DN-AMPK腺病毒载体的构建及其在小鼠成肌细胞系C2C12肌管分化中的影响

在研究AMPK的调控网络时,通常利用过表达显性失活突变型AMPK(dominant negative AMPK,DN-AMPK)作为研究手段来验证AMPK在某些重要生理病理调节通路中的关键作用。旨在利用Ad5腺病毒载体体系构建Ad-DN-AMPK表达载体,并在成肌细胞系C2C12中检测无活性AMPK高表达后对C2C12细胞分化为肌管细胞的影响。通过构建AMPKα1(D159A)和AMPKα2(K45R)的腺病毒表达载体,在HEK293细胞中成功包装并扩增出完整的腺病毒,待其感染能力基本稳定后,将腺病毒感染C2C12,利用激光定量成像仪检测其感染滴度,感染效率能高达100%,并且能够持续表达6 d。DN-AMPK高表达后,AMPK常用激活剂A769662(SN-5)不能激活AMPK,表现为AMPK下游蛋白活性丧失,如ACC磷酸化无变化。通过实时定量PCR的方法,检测DN-AMPK对C2C12分化为肌管细胞的影响,结果表明过表达DN-AMPK能够促进C2C12细胞分化为肌管细胞的标记蛋白(Myod和Myogenin)的表达,即促进C2C12分化为肌管细胞。

Construction of Recombinant Adenovirus Expression Vector of Dominant Negative AMPK and Its Evaluation in Mouse Myoblast Cell Line C2C12 Differentiation into Myotube

In order to evaluate the potency of AMPK,usualy we use dominant negative AMPK(DN-AMPK)as a tool.For this reason,we constructed recombinant adenovirus expression vector of DN-AMPK(AMPKα1(D159A)and AMPKα2(K45R)),which were transfected into mouse myoblast cell line C2C12 to detect the regulation of DN-AMPK during the C2C12 myotube differentiation.The recombinant plasmid was packaged and amplified after being transfected into HEK293 cells.After infecting C2C12 myoblast with recombinant adenovirus vector,the adenoviral infection was determined by iCys Research Imaging Cytometer.The infection efficiency was almost 100% for 6 days and Western blot indicated that the expression of AMPK in C2C12 of recombinant adenoviral infection group was signifcantly higher than the control group.At the same time,when DN-AMPK was overexpressed,SN-5(AMPK activator)could not induce activation of ACC.During the differentiation of C2C12 myoblast,DN-AMPK can increase the expression of Myod and Myogenin(myotube marker).These results indicated that the overexpression of DN-AMPK could increase the level of differentiation of C2C12 into myotube.