摘要
为研究猪Gasp1、Gasp2对成肌细胞分化的影响,采用RT-PCR技术从猪肌肉组织中扩增Gasp1和Gasp2基因片段,将其克隆入phi C31载体,构建重组质粒phi C31-Gasp1和phi C31-Gasp2。将重组质粒转染至C2C12小鼠成肌细胞,经嘌呤霉素筛选在荧光显微镜下观察,获得稳定表达Gasp1和Gasp2基因的细胞系后,应用CCK-8法检测细胞增殖能力。分别在细胞分化1、2、3、4、5 d时收集细胞,采用实时荧光定量PCR检测MyoD、MyoG、P21及MHC肌细胞分化相关基因的表达变化,进而分析Gasp1和Gasp2基因对肌肉分化的影响。结果显示,成功克隆了猪Gasp1和Gasp2基因,构建了phi C31-Gasp1和phi C31-Gasp2重组质粒,荧光显微镜下可见phi C31载体和Gasp1、Gasp2基因共转染的C2C12细胞表达强烈的绿色荧光,经CCK-8检测发现,过表达Gasp1、Gasp2基因对细胞的生长增殖影响为初期促进、后期抑制;实时荧光定量PCR检测结果表明,猪Gasp1和Gasp2可以促进细胞分化因子MyoD、MyoG、P21、MHC基因的表达。以上结果表明,Gasp1、Gasp2基因可以促进肌肉分化。
In order to study the effects of porcine Gasp1 and Gasp2 on the differentiation of C2C12 cells,Gasp1 and Gasp2 gene fragments were amplified by RT-PCR from porcine muscle tissue,and phi C31 vector was used as skeleton to construct recombinant plasmid phi C31-Gasp1 and phi C31-Gasp2. The recombinant plasmids were transfected into C2C12 cells and the cell lines stably expressing Gasp1 and Gasp2 genes were screened by puromycin and observed under the fluorescence microscope. Then the effect of the stable expression of Gasp1 and Gasp2 on the cells proliferation was detected by CCK-8 method. At last,the cells were harvested at 1,2,3,4 and 5 d,and the expression of MyoD,MyoG,P21 and MHC myoblast differentiation genes was detected by quantitative real-time PCR to analyze the influence of Gasp1 and Gasp2 genes on mouse myoblasts differentiation. The results showed that the eukaryotic expression vectors phi C31-Gasp1 and phi C31-Gasp2 were successfully constructed. The strong green fluorescence expression vectors phi C31 and phi C31-Gasp1 and phi C31-Gasp2 were transfected into C2C12 cells under the fluorescence microscope; the detection of CCK-8 showed that overexpression of Gasp1 and Gasp2 genes promotedthe growth and proliferation of cells in the early stage and inhibited them in the later stage; finally quantitative real-time PCR detection of MyoD,MyoG,P21,MHC confirmed that the Gasp1 and Gasp2 genes could promote the differentiation of C2C12 cells. In conclusion,the Gasp1 and Gasp2 genes could promote muscle differentiation.
作者
樊爽爽
张雪梅
刘晓贺
刘姣扬
明胜利
褚贝贝
杨国宇
王江
FAN Shuangshuang, ZHANG Xuemei, LIU Xiaohe, LIU Jiaoyang, MING Shengli, CHU Beibei, YANG Guoyu, WANG Jiang(Key Lab of Regulation on Animal Growth and Development of Agricultural Ministry of China, Henan Agricultural University, Zhengzhou 450002, Chin)
出处
《河南农业科学》
CSCD
北大核心
2018年第3期121-127,共7页
Journal of Henan Agricultural Sciences
基金
农业部转基因重大专项(2014ZX0801015B)
农业部948重点计划项目(2011-G35)
