Rab5a promotes expression of pro-inflammatory cytokines and type Ⅰ IFN in CpG induced macrophages / 中国免疫学杂志

ABSTRACT

Objective:Using the macrophage cell lines RAW264.7 stably expressing Rab5a and its dominant negative mutant Rab5aN133I as models to analyze the effect and the mechanism of Rab 5a,Rab5aN133I on CpG-induced production of pro-inflammatory cytokines and type Ⅰ IFN.Methods: The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7 cells by liposome,and screened with G418.The G418-resistant colonies were obtained and amplified.The transformed cell lines were i-dentified by RT-PCR,Real time-PCR and Western blot.The production of cytokines were measured after transformed cell lines of Rab5a and Rab5aN133I was stimulation with CpG for 8 h.Results: Rab5a expression in transfected cells was significantly higher than the control cell group (P<0.05).Overexpression of Rab5a significantly promoted the production of TNF -α,IL1-β(P<0.01) and IFN-β( P<0.05) in CpG stimulated RAW264.7.The production of cytokines was restored in Rab 5aN133I transfected cell line.Conclusion:Rab 5a promotes CpG-induced pro-inflammatory cytokines and typeⅠIFN in macrophages,it may be act as a positive regulator in TLR9 signaling pathway.