臺灣博碩士論文加值系統

人體骨骼肌大約佔身體重量的40-50%，主要負責運動及生理平衡，這也是惡病質主要的作用標地。惡病質經常伴隨慢性疾病的發生，像是慢性腎臟疾病、慢性心臟衰竭、慢性阻塞性肺疾病，老化、後天免疫不全症、糖尿病等，它會導致骨骼肌重量的流失以及肌肉萎縮的發生。慢性疾病誘發的肌肉萎縮會造成骨骼肌無力、發炎、衰弱，並且引致約20-30%的癌症病患死亡。 先前研究已發現促發炎細胞激素像是TNF-α、IL-1β和IL-6與這些慢性疾病相關的肌肉病變有關連。此外，肌肉生長抑制素和脂筏蛋白-3 在肌肉萎縮中亦可能扮演重要的調控角色。在本研究中，我們模擬出一細胞激素混合液誘發的離體發炎環境，以探討在骨骼肌纖維母細胞C2C12 細胞萎縮時可能的訊息調控機制。我們將TNF-α、IL-1β 和 IL-6促細胞激素的混合液稱為cytomix。首先我們先檢測個別促發炎細胞激素對於C2C12 細胞存活率及發炎的作用，在使用CCK-8 試劑組、phalloidin 細胞免疫染色、scepter assay、膜質核分離製備技術、西方墨點法、免疫細胞化學染色等處理後，結果顯示TNF-α所的造成 C2C12 細胞萎縮和發炎反應有時間和劑量相依的效果。我們接著進一步探討細胞激素混合液在 C2C12細胞的作用，結果造成細胞發生顯著的萎縮變化，亦造成特異性蛋白質的活化，如ERK、p38、CREB和NF-κB的磷酸化以及TGF-β RI和肌肉生長抑制素的上升，但脂筏蛋白-3表現量卻是減少的。這些活化的蛋白質能夠藉由預處理 MEK/ERK 抑制劑U0126而被抑制，卻不影響p38、脂筏蛋白-3和TGF-β RI的表現。另外我們也發現，預處理 p38抑制劑SB203580不但不能夠防止細胞萎縮，反而會提早細胞萎縮的出現。有趣的是，利用細胞激素混合液處理24小時的細胞培養液作用，亦會加速細胞萎縮的發生。我們的結果也證實在細胞激素混合液引起C2C12細胞萎縮的作用中，MEK/ERK/CREB訊息路徑和肌肉生長抑制素表現之間的關連性。脂筏蛋白-3似乎可以透過p38訊息路徑在防止細胞萎縮中扮演關鍵的調節角色。這些結果有助於進一步了解惡病質的病理機制，並應用於改善慢性疾病的臨床治療策略。

In human body, skeletal muscle accounts for 40% to 50% of the total body mass and is the major structure to responsible for movement and physiological homeostasis. Unfortunately, it is also the main target to be attacked from cachexia. Cachexia usually occurs with chronic diseases, such as CKD, CHF, COPD, aging, AIDS and diabetes mellitus, which causes the loss of skeletal muscle mass and muscle atrophy. Chronic diseases-induced muscle atrophy involves skeletal muscle weakness, inflammation, wasting, and leads to an estimated of 20% to 30% deaths of cancer patients. Previous studies have demonstrated that pro-inflammatory cytokines including TNF-α, IL-1β and IL-6 are associated with the cachexia-related myopathy. Additionally, myostatin and caveolin-3 may play important mediators in the muscle atrophy. In present study, we mimicked an in vitro cytokine mixture-induced inflammatory environment to investigate the possible signaling regulatory mechanisms in skeletal myoblast C2C12 cell atrophy. The pro-inflammatory cytokine mixture of TNF-α, IL-1β and IL-6 was termed as cytomix. First, we examined the effect of individual pro-inflammatory cytokine on viability and inflammatory response of C2C12 cells. After applying CCK-8 kit, the phalloidin cytochemistry, scepter assay, membranous/cytosolic/nuclear fractions preparation, western blotting and immunocytochemistry staining were carried and the results indicated that the TNF-α may cause C2C12 cell atrophy and inflammation in time- and dose-dependent manner. Moreover, we evaluated the effects of cytomix on C2C12 cells, and results displayed dramatically atrophic changes and activation of specific proteins such as phosphorylation of ERK, p38, CREB, NF-κB as well as the up-regulation of TGF-β RI and myostatin, except caveolin-3. These inductions were inhibited by pre-treatment of MEK/ERK inhibitor U0126, but no effect on p38, caveolin-3 and TGF-β RI. Otherwise, we also found that C2C12 cells not only failed to prevent atrophy but also leaded to early onset of atrophy by pre-treatment of p38 inhibitor SB203580. Interestingly, the condition medium from 24 hours cytomix-treated cell culture also could speed up the occurrence of cell atrophy. Our results identified the relationship between activation of MEK/ERK/CREB signaling pathway and expression pattern of myostatin in the cytomix-caused C2C12 cell atrophy. The caveolin-3 seems to be a crucial mediator in preventing cell atrophy through p38 signaling pathway. These findings may help in further understanding of the pathogenesis of cachexia and application in improving clinical treatment strategies on chronic diseases.

口試委員會審定書............................................................................................................I

Acknowledgement........................................................................................................II

Abstract (Chinese).......................................................................................................IV

Abstract (English).........................................................................................................V

List of abbreviations...................................................................................................VII

Chapte I. Introduction....................................................................................................1

Chapte II. Materials and Methods..............................................................................7

Chapte III. Results.......................................................................................................12

Chapte IV. Discussion..................................................................................................19

Chapte V. Summary......................................................................................................24

Chapte VI. Figures..........................................................................................................25

Chapte VII. References..................................................................................................59

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