探討bFGF與癌症惡病質症因子對於肌肉細胞分化與基因表現的影響

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姓名

李寧(Ning Li) 查詢紙本館藏

畢業系所

生命科學系

論文名稱

探討bFGF與癌症惡病質症因子對於肌肉細胞分化與基因表現的影響
(The effects of bFGF and cancer cachexia factors on myogenesis and gene expression)

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摘要(中)

在出生後肌肉發育的過程中，未活化的肌肉星狀細胞會受myogenic signals刺激而活化，basic fibroblast growth factor (bFGF, 又稱為FGF2) 是其中一個關鍵myogenic signal。肌肉在決定品系與分化時受到myogenic regulatory factors (MRF)家族嚴謹的調控，已知，FGF2會抑制MyoD表現同時促進星狀細胞的增生，然而FGF2是如何造成此影響的機制尚未被探討。本研究發現，FGF2在1.25～10 ng/ml的劑量下會促使細胞呈現dose-dependent的增生，並在10 ng/ml後效果達到飽和，在大量表現MyoD的C2C12 (C2-tTA-MyoD)中也能觀察到同樣的增生現象，這表示FGF2促進細胞增生時，MyoD表現不一定要受到抑制。利用C2-tTA-MyoD我們也發現FGF2不會直接影響MyoD mRNA，這暗示FGF2透過調控transcription level來抑制MyoD mRNA表現。實驗室先前已發現FGF2會影響MyoD promoter上游多個genomic片段，但確切是透過哪些transcription factor (TF)還有待探討。實驗室已進一步縮小推測出的調控區域，以尋找關鍵的TF，並將採用site-directed mutagenesis來刪除推測的cis-elements以確認它們是否參與調控。為確認其中一個相關的transcription factor“AP1”是否參與調控，我們利用加入AP1抑制劑sp100030，觀察被抑制的 MyoD 表現是否能被回復，但沒有發現正向的回復，這表示參與調控的是其它TF。因此未來需要進一步探討FGF2藉由哪些TFs抑制MyoD。由於在併發cancer cachexia患者的肌肉中也觀察到MyoD表現被抑制，促使我們想探討大量表現MyoD是否可挽救muscle cachexia，令我們意想不到的是，C2-tTA-MyoD 在加入從C26 colon cancer cells 收集的cachexia medium 後並沒有走向分化，顯示大量表現MyoD無法挽救cachexia抑制的肌肉分化。相反的, MyoD大量表現可挽救cachexia medium 誘導的myotube萎縮。總之, 這些結果顯示cachexia factors在抑制肌肉生成與誘導肌肉萎縮時使用了不同的機制。此外，以上證據也說明，維持myoblast與myofiber中MyoD表現量，可延緩cancer cachexia的進展。

摘要(英)

During the process of postnatal myogenesis, quiescent muscle satellite cell will be activated by myogenic signals, and basic fibroblast growth factor (bFGF, also known as FGF2) is one of the key myogenic signals. Myogenesis is critically regulated by the myogenic regulatory factors (MRF) family during the determination and differentiation of myogenic cells. FGF2 has been found to repress the expression of MyoD but promote proliferation of satellite cells at the same time, however, the mechanisms mediating FGF2 effects on both events remain to be understood. In this study, we found FGF2 increased C2C12 proliferation dose-dependently from 1.25 to 10 ng/ml and saturated effect was seen at higher doses. Similar cell proliferation effect was observed in MyoD overexpressed C2C12 cells (C2-tTA-MyoD), suggesting MyoD repression is not necessary for the cell proliferation effect of FGF2. Using C2-tTA-MyoD, we further found MyoD mRNA was not targeted by FGF2, implying the observed repression of MyoD mRNA should be regulated at transcriptional level. In our lab, a previous study has found that FGF2 affects MyoD promoter activity through several upstream genomic fragments, but the exact transcription factors mediating this effect have not been identified. These putative regulative regions have been further narrowed down for identifying key TFs mediating this effect and site-directed mutagenesis will be employed to delete putative cis-elements for confirming their involvement. The involvement of one transcription factor, AP1, was examined by rescuing the repressed MyoD expression with its inhibitor SP100030 but no positive rescue was found, suggesting involvement of other TFs. Therefore, further study is needed to identify TFs mediating the repressive effect of FGF2. Since MyoD repression was also seen in the muscle of cancer patients with cachexia, it prompted us to examine whether MyoD over-expression could rescue muscle cachexia. It was surprising to find that the myogenic differentiation of C2-tTA-MyoD treated with cachexia medium from C26 colon cancer cells was not rescued by MyoD over-expression. On the contrary, their myotube atrophy induced by cachexia medium was largely rescued by MyoD over-expression. Taken together, these observations suggest that different mechanisms are employed by cachexia factors to repress myogenesis and to induce myotube atrophy. Furthermore, these evidence also imply that maintaining a functional level of MyoD in myoblasts and myofibers might significantly attenuate the progression of cancer cachexia.

關鍵字(中)

★ 骨骼肌肉細胞
★ 肌原細胞
★ 肌原細胞決定因子
★ 纖維母細胞生長因子
★ 肌肉分化過程
★ 轉錄因子

關鍵字(英)

★ skeletal muscle
★ myoblasts
★ MyoD
★ bFGF
★ myogenesis
★ transcription factor

論文目次

摘要 i
Abstract ii
聲明(Declaration) iii
誌謝 iv
目錄 v
一、 Introduction 1
1-1. 肌肉的發展 1
1-2. 肌肉生成 (Myogenesis) 2
1-3. 肌肉調節因子(Muscle Regulatory Factor , MRF) 3
1-4. 生肌決定因子MyoD 4
1-5. 成纖維細胞生長因子 (Fibroblast growth factor) 5
1-6. 癌症惡病質與肌肉流失 6
1-7. 動機與目的 7
二、材料方法 8
2-1. 質體建構 8
2-2. 重組蛋白質純化 11
2-3. 細胞培養(Cell culture) 13
2-4. 反轉錄聚合酶鏈鎖反應 15
2-5. 西方墨點法 17
2-6. 免疫螢光染色 18
2-7. 螢火蟲冷光活性測定 19
2-8. 衛星細胞分離 19
2-9. 小鼠胚胎分離與培養 21
三、實驗結果 22
3-1. GST- FGF2為具功能性的重組蛋白 22
3-2. GST-FGF2可促進細胞增生及抑制MyoD表現 22
3-3. GST-FGF2 對大量表現MyoD的C2C12增生及分化的影響 23
3-4. GST-FGF2對不具內生性MRFs表現“10T1/2-tTA-MyoD”細胞株MyoD表現的影響 24
3-5. GST-FGF2透過潛在轉錄因子影響MyoD cis-element抑制MyoD表現 25
3-6. GST-FGF2對小鼠胚胎不同體節MyoD表現的影響 26
3-7. 惡病質症因子抑制大量表現MyoD的C2C12細胞分化 26
四、討論 28
4-1. 分析GST-bFGF影響之MyoD Promoter上游cis-element 28
4-2. GST-FGF2對肌肉星狀細胞的影響 28
4-3. GST-FGF2與真核細胞所表現之蛋白質功能一致 29
4-4. FGF2在in vivo及in vitro的對分化的影響 29
4-5. 結論 30
五、圖表 Figures 31
Fig. 3-1 GST- FGF2是具功能性的重組蛋白 31
Fig. 3-2 GST-FGF2可促進細胞增生與抑制MyoD表現 32
Fig. 3-3 GST-FGF2 對C2C12-tTA-MyoD #3增生及分化的影響 35
Fig. 3-4不具有內生性MRFs表現的10T1/2-tTA-MyoD細胞株 37
Fig. 3-5 GST-FGF2透過潛在轉錄因子影響MyoD cis-element抑制MyoD表現 38
Fig. 3-6 GST-FGF2對於小鼠胚胎不同體節MyoD表現的影響 39
Fig. 3-7癌症惡病質症因子對大量表現MyoD的C2C12分化之影響 42
六、參考文獻 43
七、附錄 47
附圖一、GST-FGF2對肌肉星狀細胞的影響 48
附圖二、GST-FGF2抑制MyoD promoter上游cis-elements片段 51
附圖三、GST-FGF2與真核細胞所表現之蛋白質功能一致 52
附錄四、Primer list 53
附錄五、溶液及溶劑配方 55

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指導教授

陳盛良(Shen-Liang Chen)

審核日期

2022-1-26

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