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| DC 欄位 | 值 | 語言 |
|---|---|---|
| dc.contributor.advisor | 黃青真(Ching-Jang Huang) | |
| dc.contributor.author | Yu-Xiang Su | en |
| dc.contributor.author | 蘇郁翔 | zh_TW |
| dc.date.accessioned | 2021-06-17T04:29:04Z | - |
| dc.date.available | 2018-08-16 | |
| dc.date.copyright | 2018-08-16 | |
| dc.date.issued | 2018 | |
| dc.date.submitted | 2018-08-13 | |
| dc.identifier.citation | Abmayr, S. M., Pavlath, G. K. (2012). Myoblast fusion: lessons from flies and mice. Development, 139(4), 641-656.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/70478 | - |
| dc.description.abstract | 目前有許多研究探討飲食調控肌肉生長或促進粒線體生合成。已有諸多文獻顯示，山苦瓜能透過多種機制進行血糖調節、改善脂肪代謝等問題。本實驗室先前研究指出，長期餵食山苦瓜之 C57BL/6J 小鼠，其骨骼肌粒線體生合成相關基因表現量增加。已知山苦瓜乙酸乙酯萃取物中含有多種活性化合物，其中共軛三烯亞麻油酸 (CLN) 的含量相當高。因此本研究之目標是以未分化的小鼠 C2C12 肌肉前驅細胞 (myoblasts) 模式更進一步探討山苦瓜萃取物促進粒線體活性之效果以及分析共軛脂肪酸 CLN 對粒線體的影響。
實驗先以1758品系之山苦瓜乙酸乙酯萃取物 (1758 EAE) 處理 C2C12肌肉前驅細胞 (myoblast) 0、1、6、24、48 小時，探討 1758 EAE 對粒線體的促進效果與時間之關係。結果顯示100 μg/mL 1758 EAE 處理細胞 24 小時後，可在不影響細胞數目的情況下，增加 MTT 讀值、促進粒線體功能指標 citrate synthase 活性、Mitrotracker 之相對螢光量。而 50 μg/mL 1758 EAE 處理細胞 6 小時後，可促進粒線體生合成相關基因之表現。此外，25 μM 的共軛脂肪酸 CLA亦可發現與 1758 EAE 有相似的效果，然而 CLN於此細胞模式下則無顯著之效果，且還發現 CLN 對於 C2C12 myoblasts 的毒性較另外兩者高，超過 10 μM 即造成細胞存活率降低。進一步以西方墨點法分析蛋白質表現量，結果顯示上述濃度之 1758 EAE 或 CLA 處理細胞 1 小時，可以促進粒線體生合成相關調控因子 AMPK 磷酸化表現量，而 CLN 於此細胞模式下無觀察到該現象。再者本研究以海馬生物能量代謝分析儀分析細胞之氧氣消耗速率 (OCR) 以及產酸速率 (ECAR)，結果顯示 100 μg/mL 1758 EAE 或 25 μM CLA 先處理細胞 24 小時後，細胞之基礎氧氣消耗速率與產酸速率明顯提升。而 CLN 則是皆無差異。 綜合以上，1758 EAE 與 CLA 於本細胞模式中，皆有促進粒線體活性的能力。然而 CLN 的部分，雖未觀察到明顯促進粒線體活性的效果，但是未來可藉由測試不同的處理條件，改善其對於 C2C12 myoblasts 毒性過高的問題後再進行粒線體之相關研究，或許可觀察到不同的結果。 | zh_TW |
| dc.description.abstract | Mitochondria play an important role in skeletal muscle metabolism. Impaired mitochondrial biogenesis is known to be associated with the development of metabolic diseases such as insulin resistance and type 2 diabetes. Previous studies showed that dietary wild bitter gourd can improve metabolic disorders such as insulin resistance and dysregulations of lipid metabolism through various mechanisms. Recent in vivo studies in our lab demonstrated that mice fed a 5% wild bitter gourd powder (BGP) diet up-regulated genes related to mitochondrial biogenesis. It is known that wild bitter gourd is rich in conjugated linolenic acid, which is one of the many bioactive compounds of this common tropical vegetable. This study aims to examine effects of wild bitter gourd extracts and conjugated fatty acid on mitochondria in C2C12 myoblasts.
In this study, C2C12 myoblasts were cultivated in 0.1%BSA/DMEM and treated with cultivar-1758 wild bitter gourd ethyl acetate extract (1758 EAE) for 0, 1, 6, 24, 48 hours. Cell numbers were determined by counting as well as the MTT assay. Compared to the vehicle-treated cells, cells treated with 50 and 100 μg/mL 1758 EAE showed significantly higher optical density in the MTT assay but did not change the cell number from counting after 24 hours of treatment. In addition, 50 or 100 μg/mL 1758 EAE enhanced the activity of citrate synthase of the cells, a marker of mitochondrial function. In addition, the relative fluorescence of mitotracker staining was significantly higher in cells treated with these concentrations of 1758EAE. Cells treated with 100 μg/mL 1758 EAE for 6 hr showed up-regulated mitochondria-related gene expression. Cells treated with 25 μM conjugated linoleic acid (CLA) showed similar effects as the 1758 EAE. However, conjugated linolenic acid (CLN) showed significant cytotoxic effects at concentrations higher than 10μM in this system. No significant effects can be observed at this concentration. Western blot analysis further showed that 100 μg/mL 1758 EAE and 25 μM CLA increased AMPK phosphorylation of C2C12 myoblasts after one-hour treatment. Moreover, 100 μg/mL 1758 EAE and 25 μM CLA can also increase the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after 24 hours of treatment. In conclusion, results of this study indicated that 50 or 100 μg/mL 1758 EAE and 25 μM CLA enhance the mitochondrial function in C2C12 myoblasts, and the regulation might be through the pathway involved AMPK and mitochondrial biogenesis. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-17T04:29:04Z (GMT). No. of bitstreams: 1 ntu-107-R05B22053-1.pdf: 2539703 bytes, checksum: caa1bcbfe8e3e749ece08f75ad283da5 (MD5) Previous issue date: 2018 | en |
| dc.description.tableofcontents | 中文摘要 i
Abstract iii 縮寫對照表 iv 總目錄 vi 圖目錄 ix 表目錄 x 第一章、緒論 1 1.1 前言 1 1.2 文獻回顧 2 1.2.1 骨骼肌 (Skeletal muscles) 2 1.2.2 肌肉幹細胞 4 1.2.3 粒線體 6 1.2.4 粒線體生合成 (Mitochondrial biogenesis) 7 1.2.5 粒線體生合成的研究方法 11 1.2.6 粒線體動態平衡 (Mitochondrial dynamic network) 14 1.2.7 去偶合作用 (Mechanism of uncoupling in mitochondria) 15 1.2.8 山苦瓜 18 1.2.9 共軛脂肪酸 (Conjugated fatty acid) 20 1.3 研究目的與架構 22 1.3.1 研究目的 22 1.3.2 研究架構 25 第二章、材料與方法 27 2.1 樣品製備 27 2.1.1 山苦瓜品系與粉末製備 27 2.1.2 山苦瓜乙酸乙酯萃取物 (Ethyl acetate extract, EAE) 製備 27 2.1.3 市售共軛脂肪酸樣品製備 27 2.2 細胞培養與繼代 27 2.2.1 C2C12 小鼠肌肉前驅細胞株培養 27 2.2.2 培養基與藥品試劑 28 2.3 實驗方法 29 2.3.1 MTT assay 29 2.3.2 細胞計數 (Cell number counting) 31 2.3.3 粒線體檸檬酸合成酶酵素活性分析 (Citrate synthase activity) 32 2.3.4 粒線體相關基因分析 35 2.3.5 粒線體複本數 (Mitochondrial copy number) 38 2.3.6 Mitotracker 螢光染色分析 40 2.3.7 蛋白質磷酸化表現量分析-西方墨點法 43 2.3.8 細胞氧氣消耗與產酸速率 48 2.3.9 統計分析 50 第三章、結果與討論 51 3.1 結果 51 3.1.1 0.1% BSA/DMEM 培養基對細胞數目之影響 51 3.1.2 以不同濃度 1758 EAE 處理細胞後之 MTT 染色結果 51 3.1.3 MTT 染色與細胞計數比較 51 3.1.4 粒線體 CS 活性分析 52 3.1.5 粒線體相關基因表現分析 53 3.1.6 粒線體複本數分析 53 3.1.7 Mitotracker 染色分析 54 3.1.8 AMPK 蛋白質及其磷酸化表現量 54 3.1.9 細胞氧氣消耗速率與產酸速率 55 3.2 討論 69 3.2.1 以 MTT assay 與細胞計數之結果找尋較佳的實驗條件 69 3.2.2 1758 EAE 與共軛脂肪酸對小鼠肌肉前驅細胞粒線體之影響 71 3.2.3 山苦瓜萃取物調控 C2C12 myoblasts 粒線體生合成之可能活性成分 77 3.2.4 比較本研究與前人研究 79 第四章、結論 82 第五章、參考文獻 83 | |
| dc.language.iso | zh-TW | |
| dc.subject | 山苦瓜 | zh_TW |
| dc.subject | AMPK | zh_TW |
| dc.subject | 粒線體 | zh_TW |
| dc.subject | 共軛脂肪酸 | zh_TW |
| dc.subject | 肌肉前驅細胞 | zh_TW |
| dc.subject | conjugated fatty acid | en |
| dc.subject | Wild bitter gourd | en |
| dc.subject | C2C12 myoblasts | en |
| dc.subject | AMPK | en |
| dc.subject | mitochondria | en |
| dc.title | 以 C2C12 小鼠肌肉前驅細胞模式評估山苦瓜萃取物與共軛脂肪酸對粒線體之影響 | zh_TW |
| dc.title | Effects of Momordica charantia Extract and Conjugated Fatty Acids on Mitochondria in C2C12 Myoblasts | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 106-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.oralexamcommittee | 呂紹俊,龔秀妮,林甫容,張美鈴 | |
| dc.subject.keyword | 山苦瓜,肌肉前驅細胞,共軛脂肪酸,粒線體,AMPK, | zh_TW |
| dc.subject.keyword | Wild bitter gourd,C2C12 myoblasts,conjugated fatty acid,mitochondria,AMPK, | en |
| dc.relation.page | 92 | |
| dc.identifier.doi | 10.6342/NTU201803153 | |
| dc.rights.note | 有償授權 | |
| dc.date.accepted | 2018-08-13 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 生化科技學系 | zh_TW |
| 顯示於系所單位： | 生化科技學系 | |
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