請用此 Handle URI 來引用此文件: http://hdl.handle.net/11455/23260
標題: 探討葡萄糖六磷酸運輸蛋白mRNA於小鼠肌肉細胞株進行另類剪接之機制
The Study of Alternative Splicing of G6pt pre-mRNA in C2C12 Muscle Cell
作者: 洪婉如
Hung, Wan-Ju
關鍵字: glycogen storage disease
肝醣儲績症
出版社: 生命科學院碩士在職專班
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Overexpression of Id1 protein inhibits the muscle differentiation program: in vivo association of Id1 with E2A proteins. Gene Dev.6:1466-79. 34. Sun, L., Trausch-Azar, J.S., Ciechanover, A., Schwartz, A.L. (2005) Ubiquitin-protease-mediated degradation, intracellar localization, and protein synthesis of MyoD and Id1 during muscle differentiation. J Biol Chem. 280:26448-56. 35. Berkes, C.A., Tapscott, S.J. (2005) MyoD and the transcriptional control of myogenesis. Semin Cell Dev Biol.16:585-95. 36. Tapscott, S.J. (2005) The circuitry of a master switch MyoD and the regulation of skeletal muscle gene transcription. Development.132:268-95. 37. Russo, S., Tomatis, D., Collo, G., Tato, F. (1998) Myogenic conversion of NIH3T3 cells by exogenous MyoD family members: dissociation of terminal differentiation from myotube formation. J Cell Sci.691-700. 38. Chen, L.Y.,Pan, C.J.,Shieh, J.J., Chou, J.Y. 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摘要: 組織中肝醣分解成葡萄糖對維持血糖濃度是很重要的。在肝醣分解與醣質新生作用的最後步驟中,G6pt攜帶著G6P從細胞質至內質網膜,再與G6Pase結合,進而水解成葡萄糖與磷酸。而缺乏G6Pase與G6pt分別可造成GSD-1a與GSD-1b病症,在臨床上會產生很多不同的病灶。人類的另類剪接機制是造成基因重組、多樣的變化使得細胞調控、組織特異及發育階段特異的不同調控。在另類剪接機制中產生G6pt及vG6pt,差異在vG6pt的第七外引子多出22個胺基酸。G6ptmRNA可在各組織中表現,而vG6pt則只在腦、心臟與肌肉中表現。MyoD是肌肉特異性轉錄因子,它可促進肌肉基因表現;Id1是抑制MyoD 的負面調控因子。利用反轉錄聚合酶連鎖反應及相關定量連鎖反應分析,我們發現vG6pt mRNA的另類剪接機制在肌肉細胞分化時可被誘發,肌肉細胞分化24小時後,可測得vG6pt。在第六天時vG6pt量可高達13倍之多,而第七天時vG6pt量卻突然直線下降。當C2C12肌微母細胞加入酸刺激的2天後,vG6pt mRNA出現且量增加,但細胞始終停留在肌微母細胞的階段。而抑制Id1基因作用及加強MyoD基因表現,也會誘發G6pt的另類剪接機制。由這些實驗結果得知,G6pt的另類剪接能藉著MyoD的相關機制而被誘發,而此另類剪接所產生較不穩定的vG6pt使在肌肉組織中有較高的汰換速率。因此我們推測G6pt與vG6pt在生理反應上可能扮演著不同的角色。
The breakdown of tissue glycogen into glucose is very important for blood glucose homeostasis. In the final steps of glycogenolysis and gluconeogenesis, intracellular glucose-6-phosphate (G6P) is transported from the cytoplasm to the lumen of the ER by glucose-6-phophate transporter (G6pt) where is hydrolysed to glucose and phosphate by glucose-6-phosphatase (G6Pase). However, lack of G6Pase and G6pt cause glycogen storage disease type I a (GSD-I a) and glycogen storage disease type I b (GSD-I b), respectively, and they can cause lots of different clinical complication. In humans, the alternative splicing is the way to cause gene reframe and diversity of protein changes which makes cell-specific, tissue-specific, and developmental stage-specific regulated manner. There are two alternatively spliced transcripts, G6pt and vG6pt, differing by the inclusion of 66-bp exon-7 sequence in vG6pt. G6pt mRNA is expressed in all human organs and tissues examined, but the vG6pt mRNA is expressed exclusively in the brain, heart, and skeletal muscle. MyoD, one of muscle-specific gene expression. And the function of MyoD is repressed by Id1.Using RT-PCR and relatively quantitative real-time PCR, we found that vG6pt mRNA was induced during myogenesis. This splicing was detected 24h after differentiation induction, after 6 days differentiation, the amount of vG6pt was increased to 13-fold, and declined immediately after 7 days differentiation. The vG6pt expression was also induced in C2C12 myoblast after changing to acidic medium, after 2 days acidic induction, the vG6pt mRNA started to appear and increase but C2C12 always stayed in undifferentiation myoblast stage. The knock-down of Id1 expression and exogenous MyoD over-expression induced the vG6pt alternative splicing. These data suggest that increasing the turnover rate of G6pt by alternative splicing may induced through a common MyoD-dependent mechanism during myogenesis of muscle cells. The alternative splicing of vG6pt is unstable and has higher turnover rate during myogenesis. Thus, we supposed that G6pt and vG6pt had play different roles in physiological consequences.
URI: http://hdl.handle.net/11455/23260
其他識別: U0005-0502200815434200
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-0502200815434200
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