FoxOs 大量表現對肌肉細胞末期分化的影響

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張儷瓊(Li-Chiung Chang) 查詢紙本館藏

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生命科學系

論文名稱

FoxOs 大量表現對肌肉細胞末期分化的影響
(Effects of FoxOs over-expression on terminal myogenic differentiation)

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摘要(中)

FoxO家族含有4名成員(FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX, FoxO6)，這些成員上均有一段consensus DNA 序列 TTGTTTAC，其功能相當於轉錄因子。FoxOs轉錄因子在調節細胞的分化、增殖、代謝及生存等相關功能上扮演重要的角色。近來研究發現FoxOs可經由PI3K的磷酸化來調控下游基因的表現，而影響肌肉的分化。一些研究指出FoxO3在骨骼肌當中表現會造成肌肉萎縮，但其他成員對肌肉造成相關性影響的功能及機制並未深入探討。為了觀察FoxOs在肌肉細胞生長及分化中所扮演的角色，因此在本研究中利用反轉錄病毒來穩定的大量表達FoxOs在肌肉纖維母細胞中。在大量表達FoxOs的肌肉細胞株中經誘導細胞分化後，觀察其型態上的差異，發現在大量表現FoxO1-AAA (FoxO1基因持續表現的突變體)、FoxO6的細胞株中，其分化的功能有被抑制的現象，其他細胞株與控制組對照之下並無明顯差異。進而抽取其不同時期 (細胞快速生長時期、細胞聚合時期、細胞分化時期) 的RNA並做RT-PCR來觀察各個FoxOs對肌肉分化相關性的特殊基因的影響。從實驗數據中發現在大量表現FoxO1-AAA的細胞株在經誘導分化之後，MRFs家族 (MyoD、MEF2C、Myf5及Myogenin) 的RNA表現量均有下降之情形，MRFs家族是調控肌肉特定基因轉錄的因子。因此可推測造成大量表現FoxO1-AAA細胞株不分化的原因可能是藉由調控MRFs基因來控制細胞生長及分化。要確定造成細胞不分化的相關機制是經由哪些訊息所影響的，因此我們在這些大量表現FoxOs而不分化的細胞株中，給予胰島素，Y27632及A23187處理，來觀察它們對於肌肉細胞末期分化所造成的影響，在實驗中發現當大量表現FoxO1-AAA時原本經誘導而不分化的型態下，在加入了胰島素時肌肉細胞反而走向分化。在實驗中觀察到葡萄糖濃度對肌肉細胞分化有差異性影響，因此我們想了解這些大量表現FoxOs並不分化的細胞株在處理胰島素之後對葡萄糖代謝是否會造成影響，所以利用glucose uptake來進行初步的實驗，並且利用流式細胞儀來觀察FoxOs在cell cycl中所造成的影響。

摘要(英)

FoxO family contains four members (FoxO1/FKHR, FoxO3/FKHRL1, FoxO4/AFX, FoxO6) that function as transcriptional activators by binding as monomer to the consensus DNA sequence TTGTTTAC. FoxO transcription factors have been implicated in regulating diverse cellular functions including differentiation, metabolism, proliferation, and survival. Recent studies have revealed that PI3K can directly regulate gene expression through phosphorylation. Some studies have shown that FoxO3 can promote atrophy in skeletal muscle. However, their regulation and function in skeletal muscle have not been thoroughly understood. In this study, we have constructed stable clones of FoxO-overexpressed myoblasts by retrovirus transduction. FoxO1-AAA、FoxO6 over-expressed cells lose their ability to differentiate into myotubes, but not the other FoxOs over-expressed stable clones. The gene expression patterns of the later after differentiation were not significantly different from those of control cells. To identify how the differentiation of FoxO1-AAA overexpressed myoblasts were inhibited, we treated them with insulin, Y27632, and A23187, and then observed their influence on terminal myogenic differentiation. Our results revealed that treating the undifferentiated FoxO1-AAA over-expressed cells with insulin would provoke their differentiation. Moreover, we also found the maturity of myotube was changed when treated with different glucose concentrations. Finally we investigated whether well-differentiation of FoxO1-AAA over-expressed cells by insulin treatment was due to effects on glucose metabolism. We also examined the glucose uptake by 2-[1,2-3H(N)]-Deoxy-D-Glucose (2-DG) and the change of cell cycle by flow cytometry.

關鍵字(中)

★ 肌肉
★ 肌肉細胞分化
★ FoxO

關鍵字(英)

★ muscle
★ differentiation
★ FoxO

論文目次

誌謝 vii
中文摘要 iv
Abstract v
目錄 viii
表目錄 xii
縮寫與全名對照表 xiii
藥品及材料 xiii
基因 xiv
第一章、序論 1
1.1 肌肉發育的起源: 1
1.2 肌肉前驅細胞的演化過程 3
1.3 衛星細胞與肌肉的再生 4
1.4 MRF與肌肉細胞發育分化的關係 5
1.5 FOXOs的功能 7
1.6 研究動機與目的 10
第二章、實驗材料與方法 12
2.1 實驗材料 12
2.2 質體建構 13
2.2.1 質體建構之基本流程 13
2.2.2 pMSCV-neo-FOXOs 表現載體之構築 17
2.3 穩定細胞株的製備 18
2.3.1 細胞培養 18
2.3.2 轉染作用 (Transfaction) 18
2.3.3 篩選穩定細胞株 18
2.3.4 建立在 C2C12 所表現的穩定細胞株 18
2.4 RT-PCR 19
2.4.1 Total RNA 的抽取 19
2.4.2 反轉錄酶反應 (Reverse Transcriptase, RT) 19
2.4.3 聚合酶鏈反應 ( Polymerase Chain Reaction, PCR ) 20
2.4.4 PCR反應在肌肉特異性基因中的各個條件 20
2.5 西方墨點實驗 (Western blot) 21
2.5.1 總蛋白質的製備 21
2.5.2 SDS-polyacryamide Gel Electropheresis 22
2.5.3 轉印 (Transfer) 22
2.5.4 Blocking 以及 Antibody 辨識 22
2.5.5 抗體脫附 (Striping) 23
2.6 在不分化的穩定細胞株中處理藥劑的實驗 (insulin、Y27632及A23187 ) 24
2.6.1 細胞培養 24
2.6.2 藥劑處理 24
2.6.3 DAPI染色 24
2.6.4 將細胞株中的細胞核量化 24
2.6.5 RT-PCR 24
2.7 將不分化的細胞株培養在不同濃度的葡萄糖中並同時處理藥劑來觀察對分化的影響 25
2.7.1 細胞培養 25
2.7.2 DAPI染色 25
2.8 流式細胞儀分析 25
2.8.1 細胞培養 25
2.8.2 細胞前置處理 25
2.8.3 流式細胞儀數據分析 26
2.9 葡萄糖攝取實驗(Glucose uptake) 26
2.9.1 細胞培養 26
2.9.2 細胞CMB時期對葡萄糖攝取之處理 26
2.9.3 細胞MT時期對葡萄糖攝取之處理 27
第三章、結果 29
3.1 建立大量表達FoxOs的穩定細胞株 29
3.2 在肌纖維母細胞 (Sol8、C2C12) 中可穩定表達FoxOs的細胞株於分化時期的形態圖 29
3.3 在大量表達FoxOs細胞株中對於肌肉細胞分化的特異性相關基因表現的影響 30
3.3.1 大量表現FoxOs對Sol8的影響 31
3.3.2大量表現FoxOs對C2C12的影響 32
3.4在insulin、Y27632及A23187處理下對FoxOs不分化細胞株所造成的影響 34
3.4.1在C2C12-FoxO1-AAA中處理insulin、Y27632及A23187之後對於細胞分化影響之形態 34
3.4.2在不分化的細胞株 (C2C12-FoxO1-AAA) 中對於肌肉細胞分化的特異性相關基因表現的影響 35
3.5 C2C12-FoxO1-AAA在不同濃度的葡萄糖環境下處理insulin、Y27632及A23187對分化所造成的影響 36
3.6各個FoxOs穩定表達在Sol8中的細胞株對於細胞週期的影響 37
3.6.1 大量表現FoxOs的PMB時期對於細胞週期的影響 37
3.6.2 大量表現FoxOs的CMB時期對於細胞週期的影響 37
3.7利用insulin induction葡萄糖吸收來觀察不分化細胞對葡萄糖攝取量的影響 38
3.7.1 利用insulin induction葡萄糖吸收來觀察在C2C12表現FoxOs不分化細胞株對其攝取量的影響 39
3.7.2 利用insulin induction葡萄糖吸收來觀察在Sol8表現FoxOs不分化細胞株對葡萄糖攝取量的影響 40
第四章、討論 41
4.1大量表現FoxO1-AAA細胞株對於肌肉終極分化過程中特異基因的相關性影響 41
4.2 FoxOs細胞株在肌肉細胞中對於細胞週期的影響 41
4.3 FoxOs對於糖類代謝的可能影響 42
第五章、參考文獻 71
附錄一 76
I. 溶液及試劑配方 76
II. 藥品試劑 79
III. 酵素和限制酶 79
IV. 抗體 79
附錄二 80
I. Primer 對照表 80
II. 與肌肉分化相關基因的primer對照表 81
III. Plasmid 對照表 82

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指導教授

陳盛良(Chen shen-liang)

審核日期

2007-7-18

推文