臺灣博碩士論文加值系統

肌肉萎縮症主要的臨床特徵為骨骼肌逐漸萎縮並且發炎，這些症狀導致病患後期失去步行能力最終死亡，到現在為止還未有任何治療可以阻止或是治癒肌肉萎縮所造成的傷害，但主要用於治療炎症的糖皮質激素像是dexamethasone(Dex)則可以延緩肌肉萎縮的速度並且穩定肌肉強度，然而在某些情況下糖皮質激素不僅無法治療肌肉病變甚至導致肌肉萎縮，因此我們想要了解Dex在抑制發炎與肌肉分化中的詳細機制。為了研究Dex在抑制發炎的詳細機制我們使用巨噬細胞Raw264.7細胞株，我們發現Dex會經由11與21羥基抑制腫瘤壞死因子的分泌而不影響蛋白或是mRNA的表現，而Dex也會經由11與21羥基降低p38 MAPK磷酸化表現近一步抑制釋放腫瘤壞死因子 tumor necrosis factor-alpha converting enzyme (TACE)的酵素活性。
另一方面為了研究Dex對於肌肉分化的影響我們使用老鼠肌母細胞C2C12細胞株，我們發現在肌肉分化過程中需要kinesin family member 5b (kif5b)，而在體外實驗中Dex會加快kif5b的速度，這也顯示Dex可能會經由kif5b影響肌肉生成，因為kif5b負責運送肌原纖維的組成蛋白與粒線體，所以我們更進一步探討在肌肉分化過程中Dex使否會經由加快kif5b的速度而影響粒線體，海馬能量測定儀的結果顯示在分化三天後Dex會促進粒線體功能但會降低細胞中ATP總量，我們推測可能因為Dex會提升kif5b的速度而消耗掉ATP，總結這些實驗我們發現Dex不只會經由降低p38 MAPK與TACE酵素活性影響腫瘤壞死因子的釋放來抑制發炎，還會經由加快kif5b速度影響肌肉分化。

Muscular dystrophy, a group of muscle diseases, contains main clinical features that are progressive loss of skeletal muscle and prominent muscle inflammation, results in loss of walking ability and death in the late stage. To date, there is no available treatment to stop or reverse any form of muscular dystrophy, while glucocorticoids, such as dexamethasone (Dex), are therapeutically applied to inhibit inflammation, slow muscle degeneration and stabilize muscle strength. However, in some cases, glucocorticoids also cause to reduce myopathy ineffectively or to promote muscle atrophy in vivo and in vitro with unknown mechanisms. Therefore, we aim to investigate the detailed mechanisms of Dex in anti-inflammation and myogenesis. To study the effect of Dex in anti-inflammation, we used macrophages RAW264.7 cells. We find that Dex inhibits the secretion of tumor necrosis factor-alpha (TNF-alpha, a pro-inflammatory cytokine, instead of protein and mRNA expression through its 11- and 21-hydroxyl groups. Dex, via its 11- and 21-hydroxyl groups, inhibits p38 MAPK phosphorylation level and further suppresses the enzymatic activity of tumor necrosis factor-α-converting enzyme (TACE) that releases membrane form of TNF-. To study the effect of Dex in myogenesis, we used murine myoblasts C2C12 cells. We find that the expression of kinesin family member 5b (kif5b) is required for myogenesis, since the motor activity of purified kif5b proteins is accelerated by Dex treatment, suggesting the important role of kif5b in Dex-regulated myogenesis. Because kif5b is responsible for localizing myofibril components and mitochondria, we further determine whether Dex functions in mitochondria-mediated energy production through kif5b motor proteins during myogenesis. We find that Dex treatment significantly increases the basal expiration and ATP production of C2C12 cells after 3 days myogenesis induction by Seahorse XFe24 Extracellular Flux Analyzer. By analyzing the amount of total ATP, we find that Dex treatment significantly suppressed the amount of ATP in C2C12 cells after 3 days myogenesis induction. These data indicate that Dex may consume more ATP because of promoting kif5b motility. In summary, we find that Dex not only function in anti-inflammation through suppressing p38 MAPK signaling and TACE activity for TNF- secretion, but also directly targets kif5b motor proteins to accelerate its motility that is required for myogenesis.

中文摘要....i
英文摘要....ii
目錄....iv
第一章 研究背景與動機....1
2.1 Dexamethasone (Dex)....1
2.2 發炎反應....2
2.3 Tumor Necrosis factor-alpha converting enzyme (TACE)....3
2.4 肌肉生成 (Myogenesis)....3
2.5 肌肉再生 (Muscle regeneration)....4
2.6 Kinesin family membrane 5B（Kif5b）....5
2.7 研究動機....6
第二章 實驗材料與方法....7
2.1 細胞株 (Cell line)....7
2.2 抗體 (Antibody)....7
2.4 西方墨點法 (western blot)....9
2.5 RNA萃取....9
2.6 Genomic DNA萃取....10
2.7 即時聚合酶鏈鎖反應 (Quantitative real time polymerase chain reaction)....10
2.8 酵素免疫分析法 (Enzyme-Linked ImmunoSorbent Assay)....10
2.9 Tumor Necrosis factor-alpha converting enzyme 活性測試....11
2.10 TACE Inhibitor Screening Assay Kit....11
2.11 ATP 定量....12
2.12 吉姆薩染色 (Giemsa stain)....12
2.13 粒線體功能分析....13
第三章 實驗結果....15
3.1 Dex不影響腫瘤壞死因子-蛋白表現但影響分泌出細胞外的量....15
3.2 Dex可能經由抑制p38磷酸化降低TACE酵素活性....15
3.3 Dex會經由11與21羥基降低p38磷酸化與TACE酵素活性進而影響腫瘤壞死因子-....16
3.5 Dex短時間不會經由kif5b影響粒線體....17
3.6 Dex可能影響NMHC-II-A、NMHC-II-B與Clip170蛋白運送而促進肌肉分化....19
3.7 肌肉分化過程中Dex會影響粒線體功能....19
3.8 Kif5b為Dex影響肌肉分化的分子之一....20
第四章 實驗討論....21
第五章 圖和圖例....24
Figure 1. Dex抑制腫瘤壞死因子-分泌到細胞外....25
Figure 2. Dex抑制p38磷酸化並且降低TCAE酵素活性....26
Figure 3. 結構示意圖....28
Figure 4. Dex失去21號與11號碳上的羥基後，抑制腫瘤壞死因子-分泌的能力會下降....29
Figure 5. Dex失去21號與11號碳上的羥基後無法抑制腫腫瘤壞死因子-回饋刺激產生的mRNA....30
Figure 6. Dex失去21號與11號碳上的羥基後降低p38磷酸化與抑制TACE酵素活性的能力降低....31
Figure 7. Dex會促進C2C12肌母細胞分化與融合....32
Figure 8. Dex短時間不經由kif5b影響粒線體功能與ATP總量....33
Figure 9. Dex會影響肌肉分化、粒線體與微管相關蛋白的表現....35
Figure 10. 在肌肉分化過程中Dex會影響ATP總量與粒線體耗氧率....36
Figure 11. 缺乏kif5b會影響肌肉分化....37
第六章 參考資料....38
第七章 附錄....41

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