[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)。
Construction of 3T3-L1 cell line with overexpression of AMPKα1 based on lentivirus system
[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.
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