基于慢病毒系统构建AMPKα1过表达的3T3-L1细胞系  

Construction of 3T3-L1 cell line with overexpression of AMPKal based on lentivirus system

作　　者：段文婷 李倩[1] DUAN Wenting;LI Qian(Department of Pharmacy,The Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010010,China)

机构地区：[1]内蒙古医科大学附属医院药学部,内蒙古呼和浩特010010

出　　处：《生物技术》2024年第3期269-274,共6页Biotechnology

摘　　要：[目的]采用慢病毒系统构建AMP依赖的蛋白激酶α1(AMP activated protein kinase α1,AMPKα1)基因过表达的3T3-L1细胞系,探讨其对炎症因子表达水平的影响。[方法]在美国国家生物技术信息中心(NCBI)上查找到AMPKα1的基因序列,利用SnapGene软件设计引物,进行目的基因扩增、纯化。将纯化后的AMPKα1片段克隆到PLJM1-EGFP载体质粒上,构建好的质粒与辅助质粒共同转染到HEK293T细胞中,收集病毒液。用含有病毒液的培养基培养3T3-L1细胞,待抗性蛋白表达后用嘌呤霉素筛选,杀死未成功转染的3T3-L1细胞,更换新鲜的完全培养基继续培养。[结果]成功构建稳定过表达AMPKα1的3T3-L1细胞系(P<0.05);AMPKα1过表达的3T3-L1细胞系的炎症因子MCP-1蛋白表达水平降低(P<0.05),炎症减轻。[结论]过表达3T3-L1细胞中的AMPKα1,减轻炎症因子MCP-1的表达水平(P<0.05)。[Objective]The 3T3-LI cell line overexpressing AMPKal gene was constructed by lentivirus system,and its effect on the expression of inflammatory factors was investigated.[Method]The gene sequence of AMPKal was found in the NCBI,and primers were designed by SnapGene software to amplify and purify the target gene.The purified AMPKal fragment was cloned into PLJM1-EGFP vector plasmid,and the constructed plasmid and helper plasmid were co-transfected into HEK293T cells to collect virus solution.3T3-L1 cells were cultured in the medium containing virus solution.After the resistance protein was expressed,the 3T3-L1 cells were screened with puromycin,and the unsuccessfully transfected 3T3-L1 cells were killed and cultured in fresh complete medium.[Result]The 3T3-Ll cell line stably overexpressing AMPKal was successfully constructed,and the inflammatory cytokine MCP-1 protein expression was decreased in the 3T3-Ll cell line with overexpression of AMPKal.[Conclusion]J Overexpression of AMPKal in 3T3-Ll cells reduced the expression level of inflammatory cytokines MCP-1.

关 键 词：慢病毒系统 基因过表达 AMP依赖的蛋白激酶α1 3T3-L1细胞 稳转 单核细胞趋化蛋白-1 肥胖 质粒 

分 类 号：R364[医药卫生—病理学]