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 AMPKα1 based on lentivirus system
段文婷 1李倩1
作者信息
- 1. 内蒙古医科大学附属医院药学部,内蒙古呼和浩特 010010
- 折叠
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).
Abstract
[Objective]The 3T3-L1 cell line overexpressing AMPKα1 gene was constructed by lentivirus system,and its effect on the expression of inflammatory factors was investigated.[Method]The gene sequence of AMPKα1 was found in the NCBI,and primers were designed by SnapGene software to amplify and purify the target gene.The purified AMPKα1 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-L1 cell line stably overexpressing AMPKα1 was successfully con-structed,and the inflammatory cytokine MCP-1 protein expression was decreased in the 3T3-L1 cell line with overexpression of AMPKα1.[Conclusion]Overexpression of AMPKα1 in 3T3-L1 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Key words
lentivirus system/gene overexpression/AMPKα1/3T3-L1 cell/stable rotation/MCP-1/obesity/plasmid引用本文复制引用
出版年
2024
NETL
NSTL
万方数据