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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃青真 | |
| dc.contributor.author | Yii-Tzu Huang | en |
| dc.contributor.author | 黄苡慈 | zh_TW |
| dc.date.accessioned | 2021-05-19T17:47:19Z | - |
| dc.date.available | 2023-08-23 | |
| dc.date.available | 2021-05-19T17:47:19Z | - |
| dc.date.copyright | 2018-08-23 | |
| dc.date.issued | 2017 | |
| dc.date.submitted | 2018-06-11 | |
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/7592 | - |
| dc.description.abstract | 肌少症 (Sarcopenia) 是指隨著年紀上升，肌肉質量、力量及功能逐漸下降，大致是由於神經退化、長期活動量降低、體內內分泌賀爾蒙雄激素睪固酮 (testosterone)、類胰島素生長因子-1 (Insulin-like growth factor-1, IGF-1) 分泌減少所導致骨骼肌修復能力降低或肌纖維蛋白質分解加速。目前改善肌少症的方針主要為運動及營養補充，亦有許多研究朝向調控肌肉生長並壯大肌肉的路徑著手。山苦瓜可調節諸多生理活性，包括血糖、血脂、免疫及改善代謝異常，然而其對於肌肉生長的研究至今甚少。因此，本研究目標為以細胞模式及動物模式探討山苦瓜對於骨骼肌生長與功能的影響。
在細胞模式中，本實驗以山苦瓜萃物處理 C2C12 肌肉前驅細胞 (myoblast) 48 小時，探討山苦瓜是否可以促進細胞增生。結果顯示，1758 品系山苦瓜乙酸乙酯萃物 (1758 EAE) 單獨處理 myoblast 雖無法使細胞數目增加，但可以促進粒線體功能指標 citrate synthase 活性。再者，1758 EAE 與 IGF-1 共同處理之下，可以顯著增加 myoblast 細胞數目。以 PI3K 抑制劑 LY294002 抑制 IGF-1 傳遞路徑後，1758 EAE 協助 IGF-1 促進細胞增生之效應消失。進一步以西方墨點法分析蛋白質表現量，結果顯示，共處理 myoblast 一小時，1758 EAE 可以幫助 IGF-1增加下游傳遞路徑 Akt 磷酸化表現量。在動物模式中，本研究將不同品系 4 % 山苦瓜全果凍乾粉添加於高脂飲食中，並餵食 C57BL/6J 公鼠進行短期 4 週動物試驗。結果顯示，山苦瓜可以減低高脂飲食誘導之體重、白色脂肪堆積及胰島素阻抗情形，並能增加腓腸肌相對重量，而 1758 品系能進一步促進小鼠腓腸肌 IGF-1、Igf1r、Akt2、Hk2 基因表現。綜合以上，1758 山苦瓜可以協同 IGF-1 促進肌肉前驅細胞增生，並具有調控骨骼肌 IGF-1 下游傳遞路徑的潛力。 | zh_TW |
| dc.description.abstract | Sarcopenia is a gradual loss of skeletal muscle mass, function and strength associated with ageing. The main causes of sarcopenia include loss of motor neurons, physical inactivity or a decline in anabolic hormones such as testosterone and insulin-like growth factor-1 (IGF-1), which lead to reduction in regenerative capacity or increased protein degradation in skeletal muscle. Currently, the validated strategies are exercise and nutritional supplementation. On the other hand, the pathways which stimulate muscle hypertrophy have been actively investigated. Wild bitter gourd (WBG) has been shown to ameliorate metabolic disorders such as hyperglycemia and hyperlipidemia. However, little research was focussed on muscle growth. Hence, the aim of this study is to examine the effect of WBG on skeletal muscle growth and function as an initial approach.
In the cell model, C2C12 myoblasts were treared with WBG extracts for 48 hours to determine the proliferation ability of WBG. The results showed that the WBG cultivar 1758 ethyl acetate extract (EAE) enchanced the activity of citrate synthase, a marker of mitochondria, and, in the presence of IGF-I, promoted myoblasts proliferation. The effect of 1758 EAE on IGF-1-mediated proliferation was diminished in the presence of PI3K inhibitor LY294002. Western blot analysis further showed that 1758 EAE increased Akt phosphorylation of C2C12 myoblasts in the presence of IGF-I after 1 hour of treatment. In the short-term animal study, C57/BL/6J male mice were fed a high-fat diet supplemented without or with 4% various cultivars of WBG for 4 weeks. WBG reduced total body weight, white adipose mass, insulin resistance, and increased relative gastrocnemius muscle mass. 1758 WBG specifically enhanced mRNA expression levels of IGF-1, Igf1r, Akt2, Hk2 in gastrocnemius muscle. These results indicate that 1758 WBG may enhance the proliferation effect of IGF-1 in myoblasts, and might regulate IGF-1 signaling pathway in skeletal muscle. | en |
| dc.description.provenance | Made available in DSpace on 2021-05-19T17:47:19Z (GMT). No. of bitstreams: 1 ntu-106-R04b22024-1.pdf: 6878557 bytes, checksum: 27faa3d87b35685e1b5a6f740d438620 (MD5) Previous issue date: 2017 | en |
| dc.description.tableofcontents | 總目錄
第一章 緒論 1 第一節 前言 1 第二節 文獻回顧 3 一、 肌少症 (Sarcopenia) 3 二、 骨骼肌 (Skeletal muscles) 6 1. 肌纖維 (Muscle fibers) 6 2. 肌肉幹細胞與肌肉再生能力 7 三、 肌肉生長調控路徑 8 1. 類胰島素生長因子-1 IGF-1 (Insulin-like growth factor-1) 8 2. PI3K/Akt 路徑 (圖1-5) (Gonzalez-Franquesa et al., 2012) 9 3. 其他調控路徑 10 四、 粒線體 13 1. 粒線體生合成 (Mitochondrial biogenesis) 與粒線體動態 (mitochondrial dynamics) 13 2. 粒線體生合成調控因子 14 五、 肌肉再生能力與功能失常 16 1. 肌肉幹細胞衰老 16 2. 粒線體功能缺失與代謝異常 17 3. 肌肉與胰島素阻抗 20 六、 山苦瓜 21 第三節 研究假說及實驗架構 23 一、 研究假說 23 二、 實驗架構 23 第二章 山苦瓜萃物對 C2C12 myoblasts 增生能力之影響 24 第一節 前言與實驗設計 24 一、 前言 24 二、 實驗設計 27 第二節 實驗材料與方法 28 一、 材料 28 1. C2C12肌肉前驅細胞 (myoblast cells) (ATCC CRL-1772) 28 2. 培養基與藥品試劑 28 二、 細胞培養與繼代 29 三、 山苦瓜樣品製備 29 1. 山苦瓜樣品 29 2. 山苦瓜樣品水解處理 29 3. 山苦瓜樣品萃取 29 四、 細胞存活率分析—MTT assay 30 五、 細胞增生試驗—Trypan Blue Exclusion 細胞計數 30 六、 粒線體檸檬酸合成酶酵素活性分析 31 1. 樣品製備 31 2. 藥品試劑 31 3. 原理 31 4. 方法步驟 32 七、 PI3-Kinase抑制劑 LY294002 介入試驗 33 八、 蛋白質表現量分析—西方墨點法 33 1. 樣品製備 33 2. 藥品試劑 33 3. 儀器設備 36 4. 實驗方法 36 九、 統計分析 38 第三節 實驗結果 39 一、 細胞存活率分析──MTT assay 39 二、 細胞增生試驗──Trypan Blue Exclusion 細胞計數 39 三、 粒線體檸檬酸合成酶 (Citrate synthase) 酵素活性分析 40 四、 PI3-Kinase抑制劑 LY294002 介入試驗 40 五、 蛋白質表現量分析—西方墨點法 41 第四節 討論 53 一、 不同品系山苦瓜暨水解乙酸乙酯萃取物對 C2C12 myoblasts 增生能力的影響 53 二、 山苦瓜萃取物調控 C2C12 myoblasts 增生能力之可能活性成份 57 1. 三萜類 (Triterpenoids) 57 2. 共軛次亞麻油酸 (9c, 11t, 13t-Conjugated Linolenic Acid, CLN) 58 三、 山苦瓜萃取物調控 C2C12 myoblasts 增生能力之調節因子相關性探討 61 1. PPAR-δ (Peroxisome proliferator-activated receptor δ) 61 四、 1758 山苦瓜萃取物促進 IGF-1 對 C2C12 myoblasts 增生能力之作用路徑 64 五、 1758 山苦瓜萃取物促進 C2C12 myoblasts 之粒線體功能 66 第五節 結論 66 第三章 不同品系山苦瓜對高脂飲食模式小鼠骨骼肌功能的影響 67 第一節 前言與實驗架構 67 一、 前言 67 二、 實驗設計 70 第二節 實驗材料與方法 72 一、 動物飼養 72 二、 飼料配製 72 1. Chow diet 飼料 72 2. 高脂飲食 72 3. 添加山苦瓜之高脂飲食 73 三、 禁食血清樣本收集 76 四、 動物犧牲與樣品收集 76 1. 器具準備 76 2. 實驗流程 76 3. 組織保存 76 五、 血清生化分析 77 1. 葡萄糖 (glucose) 分析 77 2. 三酸甘油酯 (triglycerides) 測定 77 3. 膽固醇 (cholesterol) 測定 78 4. 胰島素 (insulin) 分析 79 六、 肌肉基因 mRNA 表現分析 80 1. 組織 total RNA 抽取 80 2. Total RNA反轉錄成 cDNA 81 3. Quantitative Real-Time PCR 81 六、 統計分析 83 第三節 實驗結果 84 一、 生長情形 84 1. 生長曲線與體重變化 84 2. 攝食量、攝食利用率、能量攝取與能量利用率 84 3. 器官與組織重量 85 二、 血清生化分析 86 三、 骨骼肌基因 mRNA 表現分析 87 第四節 討論 111 一、 山苦瓜有助於減緩短期高脂飲食模式小鼠之代謝症候群指標 111 1. 體重變化情形、器官組織重量及血清生化分析 111 2. 短期誘發肥胖模式與過去研究之比較 113 二、 山苦瓜對於小鼠骨骼肌基因表現之影響 115 1. 粒線體生合成、融合分裂與代謝 115 2. 骨骼肌生長 116 3. IGF-1、Akt 及下游調控路徑 117 第五節 結論 118 第四章 綜合討論與總結論 124 第一節 綜合討論 124 第二節 總結論 126 第五章 參考文獻 127 第六章 附錄 139 圖目錄 圖 1- 1. 造成肌少症的可能原因 5 圖 1- 2. 導致肌肉萎縮的路徑 5 圖 1- 3.靜止的衛星細胞、啟動的衛星細胞以及肌肉再生 11 圖 1- 4. 哺乳類肌肉發育與成熟 11 圖 1- 5. Insulin & IGF-1 訊息傳遞路徑 12 圖 1- 6. PGC-1α 調控途徑 16 圖 1- 7. 粒線體融合分裂失調導致肌肉萎縮 19 圖 1- 8. 粒線體功能失常與胰島素阻抗 19 圖 2- 1. 肌肉前驅細胞分化為成熟肌小管 25 圖 2- 2. 實驗設計－細胞模式 26 圖 2- 3. 檸檬酸合成酶酵素活性分析原理 32 圖 2- 4. 各品系山苦瓜乙酸乙酯萃物單獨處理或與 IGF-1 共處理對 C2C12 myoblasts 增生的影響 (MTT assay) 43 圖 2- 5. 經不同溫度水解之1758山苦瓜乙酸乙酯萃物單獨處理及與 IGF-1 共處理對 C2C12 myoblasts 增生的影響 (MTT assay) 45 圖 2- 6. 1758山苦瓜乙酸乙酯萃物單獨處理及與 IGF-1 共處理對 C2C12 myoblasts 增生的影響 (Trypan Blue Exclusion 細胞計數) 46 圖 2- 7. 1758山苦瓜乙酸乙酯萃物對 C2C12 myoblasts粒線體檸檬酸合成酶酵素活性的影響 48 圖 2- 8. 抑制劑 LY294002 介入對 1758 山苦瓜乙酸乙酯萃物造成 C2C12 myoblasts 增生效果之影響 50 圖 2- 9. C2C12 myoblasts 給予 1758 EAE 處理 1 小時之 Akt 蛋白質及其磷酸化表現量 51 圖 2- 10. 各品系山苦瓜三萜類含量與促進 C2C12 myoblasts 增生能力之相關性 59 圖 2- 11. 各品系山苦瓜乙酸乙酯萃取物之 CLN 吸收光譜 60 圖 2- 12. 各品系山苦瓜乙酸乙酯萃取物之 CLN 含量 60 圖 2- 13. 各品系山苦瓜乙酸乙酯萃取物 CLN 含量與促進 C2C12 myoblasts 增生能力之相關性 61 圖 2- 14. 各品系山苦瓜乙酸乙酯萃取物活化 PPAR-δ 的轉錄活性與促進 C2C12 myoblasts 增生能力之相關性 63 圖 2- 15. 山苦瓜水解乙酸乙酯萃取物活化 PPAR-δ 的轉錄活性與促進 C2C12 myoblasts 增生能力之相關性 63 圖 2- 16. 胰島素受體、IGF-1 受體與雜合受體 65 圖 3- 1. 肥胖對肌肉再生能力影響之機制 69 圖 3- 2. C57BL/6J 公鼠餵食實驗飼料 4 週之生長曲線 91 圖 3- 3. C57BL/6J 公鼠餵食實驗飼料 4 週之體重增加量 92 圖 3- 4. C57BL/6J 公鼠餵食試驗飼料 4 週之能量攝取與能量效率 95 圖 3- 5. C57BL/6J 公鼠餵食試驗飼料 4 週後之脂肪組織之絕對與相對重量 100 圖 3- 6. 小鼠餵食試驗飼料 4 週後腓腸肌、比目魚肌與脛前肌之絕對及相對重量 101 圖 3- 7. C57BL/6J 小鼠餵食試驗飼料 4 週之血糖、血脂、血膽固醇之變化 103 圖 3- 8. C57BL/6J 小鼠於正式實驗滿 4 週之禁食血清胰島素、血糖及胰島素阻抗指標 HOMA-IR index 104 圖 3- 9. C57BL/6J 公鼠正式實驗滿四週之腓腸肌粒線體生合成、融合分裂、氧化磷酸化相關基因表現 105 圖 3- 10. C57BL/6J 公鼠正式實驗滿四週之腓腸肌肌肉增生、分化相關基因表現 106 圖 3- 11. C57BL/6J 公鼠正式實驗滿四週之腓腸肌 IGF-1、IGF-1R、Akt 及下游調控路徑基因表現 107 圖 3- 12. C57BL/6J 公鼠正式實驗滿四週之比目魚肌粒線體生合成、融合分裂、氧化磷酸化相關基因表現 108 圖 3- 13. C57BL/6J 公鼠正式實驗滿四週之比目魚肌肌肉增生、分化相關基因表現 109 圖 3- 14. C57BL/6J 公鼠正式實驗滿四週之比目魚肌 IGF-1、IGF-1R、Akt 及下游調控路徑基因表現 110 圖 3- 15. 脂肪組織慢性發炎導致肌肉組織胰島素阻抗 114 圖 3- 16. C57/BL/6J 公鼠餵食實驗飼料 4 週後體重、血清胰島素、HOMA-IR Index 與比目魚肌 Mstn 基因表現之相關性 123 表目錄 表 2- 1. 不同品種山苦瓜乙酸乙酯萃物對 C2C12 myoblasts 增生的影響 55 表 2- 2. 未經水解處理與經 37℃、50℃以及60℃ 水解之山苦瓜乙酸乙酯萃物對 C2C12 myoblasts 增生的影響 56 表 3- 1. C57BL/6J 公鼠各肌群的纖維型態 69 表 3- 2. 市售 Chow diet 組成 74 表 3- 3. 實驗飼料配方 75 表 3- 4. 本研究 qPCR 分析使用引子整理表 82 表 3- 5. C57BL/6J 公鼠餵食試驗飼料 4 週之初始體重、最終體重及體重增加量 93 表 3- 6. C57BL/6J 公鼠餵食試驗飼料 4 週之攝食量、攝食利用率、能量攝取與能量利用率 94 表 3- 7. C57BL/6J 公鼠餵食試驗飼料 4 週後之體重與器官絕對重量 96 表 3- 8. C57BL/6J 公鼠餵食試驗飼料 4 週後之骨骼肌、脂肪組織絕對重量 97 表 3- 9. C57BL/6J 公鼠餵食試驗飼料 4 週後之器官相對重量 98 表 3- 10. C57BL/6J 公鼠餵食試驗飼料 4 週後之骨骼肌、脂肪組織相對重量 99 表 3- 11. C57BL/6J 小鼠餵食試驗飼料 4 週之禁食血清生化分析 102 表 3- 12. 各品系山苦瓜對小鼠生長情形、組織重量及血清生化值及整理表 119 表 3- 13. 本研究與過去研究結果比較 120 表 3- 14. 各品系山苦瓜對小鼠腓腸肌相關基因表現整理 121 表 3- 15. 各品系山苦瓜對小鼠比目魚肌相關基因表現整理 122 | |
| dc.language.iso | zh-TW | |
| dc.title | 山苦瓜萃取物協助 IGF-1 透過 PI3K/Akt 路徑促進小鼠骨骼肌前驅細胞生長 | zh_TW |
| dc.title | Wild Bitter Melon Extract Assists IGF-1 in Promoting Mouse Skeletal Muscle Cell Proliferation Through PI3K/Akt Pathway | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 林甫容,褚志斌,蕭培文,呂紹俊 | |
| dc.subject.keyword | 山苦瓜,肌肉細胞,類胰島素生長因子-1,粒線體, | zh_TW |
| dc.subject.keyword | Wild bitter gourd,C2C12 myoblasts,IGF-1,Mitochondria, | en |
| dc.relation.page | 140 | |
| dc.identifier.doi | 10.6342/NTU201704166 | |
| dc.rights.note | 同意授權(全球公開) | |
| dc.date.accepted | 2018-06-12 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| dc.date.embargo-lift | 2023-08-23 | - |
| 顯示於系所單位： | 生化科技學系 | |
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