新生小鼠骨骼肌成肌细胞分离和分化方法研究  

Culture and differentiation of neonatal mouse skeletal myoblasts

作　　者：刘文斌[1] 吴丽姿 李睿[3] 曾驰[3] 史艳丽[1] 

机构地区：[1]武汉轻工大学医学技术与护理学院,湖北武汉430023 [2]佛罗里达大学医学院,佛罗里达甘城fl32610 [3]武汉轻工大学生物与制药工程学院,湖北武汉430023

出　　处：《武汉轻工大学学报》2015年第1期21-25,36,共6页Journal of Wuhan Polytechnic University

摘　　要：建立一个高效的分离和分化小鼠成肌细胞的方案,以便探讨MAML1在转基因小鼠中肌管的形成和肌肉发生中的作用。使用细胞分化方法鉴定MAML1在肌肉发生中的作用。使用Western blot检测肌肉发生的标志蛋白肌球蛋白Myosin的表达。研究发现,传代次数少于5次及卫星细胞对成肌细胞在体外形成正确的肌管非常重要。不传代直接进行诱导分化,能促使成肌细胞形成更多的肌管。在MAML1转基因小鼠中,MAML1通过促进形成更多、更粗壮而长的肌管,提高肌球蛋白的蛋白水平来促进肌肉发生。这些MAML1转基因的肌管能够持续存在超过5天,然后才会降解。Myoblast is a type of embryonic progenitor cell that gives rise to myocytes. MAML1（ mastermind- like1） is a co- activator of NOTCH1 in the process of activation of NOTCH1 target genes. The purpose of this study is to develop an efficient protocol to isolate and differentiate murine myoblasts and to explore the roles of MAML1 in myotube formation or myogenesis in transgenic animals. PCRs were performed to identify MAML1- transgenic mice,immunofluoresencence was performed to identify the expression of myoblast representative protein Myo D,and cell differentiation assays were performed to check the roles of MAML1 in myogenesis. Western blot was applied to check expression of myogenesis marker gene,myosin. Less passage time（ 〈5） and satellite cells are very important for myoblasts to form proper myotubes in vitro. Direct differentiation without passage can induce more myotubes to form. In the culture of MAML1- transgenic murine myoblasts,MAML1 promotes myogenesis through its enhancing formation of more myotubes,making myotubes stronger and longer,and improving protein level of myosin.These MAML1- transgenic myotubes can last for more than five days. This study helps to study myoblast differentiation and understand further the roles of MAML1 gene in myogenesis of mouse.

关 键 词：成肌细胞 肌肉发生 转基因 分化 

分 类 号：Q2[生物学—细胞生物学]