小鼠SOCS3基因真核表达载体的构建及其对3T3-L1细胞凋亡的影响  

Construction of mice SOCS3 eukaryotic vector and its effects on 3T3-L1 cells apoptosis

作　　者：安磊[1] 孙婵[2] 解芸菲[1] 王勇[2] 吕彬[2] 孙超[2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]西北农林科技大学动物科技学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2011年第9期17-23,共7页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(30871785);教育部新世纪优秀人才计划项目(NCET-06-0865)

摘　　要：【目的】探讨SOCS3对3T3-L1细胞凋亡的影响。【方法】以小鼠脂肪组织提取的RNA为模板,用RT-PCR扩增SOCS3基因,将其克隆至pMD18-T Simple,构建pMD18-T-SOCS3重组质粒,经测序验证后,以pMD18-T-SOCS3为模板扩增SOCS3基因,将其克隆至pEGFP-N1,构建真核重组质粒pEGFP-N1-SOCS3,测序正确后,将重组质粒pEGFP-N1-SOCS3转染3T3-L1细胞,荧光下观察GFP的表达,RT-PCR和Western blotting检测细胞内SOCS3mRNA和蛋白的表达,观察细胞凋亡的变化。【结果】测序表明,pMD18-T-SOCS3和pEGFP-N1-SOCS3的核苷酸序列正确率为100%;在荧光显微镜下发现,脂质体组和SOCS3组均有GFP的表达;RT-PCR和Westernblotting检测表明,3T3-L1细胞中SOCS3mRNA表达显著提高(P<0.05);Hoechst 33258染色发现,SOCS3组的细胞凋亡显著;Bax和c-myc基因的mRNA表达水平显著升高(P<0.05),bcl-2和mcl-1基因表达水平显著降低(P<0.05)。【结论】SOCS3对脂肪细胞凋亡的发生有促进作用。【Objective】 The research is aimed at discussing the effects of SOCS3 on 3T3-L1 Lipoapoptosis.【Method】 With RNA extracted from mice adipose tissue as a template,SOCS3 was amplified to pMD18-T by RT-PCR and recombinant plasmid pMD18-T-SOCS3 was constructed.After sequencing,SOCS3 was amplified to pEGFP-N1 with pMD18-T-SOCS3 as a template and the eukaryotic recombinant plasmid pEGFP-N1-SOCS3 was contructed,after sequencing the pEGFP-N1-SOCS3.GFP under the fluorescence microscope is expressed and the expressions of SOCS3 mRNA and protein by RT-PCR and Western blotting was used to detect the change of apoptosis.【Result】 The recombinant plasmid pMD18-T-SOCS3 and pEGFP-N1-SOCS3 are qualified to be right by sequence identification in genbank;Lipofectamine group and SOCS3 group show that GFP is expressed under the fluorescence microscopy;SOCS3 mRNA and protein in the cells detected by RT-PCR and Western blotting express significantly（P〈0.05）.The apoptosis of SOCS3 group increased significant after Hoechst 33258 staining;Compared with the control group,SOCS3 group significantly increased apoptosis,the mRNA expression level of Bax and c-myc gene was significantly increased（P〈0.05）,while bcl-2 and mcl-1 was significantly decreased（P〈0.05）.【Conclusion】 SOCS3 can promote fat cell apoptosis.

关 键 词：细胞凋亡 SOCS3 PEGFP-N1 

分 类 号：Q786[生物学—分子生物学]