Construction of a RAW264.7 cell line overexpressing AhR and its regulatory effects on inflammatory cytokines
ZHU Junyu
YIN Shangqi
LUO Li
TIAN Lixing
TANG Wanqi
MA Wei
FAN Xia
LIANG Huaping
Abstract：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-FIag-EGFP gene was constructed and then packaged in HEK293T 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.
Keywords：Aryl hydrocarbon receptorRAW264.7Stable expressionCytokine
Publication Date：2017-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：6( 1082-1087 )