| 摘要: | 中文摘要
在脊椎動物中，軀幹體節(somite)的發育會受到周圍生長因子的影響。位於體節dermomyotome中的幹細胞受到從細胞外基質和神經管底層釋放的肌肉生成因子的影響，會走向表現Pax3和Pax7的肌肉幹細胞 (myogenic stem cells, MSC)。 之後，MSC細胞中的肌肉專一性調節因子(Muscle Regulatory Factors, MRFs) MyoD及Myf5的表現被誘發後，可活化下游與肌肉分化有關的基因表現，使MSC成為肌原母細胞 (myoblasts)。由於MyoD可以促使幹細胞走向肌肉發育，因此被稱為肌肉生成的主要調節因子。目前有文獻已知在體節附近分泌的纖維細胞生長因子 (bFGF)會響肌肉發育的過程，但是它對MyoD表達的影響仍然值得探討。本研究主要透過實驗室建立的MyoD promoter以及其上游的片段 (cis-element, -20~-6 kb)，分析bFGF在肌肉分化過程中潛在的調控機制。我們的結果顯示經由處理bFGF的myoblasts，其MyoD表現量會被抑制且提高Pax3與Myf5的表現量，並促進細胞增生。透過分段測試MyoD promoter上游的cis-element，發現bFGF會藉由C片段 (-18 ~ -17kb)、G片段 (-14~ -13kb)、M片段 (-8 ~ -7kb) 影響MyoD promoter的活性，我們推測這些片段可能有影響MyoD promoter活性的轉錄因子結合區域。經由生物資訊軟體分析預測，篩選出AP1、AP4及Sox5為潛在的可受到bFGF影響而調控MyoD表現的轉錄因子。bFGF也透過活化MAPK (JNK, p38和Erk)與Akt訊息傳遞路徑調控AP1、AP4及Sox5的表現，之後的研究可以再探討潛在的轉錄因子與MyoD之間的調控關係。同時也發現bFGF雖然抑制MyoD promoter活性，但卻不影響MyoD調控下游的基因功能，所以bFGF應該不會干擾MyoD的正向自我活化。此外，我們也探討了Wnt3a和bFGF之間的關係，因為這兩種因子都對肌肉幹細胞生成和MyoD表達有很重要的影響。實驗結果顯示Wnt3a和bFGF同時存在對胚胎和成體肌肉細胞的肌肉發育信號的影響是一致，它們皆誘導Pax3、Myf5的表現去維持肌原細胞的特異性，並具有共同促進肌肉細胞再生及分化的功能。綜合以上結果，證明bFGF是透過多方面的信號傳遞途徑調控MyoD上游的轉錄因子，而這些不同的轉錄因子會結合在MyoD上游不同的cis-elements上，抑制MyoD的表現。
;Abstract
In response to local myogenic signals transmitted from their surrounding tissues, some stem cells in dermomyotome of somites will become Pax3- and Pax7- expressing myogenic stem cells (MSC). These stem cells will be further confined within the myogenic lineage by the expression of muscle-specific transcription factors, either MyoD or Myf5 that will activate their downstream transcription factors and other muscle-specific genes to drive MSC to become myoblasts. MyoD is called as the master regulator of myogenesis and it can turn on the whole myogenic program in MSC or cells of other lineages, so induction of MyoD expression leads to determination and development of myogenic cells. A few signals found in the somite neighborhood, such as basic fibroblast growth factor (bFGF), released from extracellular matrix and the floor plate of neural tube have been found to promote myogenic program but their effects on MyoD expression has not been analyzed thoroughly. In this study, the MyoD-reporters driven by MyoD promoter and its upstream fragment (-20 ~ -6 kb) were used to analyze the potential regulatory mechanism of bFGF during muscle differentiation. Our results showed that myoblasts treated with bFGF not only inhibited the expression of MyoD but also increased the expression of Pax3 and Myf5 and promoted cell proliferation. Through screening a series of genomic fragments upstream of MyoD promoter, we found that bFGF affected the activity of MyoD promoter through the C fragment (-18~-17kb), G fragment (-14~-13kb) and M fragment (-8~-7kb). Based on the analysis and prediction of bioinformatics software, AP1, AP4, and Sox5 were identified as potential transcription factors that might be induced by bFGF to regulate MyoD expression. Later studies should explore the regulatory relationship between these potential transcription factors and MyoD. We also found that bFGF could activate MAPK (JNK, p38 and Erk) and Akt signaling pathways to trigger downstream gene expression. At the same time, we also found that although bFGF does not affect the transactivational activity of MyoD, implying that FGF does not interfere with the positive feedback loop of MyoD. In addition, we also explore the cooperation between Wnt3a and bFGF, because they have important effects on muscle stem cell proliferation and MyoD expression. We found the same effects in embryo and adult muscle cells that they both induce the expression of Pax3 and Myf5 to maintain the myogenic cell lineage and have the function of promoting muscle cell regeneration and differentiation. Based on the above results, it is proved that bFGF regulates the transcription factors upstream of MyoD through various signal transduction pathways, and these different transcription factors will bind to different cis-elements upstream of MyoD to inhibit its expression. |
