摘要
为研究脑信号蛋白家族(Semaphorins)成员Sema7A对成肌细胞增殖和分化的影响,本文设计并合成了Sema7A基因的小干扰RNA(small interfering RNA,siRNA),用此siRNA转染C2C12成肌细胞.通过Hoechst核染和流式细胞术检测细胞增殖情况,免疫荧光检测肌管的形成情况,realtime qPCR和Western印迹技术检测成肌标记基因的变化.结果显示,干扰Sema7A后,C2C12成肌细胞增殖减慢,处在G2和S期的细胞所占的比例明显下降,而G1期细胞的比例升高.免疫荧光检测结果显示,干扰Sema7A后,肌管的直径及MyHC+细胞所占比例均显著降低.Real-time qPCR和Western印迹结果也显示,肌肉分化标志基因MyoD、MyoG、MyHC的mRNA及蛋白质表达均下降.进一步检测Sema7A受体下游信号通路发现,干扰Sema7A后,其下游信号分子PI3K和AKT的磷酸化水平被下调.以上结果表明,Sema7A可以调节C2C12成肌细胞的增殖和分化,可能是通过其受体作用于PI3K/AKT信号通路实现的,这为进一步研究Sema7A在骨骼肌发育中的作用提供实验基础.
To explore the role of Semaphorin family member Sema7A in myoblast proliferation and differentiation, small interfering RNA（siRNA）of Sema7A was introduced into C2C12 myoblasts. Hoechst staining and flow cytometry were performed to determine the proliferation of C2C12 myoblast; and myogenic differentiation was characterized by immunofluoreseence of myotubes. Real-time qPCR and Western blotting were used to measure the expression of myogenic marker genes. The results showed that the C2C12 myoblasts proliferation was inhibited by Sema7A interference with marked decrease in G2 or S phase cell proportions and increased G1 phase cells. Immunofluorescence showed that the diameter of myotubes and MyHC-positive cells were decreased. RQ-PCR and Western blotting showed that the expression of myogenic marker genes of MyoD, MyoG, and MyHC were decreased following Sema7A interference. Further analyses revealed that interference of Sema7A ablated the phosphorylation of PI3K and AKT. These findings suggested that Sema7A modulated the proliferation and differentiation of C2C12 myoblasts via the PI3 K/AKT signal pathway; and our observations indicated the importance of Sema7A in skeletal muscle development.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
北大核心
2014年第2期170-178,共9页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金项目(No.31272410)
西北农林科技大学青年科技创新项目(No.ZD2012007)~~
