研究過氧化氫體和粒線體在肌肉細胞生成過程中的功能變化;Investigating the functional changes of peroxisomes and mitochondria during myogenesis

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题名:	研究過氧化氫體和粒線體在肌肉細胞生成過程中的功能變化;Investigating the functional changes of peroxisomes and mitochondria during myogenesis
作者:	陳威承;CHEN, WEI-CHENG
贡献者:	生命科學系
关键词:	肌肉生成作用;過氧化氫體;粒線體;氧化代謝;肌纖維母細胞;C2C12;Myogenesis;Oxidative metabolism;Peroxisome;Mitochondiron
日期:	2019-07-29
上传时间:	2019-09-03 14:33:10 (UTC+8)
出版者:	國立中央大學
摘要:	在骨骼肌中，過氧化氫體和粒線體為兩個負責氧化代謝及活性氧物質移除的重要胞器，但現今這兩個胞器在肌肉生成過程中，生理上所扮演的角色或功能並未被清楚了解。我們在有關過氧化氫體的初步數據顯示，透過穩定細胞株C2C12-RFP-PTS1 monoclone 13能讓我們觀察肌肉生成作用中過氧化氫體的數量變化，並發現分化後的肌管其過氧化氫體數量有顯著增加，我們也發現在PMB (proliferative myoblast)時期有較高的過氧化氫酶活性。而在有關粒線體的初步數據顯示，我們透過陽離子的親脂性粒線體膜電位染劑MitoTracker Red CMXRos和JC-1，發現在MT (myotube)時期的粒線體膜電位有顯著的下降，但在粒腺體內膜上的重要酵素琥珀酸去氫酶(succinate dehydrogenase, SDH)，其活性也隨著肌肉生成過程中有明顯上升。與氧化代謝有關的活性氧物質(ROS)和ATP含量在肌肉生成過程中分別顯著上升和下降，而氧化還原指數和NADH/FAD的比值在肌肉生成過程中也沒有太大的影響。過去實驗室對Bhlhe40 (basic helix-loop-helix family, membrane e40)在骨骼肌的氧化代謝上有深入了解。我們發現在CMB時期過量表現Bhlhe40並不會影響過氧化氫酶的活性，在CMB和MT時期過量表現Bhlhe40會稍微增加SDH活性而基因減弱(knockdown, KD)則會降低SDH活性，超氧化物歧化酶(superoxide dismutase, SOD)的活性則沒有任何變化。此外我們也觀察到Bhlhe40會降低MTCO1、SDHB和NDUFB8 的蛋白質含量。在未來我們會著重在分子生物機制，觀察在肌肉生成過程中有關氧化代謝基因的RNA和蛋白質表現量的變化，並可以透過穩定細胞株C2C12-RFP-PTS1 monoclone 13應用在藥物篩選等初步實驗，且透過基因減弱技術觀察過氧化氫體的重要生物合成基因pex3、pex16和pex19在骨骼肌內的過氧化氫體在肌肉生成過程中的影響，以模擬過氧化氫體缺陷的相關疾病在骨骼肌的影響，未來也會持續深入了解Bhlhe40在肌肉生成過程中在氧化代謝中的生理意義及分子機制。;Peroxisomes and mitochondria are two crucial organelles responsible for oxidative metabolism and reactive oxygen species (ROS) removal in skeletal muscle (SKM). To date, the physiological roles/functions of both organelles in myogenesis have not been well-established yet. Our preliminary data found that the stable clone C2C12-RFP-PTS1 monoclone 13 allowed us to trace the homeostasis of peroxisome number, and significant increase was found after terminal differentiation. We also found that there was higher catalase activity at proliferative myoblast (PMB) stage. Mitochondrial membrane potential (Δψm) determined by MitoTracker Red CMXRos and JC-1 staining was found decreased at myotube (MT) stage. Mitochondrial succinate dehydrogenase (SDH) activity was increased during myogenesis. ROS was increased but ATP content was decreased during myogenesis. The redox index NADH/FAD ratio didn’t change too much during myogenesis. Our recent studies have demonstrated the deep involvement of basic helix-loop-helix family, membrane e40 (Bhlhe40) in SKM oxidative metabolism. Here we found overexpression of Bhlhe40 didn’t change the catalase activity at CMB stage. We also found overexpression of Bhlhe40 slightly increased SDH, but not SOD, activity at CMB and MT stages. On the contrary, reduced SDH activity was observed in Bhlhe40 knockdown clone. We also found overexpression of Bhlhe40 decreased protein levels of MTCO1 (complex IV), SDHB (complex II) and NDUFB8 (complex I). In the near future, we will do qRT-PCR and Western blot to observe RNA and protein expression of genes related to oxidative metabolism. We will also do gene knockdown of important peroxisome biogenesis gene (pex3, pex16, pex19) on C2C12-RFP-PTS1 monoclone 13 to monitor peroxisome deficient disease during myogenesis by lentivirus infection, and there will be more experiments to investigate the further influence by Bhlhe40.
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