| 摘要: | 中文摘要 Fox-O 家族（foxo family）是一群擁有conserved的 domain（Fork domain）的轉錄調控因子，屬於fox家族(forkhead family)中的分支家族(subfamily)。foxo 家族的成員包括了foxo1(fkhr)、 foxo3(fkhrl1)、 foxo4(afx)、及2003年發現的foxo6。在之前的研究中指出FOXO1會受到PI3K pathway的影響，進而影響到肌肉細胞的分化，然而其中的分子機制卻並沒有深入的探討，且Fox-O 家族的其他成員(FOXO3、FOXO4、FOXO6)在肌肉分化中扮演的角色依然未知。我們建構了FOXOs的驅動子，並觀察其在肌肉分化的過程中扮演的角色，在本實驗中我們發現FKHR、MyoD這些影響肌肉分化的因子會活化我們建構的FOXO4驅動子。同時我們也發現，FOXOs的表現量會隨著肌肉生長分化的過程而有所提升，因此我們猜測FOXOs在肌肉細胞生長分化中扮演重要的角色。基於此點，我們利用反轉錄病毒大量表現FOXOs於肌肉細胞中，同時也利用siRNA將肌肉細胞中的FOXOs抑制，藉此來觀察大量表現或是抑制FOXOs對肌肉細胞生長與分化的影響。在大量表現FOXOs的肌肉細胞被誘導分化之後，我們觀察其型態上的差異，發現到大量表現FOXO1-AAA(FOXO1基因持續活化的突變體)的肌肉細胞，其分化的功能似乎被抑制或是延緩了，而FOXO3、FOXO4大量表現的細胞，在分化後相對於控制組則沒有太大的改變。我們將大量表現FOXOs的肌肉細胞於快速增生時期、細胞緊貼相連時期、細胞分化後時期的RNA抽出並做RT-PCR，觀察大量表現FOXOs對肌肉分化相關基因的影響。從實驗中我們發現大量表現FOXO1-AAA的細胞，由細胞快速增生時期乃至於細胞被誘導分化後，Msx1、Atrogin-1的表現量上升，而Mef2C的表現量則被抑制了；另外在大量表現FOXO1-AAA的細胞被誘導分化後，相對於控制組，MyoD的表現量被大量的抑制住。MyoD、Mef2C為調控肌肉細胞分化重要的因子，而Msx1、Atrogin-1大量表現時則肌肉細胞無法走向分化，因此從我們的實驗中可以看出大量表現FOXO1-AAA的肌肉細胞可以藉由調控MyoD、Mef2C、Msx1、Atrogin-1等因子，控制肌肉細胞的生長與分化。 Abstract FKHR（FOXO1）,FKHRL1（FOXO3）, AFX（FOXO4）, and FOXO6 constitute an evolutionarily conserved subgroup within the larger family known as winged helix/fork-head (fox) transcription regulators. Previous studies have indicated that PI3K pathway and FKHR regulate terminal muscle differentiation; however, the molecular mechanisms remain to be determined. Besides, the functions of other members were not examined to date. So far, we have cloned the promoter of FOXOs for analyzing their activity during myogenesis. In our experiment, it appeared that MyoD and FKHR, the regulators of muscle terminal differentiation may activate the promoter of FOXO4. We also found out that when myoblasts differentiate into myotube, the expression of FOXOs are up-regulated, especially FOXO4 and FOXO6. Hence, FOXOs are suggested to be involved in the process of muscle terminal differentiation. Based on this, individual FOXO-overexpression mediated by retrovirus infection and FOXO knocked-down by siRNA in myoblast have been established. We induced the stable clones of FOXOs-overexpressed and FOXO4 siRNA-treated myoblasts differentiate into myotubes by 5％HS in DMEM. We found that FOXO1-AAA over-expressed cells, lose the ability to differentiate, but the patterns of others after differentiation are not markedly different relative to control cells. We then collected the RNA from stable clones with stage of proliferating myoblast, confluent myoblast and myotube, and the expression level of relative genes in myogenesis were detected. We found that Atrogin-1 and Msx1 are upregulated, Mef2C is down-regulated in FOXO1-AAA-overexpressed cells in three stages, and the expression levels of MyoD are down-regulated significantly in the stage of myotube. Since MyoD, Mef2C and Msx1 are suggested to be the principal factors regulating myogenesis, our results indicate that FOXO can inhibit muscle terminal differentiation by regulating these genes relative to myogenesis. |
