| 摘要: | Forkhead 轉錄因子由於都具有一段稱為 Forkhead box 的 DNA 結合位，所以被分類為同一族群，而且此族群的 DNA 結合位具高度保留性，其中的亞族 FoxO，包含了 FoxO1、FoxO3、FoxO4、FoxO6 都和許多生理過程有關，像是細胞週期的調控、DNA 的修復、細胞的分化以及凋亡。在這些 FoxO 中，近期才被發現的 FoxO6 在許多生理反應過程中所扮演的角色以及其表現情形都尚未被研究，因此我們想要探討在發育中或是已經發育成熟的肌肉中，分別觀察其 FoxO6 的 mRNA 或是蛋白質的表現情形。首先以 Q-PCR 去觀察 FoxO6 的 mRNA 表現情形，我們發現在心臟、肝臟及腎臟中，FoxO6 的表現量遠低於腦部及肌肉，而且腦下垂體的 FoxO6 是表現量最多的部位；同時我們也注意到 FoxO6 的 mRNA 表現會隨著年紀的增加而減少。另外我們也想知道在不同時期的肌纖維母細胞中，FoxO6 在細胞中的表現位置。為了達到這些目的，我們建構了一個含有 FoxO6-specific domain 的蛋白質，用來當作抗原，使我們能夠在兔子身上製造出 FoxO6 的多株抗體。在測試抗體效價後，我們確定此抗體會專一的辨認 FoxO6 而不會辨認到其他的 FoxOs。利用免疫螢光染色，我們發現在增生中的肌纖維母細胞，其 FoxO6 會多數聚集在核中；當細胞長滿後，FoxO6 則會幾乎全數進入核內；但是在分化形成肌管後，FoxO6 則會散布在細胞質與細胞核中。因為 FoxO6 的表現情形類似於其他的 FoxOs，故我們推論在肌肉生成的過程中，FoxO6 可能和其他 FoxOs 扮演著相同的角色。當了解 FoxO6 的表現情形後，我們將會進一步的利用 promoter assay 去找出 FoxO6 在肌肉中是如何被調控的。 Forkhead transcription factors constitute a family that share a conserved DNA binding domain, the Forkhead box. Members of the subfamily O, including FoxO1, FoxO3, FoxO4, and FoxO6, are involved in many physiological processes, such as cell cycle arrest, DNA damage repair, cell differentiation and apoptosis. Among these FoxOs, the expression and function of the recently discovered FoxO6 in most physiological processes has not been studied before. Therefore, we want to delineate the expression of FoxO6 at mRNA and protein levels respectively in developing muscles in utero and in various post-natal mature muscles. So we detected FoxO6 mRNA in mouse tissues by Q-PCR and observed that the heart, liver, kidney have lower FoxO6 expression than brain and muscle. The highest expression of FoxO6 was found in pituitary. We also found that FoxO6 expression decreased with increasing age. It is also of interest to know the Foxo6 localization at the different stages of myoblasts. To achieve these goals, we used FoxO6-speific domain (amino acid 229~492) as antigen to produce FoxO6 polyclonal antibody in rabbits. After titering, we are sure that this antibody can recognize FoxO6 specifically without cross-reacting with other FoxOs. Using immunofluorescence, we demonstrated that FoxO6 accumulated mostly in nucleus of proliferating myoblasts, and this nuclear localization was further increased when myoblasts became confluent. After terminal differentiation, homogeneous distribution of FoxO6 was detected in the cytoplasm and nucleus of myotubes. Since the expression pattern of FoxO6 is similar to other FoxOs, it suggests that the role played by FoxO6 in myogenesis is probably the same as other FoxOs do. After the delineation of its expression, we will perform promoter assay to find out how FoxO6 expression is regulated in muscle. |
