SOCS3过表达细胞模型的建立

摘要

目的 建立体外高表达细胞因子信号转导负调控蛋白3( suppressor of cytokine signaling 3,SOCS3)的细胞模型,为进一步研究SOCS3在炎症因子信号通路中的作用机制提供研究手段.方法 通过RT-PCR克隆SOCS3的编码片段,连接至pIRES2-EGFP真核表达质粒,经测序鉴定后,转染至293T细胞.通过荧光显微镜观测转染效率,RT-PCR和Western印迹法检测SOCS3表达情况,同时检测SOCS3高表达对脂多糖(lipopolysaccharides,LPS)诱导的信号转导和转录激活因子3 (signal transducers and activators of transcription 3,STAT3)磷酸化的影响.结果 经测序证实,pIRES2-EGFP-SOCS3重建质粒中的SOCS3序列完全正确,转染后细胞中可见明显绿色荧光,且SOCS3在mRNA和蛋白水平表达均显著增加,过表达SOCS3可显著抑制由LPS诱导的STAT3的磷酸化.结论 成功构建SOCS3基因重组质粒,转染293T细胞,获得高表达具有功能性SOCS3分子.该细胞模型建立为进一步研究SOCS3的调节机制提供了有利工具.%Objective To establish an overexpressing-suppressor of cytokine signaling 3 ( SOCS3) cell model for study the mechanism of SOCS3 in inflammatory signal transduction. Methods Human SOCS3 gene fragment was obtained from THP-1 cells by RT-PCR and subcloned into eukaryotic expression pIRES2-EGFP plasmid. After sequencing, the recombinant plasmid was transfected into 293T cells by lipidosome. The enhanced green fluorescent protein, expression in the cell was detected by inverted fluorescence microscope. The expression level of SOCS3 in 293T cells was detected by RT-PCR and Western blotting. The function of SOCS3 overexpressed in 293T cells was also detected by Western blotting. Results The cloned open reading frame (ORF) of SOCS3 gene fragment in recombinant pIRES2-EGFP-SOCS3 plasmid was confirmed by DNA sequencing. After transfection, the EGFP was highly expressed in the 293T cell. And there were abundant SOCS3 mRNA and protein expressed in the cells transfected with pIRES2-EGFP-SOCS3 plasmid. After stimulation with LPS, the P-STAT3 expression was significantly reduced in 293T cells transfected with pIRES2-EGFP-SOCS3 plasmid compared to that of untransfected 293 T cells and 293 T cells transfected with pIRES2-EGFP plasmid. Conclusion Human SOCS3 gene was cloned successfully and expressed effectively in 293 T cells which provides a useful tool for further study of SOCS3 function.