology,dietary restriction,dietary restriction mimic,skeletal muscle aging,mitochondrial quality control,curcumin, | en |
| dc.relation.page | 91 | |
| dc.identifier.doi | 10.6342/NTU202201554 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2022-07-21 | |
| dc.contributor.author-college | 生物資源暨農學院 | zh_TW |
| dc.contributor.author-dept | 動物科學技術學研究所 | zh_TW |
| dc.date.embargo-lift | 2022-07-22 | - |
| 顯示於系所單位： | 動物科學技術學系 | |
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