- 學位論文
老鼠骨骼肌肉細胞株C2C12之肌肉分化因子研究
Analysis of myogenesis factors in mouse skeletal muscle C2C12 cells
摘要
骨骼肌細胞的分化是經由許多因子協調控制來完成，其中包括肌肉特異性轉錄因子群(MRFs)。在體外實驗，當肌肉纖維母細胞株(myoblast)培養在低血清的培養基時，會誘使這些肌肉特異性轉錄因子群的蛋白質表現，進ㄧ步促使肌肉纖維母細胞進行分化融合形成多核的肌小管(myotubes)。在本實驗中，利用老鼠骨骼肌肉纖維母細胞株C2C12進行肌肉分化(myogenesis)。首先以西方墨點法(western Blot)分析myogenin及p21表現量隨著分化時間增加而增加，而Cyclin D則相對的遞減至消失；再從流式細胞儀(Flow cytometry)分析細胞停留在細胞週期的的G0/G1期之比例隨著分化天數的增加而遞增，顯示細胞正在進行分化。接著以二維膠體電泳分析肌肉纖維母細胞C2C12(對照組)及分化1到5天的細胞(實驗組)，利用軟體分析比對結果發現78個蛋白質點有差異，其中有45個蛋白質點經質譜儀被鑑定出來，在這45個蛋白質中有37個是表現增加(包括VDAC-1及TCTP)，有8個是表現減少的。而這些蛋白質在肌肉分化的過程中牽涉到了細胞內外訊息的傳遞、細胞的形態、增生、凋亡、能量的代謝等等。為了瞭解這些表現差異的蛋白質是否真正參與肌肉細胞的分化，我們利用分子選殖(cloning)技術初步選殖了VDAC-1及TCTP二個基因，利用細胞轉殖(transfection)、RNA干擾(RNAi)及西方墨點分析法探討這二個基因在肌肉細胞分化所扮演的角色，結果顯示出這些在肌肉分化的過程中表現量有差異的基因並沒有明顯的改變myogenin的表現量，可是卻能影響肌肉細胞進行分化時形態的變化(morphology change)。另一方面，在肌肉細胞進行分化的過程中，會伴隨著細胞凋亡的產生，可是引起肌肉細胞凋亡的真正原因還不是很清楚。而VDAC-1和TCTP又分別和凋亡以及抗凋亡有關，所以我們分別將VDAC-1和TCTP這兩個基因送入C2C12肌肉纖維母細胞，使其在細胞分化中過度表現，結果發現VDAC-1會造成DNA ladder的產生，顯示VDAC-1確實參與肌肉細胞分化中所伴隨的細胞凋亡現象，然而TCTP並沒有辦法幫助細胞抵抗凋亡。至於VDAC-1是否為肌肉細胞進行分化時引起凋亡的真正原因，將值得進ㄧ步深入探討。
關鍵字
老鼠骨骼肌肉細胞株C2C12並列摘要
The skeletal muscle cells differentiation is a complicated regulatory process via a variety of factors, including the muscle-specific regulatory factors (MRFs). In vitro study, when myoblast cultured in the low serum-containing medium, the MRFs will be induced, and then trigger differentiation process and finally fusion into multinucleated myotubes. In the present experiment, we use mouse C2C12 myoblast as a model to study the factors involving the process of myogenesis. First, western blotting was applied to show the expression level of myogenin and p21 increasing, and of cyclin D1 decreasing progressively to undetectable during myogenesis. Furthermore, the proportion of cells arrested at G0/G1 phase of cell cycle was increasing progressively along with the time of differentiation medium (DM) incubation to demonstrate that the C2C12 cells were undergoing the process of differentiation. Two-dimensional gel electrophoresis was performed to compare the proteins expression level in C2C12 myoblast (as control group) with that in differentiation cells for 1 to 5 days (as experiment groups). Seventy eight of spots with differential expression were obtained by using Image MasterTM 2D Platinum software. Among them, some 45 proteins (including previous data) were identified by ESI-Q-TOF, 37 of these 45 proteins displaying up-regulation and only 8 proteins with down-regulation. These proteins have been studied to involve in the signal transduction, morphology, proliferation, apoptosis, energy metabolism and so on. In order to understand whether they participate in muscular differentiation, we have cloned two of interesting up-regulation genes named, VDAC-1 and TCTP. Neither overexpression nor knockdown of VDAC-1 and TCTP affects the expression of myogenin, indicating that these two genes may not play the central role during myogenesis. However, DNA ladder, the hallmark of apoptosis, and some morphology changes of forming myotubes were observed on the VDAC-1-transfected cells, indicating that it could play an important role on mygenesis. Due to the appearance of an apoptosis phenotype is accompanied by the induction of the myogenesis, whether VDAC-1 really involving in this process is under investigated.