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標題: | TGF-β1誘導的線粒體功能障礙與肌肉生成有關 TGF-β1-induced Mitochondrial Dysfunction Implicated in the Myogenesis |
作者: | Yi-Ting Wu 吳宜庭 |
指導教授: | 姜至剛(Chih-Kang Chiang) |
關鍵字: | 肌少症,乙型轉化生長因子,肌肉細胞生成,細胞自噬,粒線體,粒線體自噬, Sarcopenia,Transforming growth factor-beta 1,Myogenesis,Autophagy,Mitochondria,Mitophagy, |
出版年 : | 2020 |
學位: | 碩士 |
摘要: | 肌少症為老化相關疾病,其特徵骨骼肌肉質量、力量及功能的下降。肌少症的發生不只影響生活品質,也是全因性死亡率和發病率的預測指標。因此,近年來許多研究探討肌肉流失過程中的參與的相關機制。先前研究指出:粒線體的生成、粒線體動態平衡及粒線體自噬在骨骼肌細胞分化過程中扮演重要的角色。乙型轉化生長因子(transforming growth factor-beta 1, TGF-β1)是參與共同調控老化的一種細胞激素,參與細胞分化、增殖與胞外基質產生等反應;然而,許多研究指出過多的TGF-β1與肌肉流失和纖維化有關。另外,在我們先前的研究中證實TGF-β1會抑制骨骼肌細胞分化,但其中透過的機制尚未明瞭,因此,本篇研究目的為探討肌肉細胞分化過程中TGF-β1抑制細胞分化的可能機制。首先,我們證實年老的老鼠肌肉中含有更高的TGF-β1 mRNA表達,並有較差的肌耐力表現。接著,在C2C12細胞實驗中利用LC3 turnover assay 證實TGF-β1會誘發細胞自噬,而且透過同時處理自噬體與溶酶體融合的抑制劑氯喹(chloroquine)說明TGF-β1抑制細胞分化的過程中autophagy flux被過度誘發但並未受到阻斷。由於粒線體功能異常被認為是老化所致的肌肉退化的主要原因且粒線體也是細胞自噬的受質之一,因此,我們進一步由特定螢光探針染色並以共軛焦螢光顯微鏡觀察後發現,TGF-β1增加細胞中粒線體與自噬小體的共定位,表示TGF-β1誘發粒線體自噬的現象。接著,在分化後三天的C2C12中以海馬儀測量粒線體功能,發現處理TGF-β1顯著降低耗氧率(oxygen consumption rate, OCR)及ATP的產生,並以JC-1染劑偵測發現其膜電位顯著低於控制組。為了探討其粒線體功能下降的原因,我們以西方墨點法證實在C2C12分離出的粒線體蛋白中,與粒線體生成及動態平衡相關的蛋白表現量降低。綜上所述,根據我們目前的研究結果顯示,TGF-β1可能是透過過度誘發的細胞自噬及粒線體自噬與抑制粒線體合成與動態平衡蛋白表現,而使粒線體功能下降。未來仍需要更多研究證實TGF-β1抑制肌肉細胞分化的機制,發展做為治療肌少症的治療的標的。 Sarcopenia has been defined as an age-related disease that features loss of skeletal muscle mass, strength, and function. It not only affects the quality of lives, but also a predictor of all-cause mortality and morbidity. Thus, many studies focus on the skeletal muscle-specific mechanisms underlying muscle loss progression. Previous research has shown that mitochondrial biogenesis, dynamics and mitophagy, which means the selective degradation of mitochondria via autophagic pathways, contribute to the skeletal muscle cell differentiation. Many cytokines take part in the aging process, one of them is transforming growth factor-beta 1 (TGF-β1), which is a multifunctional growth factor participated in a variety of biological process such as cell differentiation, proliferation, and extracellular matrix production. However, aberrant TGF-β1 production or stimulation always correlated with muscle loss and fibrosis. Our previous study demonstrated that TGF-β1 impaired skeletal muscle myogenesis. However, underlying mechanisms of TGF-β1-induced myogenic impairment still not elucidated. In this study, we aim to investigate the mechanisms of TGF-β1-induced myogenic differentiation impairment. Here we confirmed that muscle from old mice increase the TGF-β1 mRNA expression while and the muscle endurance was lower compared with young mice. In C2C12 cells, TGF-β1 treatment induced completely autophagy by LC3 turnover assay through co-treatment with chloroquine, the inhibitor of the last stage autophagy. The results suggested that myogenesis inhibition might be associated with hyperactivated autophagy but not blocked and accumulative autophagy. Because mitochondrial dysfunction of skeletal muscle cells was considered as the main factor leading to age-dependent muscle degeneration, and mitochondria was also a subtract of autophagy. We further investigated whether TGF-β1 interfered mitophagy. We observed TGF-β1 increased the colocalization between mitochondria and autophagosome under confocal fluorescence microscopy, indicating that TGF-β1 induces mitophagy. Then, we assessed the mitochondrial function with Seahorse analyzer and confirm that TGF-β1 significantly declined the oxygen consumption rate and ATP production after exposure for 3 days in differentiation medium and enhanced the depolarization of mitochondrial membrane potential which was monitored by membrane potential sensitive dye JC-1. To explore the reason of mitochondrial dysfunction, the expression of mitochondrial biogenesis and dynamics related proteins were found to decrease in the mitochondrial fraction in TGF-β1-treated cells with immunoblotting. Based on the current finding, our results, at least in part, demonstrated that TGF-β1 impaired myogenesis through inducing hyperactivated autophagy/mitophagy, suppressing mitochondrial function and inhibiting the mitochondrial biogenesis and dynamics. Further studies are indicated to confirm the underlying mechanisms involved in the TGFβ1-induced myogenic impairment to find a potential therapy of sarcopenia. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/18855 |
DOI: | 10.6342/NTU202003956 |
全文授權: | 未授權 |
顯示於系所單位: | 毒理學研究所 |
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