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標題: | lmbrd1基因參與肌細胞生成的角色 The role of lmbrd1 gene involved in myogenesis |
作者: | Po-Fan Liu 柳柏樊 |
指導教授: | 張明富(Ming-fu Chang) |
關鍵字: | lmbrd1,肌肉生成, lmbrd1,myogenesis, |
出版年 : | 2016 |
學位: | 碩士 |
摘要: | 肌細胞由肌纖維母細胞(myoblasts)分化而來,可分為平滑肌 (smooth muscle)、心肌 (cardiac muscle)以及骨骼肌 (skeletal muscle)細胞。骨骼肌是由肌纖維母細胞融合而成的,所以造就了骨骼肌多核的特性。肌纖維母細胞的融合在生物體生長以及組織受損的修復扮演著重要的角色。lmbrd1基因表現四種異體蛋白質 (protein isoform) ,分別是有540、467、392及188個胺基酸異體蛋白質。先前我們實驗室發現lmbrd1-/-的老鼠均胎死腹中,且lmbrd1+/-基因剔除老鼠在步行時有平衡不良之情形,其骨骼肌有細胞排列不整的現象。另外,利用注射lmbrd1 morpholino antisense oligonucleotide (lmbrd1-MO) 的方式阻斷lmbrd1基因的轉譯之lmbrd1-MO斑馬魚,在外觀上其軀幹部位之肌肉生長呈現彎曲、萎縮。利用F59、phalloidin、α-actin、dystrophin 一系列之肌肉與細胞骨架之標記抗體進行染色觀察,可以觀察到斑馬魚胚胎的肌纖維排列混亂、崩解,肌動蛋白排列不規則的現象,這些發現都暗示著lmbrd1基因可能參與在肌肉發育的過程中。本研究探討LMBD1參與在肌纖維母細胞的分化,利用小鼠的肌纖維母細胞C2C12模擬肌肉細胞分化的系統,發現當細胞開始進行分化時,LMBD1蛋白質的表現量開始上昇。利用表現lmbrd1 shRNA的VSV-G pseudotyped慢病毒感染C2C12細胞,無論是西方墨點法偵測分化的指標蛋白質 (myosin heavy chain)或是免疫螢光顯微鏡觀察細胞形態都可以發現細胞的分化被抑制,且進一步研究發現到lmbrd1 knockdown會增加C2C12細胞的蛋白激酶A (protein kinase A)的磷酸化,可能再透過myocyte enhancer factor-2 (MEF2)調控骨骼肌的分化。在已知的LMBD1蛋白質可以協助維生素B12從溶酶體內釋放到細胞質以及參與胰島素受體的內吞作用之後,本研究新發現了LMBD1蛋白質參與在肌細胞分化的生物功能。 Myocytes developed from myoblasts to form muscles in a process known as myogenesis. There are specialized forms of myocytes: smooth, cardiac and skeletal muscle cells, with different properties. Skeletal muscle are formed from the fusion of myoblasts, giving the multi-nucleus characteristics. Myoblasts play important roles in growth and tissue repair. lmbrd1 gene encods four protein isoforms, including the LMBD1 (540 amino acids) and NESI (467 amino acids). Our laboratory has previously demonstrated that lmbrd1-/- mice is lethal at early embryonic stages and lmbrd1+/- mice have characteristics of stumbling, lifting the hind-legs, and dragging tails during movement. Moreover, the lmbrd1 knockdown zebrafish displayed curved-body phenotypes. Embryo stained by muscle specific markers, such as F59, phalloidin, α-actin and dystrophin, demonstrated disordered muscle fibers alignement and loss of integrity. These data suggested that LMBD1 may function to regulate muscle fiber organization and play an important role in myogenesis. In this study, the role of lmbrd1 gene involved in myogenesis was investigated. The results showed a temporary increase of LMBD1 expression when C2C12 cells underwent differentiation. In lmbrd1-knockdown C2C12 cells, the expression of myosin heavy chain (MHC), a differention marker, was decreased and the formation of myotubes were inhibited when C2C12 cells were induced to differentiation. Besides, lmbrd1 knockdown increased protein kinase A phosphorylation that may regulate myogenesis through myocyte enhancer factor-2 (MEF2), suggesting that C2C12 myoblasts differentiation depends on LMBD1 expression. In addition to the function of LMBD1 protein as a putative lysosomal exporter of vitamin B12 and a regulator of insulin receptor endocytosis, this study identifies a novel function of LMBD1 protein involved in myogenesis. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/19041 |
DOI: | 10.6342/NTU201603137 |
全文授權: | 未授權 |
顯示於系所單位: | 生物化學暨分子生物學科研究所 |
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