摘要
目的构建稳定高表达芳香烃受体(AhR)的小鼠RAW264.7细胞株,观察AhR对脂多糖(LPS)刺激的RAW264.7细胞炎症细胞因子表达的影响。方法构建含AhR-Flag-EGFP基因的慢病毒重组质粒载体并获得病毒上清,感染RAW264.7细胞后经嘌呤霉素筛选获得稳定表达的单克隆细胞株,通过荧光显微镜和流式细胞仪检测绿色荧光蛋白表达情况,qRT-PCR和Western blot法验证AhR基因及蛋白水平表达情况。qRT-PCR和ELISA检测LPS刺激后炎症细胞因子的表达情况。结果获得1株稳定高表达AhR的RAW264.7细胞,荧光显微镜和流式细胞仪显示其荧光率达100%,qRT-PCR和Western blot结果表明RAW/AhR稳转组相对于RAW/Vector空转组,AhR的m RNA和蛋白水平明显上调(P<0.05)。LPS刺激后炎症相关细胞因子表达升高,相对于空转组,稳转组的促炎细胞因子IL-6、IL-1β的表达更低(P<0.05),抗炎细胞因子IL-10的表达更高(P<0.05)。结论成功构建稳定表达AhR的RAW264.7单克隆细胞株,证实了AhR对LPS刺激的巨噬细胞炎症反应具有负性调控作用,为后续进一步研究巨噬细胞中AhR的免疫调节及其他功能奠定基础。
This study was performed to establish a murine macrophage RAW264.7 cell line stably overexpressing AhR for evaluating the regulatory effects of AhR in inflammation induced by LPS stimulation.A lentiviral vector containing AhR-Flag-EGFP gene was constructed and then packaged in HEK293 T cells;RAW264.7 cells were infected with collected supernatant and screened by puromycin.The gene and protein expression of AhR were analyzed by qRT-PCR and Western blotting,respectively; the expression of inflammatory cytokines were detected by qRT-PCR and ELISA.Data showed that a RAW264.7 cell strain with stable expression of AhR-Flag-EGFP was successfully constructed.Fluorescent microscopy and flow cytometry demonstrated that the expression rates of EGFP reached to 100%; the qRT-PCR and Western blot showed that the gene and protein expression of AhR were significantly upregulated in RAW/AhR group compared to RAW/Vector group.The expression of IL-6 and IL-1β in LPS-induced RAW/AhR group were much lower than that in LPS-induced RAW/Vector group.In contrast,the level of IL-10 in LPS-induced RAW/AhR group was much higher than that in LPS-induced RAW/Vector group.In conclusion,a RAW264.7 cell line stably expressing AhR has constructed,and AhR negatively regulates the LPS-induced inflammatory response in macrophages,which lay a foundation for further study AhR function.
出处
《免疫学杂志》
CAS
CSCD
北大核心
2017年第12期1082-1087,共6页
Immunological Journal
基金
国家自然科学基金项目(81671906)
第三军医大学成果转化基金项目(2016XZH11)
创伤烧伤与复合伤国家重点实验室开放课题(SKLKF201601)
