Research On The Regulation Of Rab Protein On The TLR9Signaling Pathways In Macrophages

Toll-like receptors are important pattern recognition receptors. Cytokines and kemokines are produced by innate immune cells through identification of pathogen-associated molecular patterns of pathogenic microorganisms, and then then innate immune responses were initiated. Such responses constitute. However, Excessive immune response has reported to bring adversely affect, such as systemic lupus erythematosus, endotoxic shock and autoimmune diseases. Therefore, TLRs signaling pathways need to be tightly controlled. Objective:To construct Rab5a and Rab9eukaryotic expression vector, establish transformed macrophage cell lines stably expressing Rab5a and its dominant negative mutant Rab5aN133I, and analyze the effect and the mechanism of Rab5a, Rab5aN133I, Rab7and dominant negative mutant form of Rab7(Rab7T22N) on the production of cytokines in RAW264.7macrophages induced by CpG Methods:Using the cDNA of RAW264.7cells as templates, the open reading frame of Rab5a and Rab9was amplified by primers designed according to the full-length sequence of Rab5a in Genbank. To amplify the open reading frame of Rab5a and Rab9, the target gene were connected with the eukaryotic expression vector pcDNA3.1, and transformed into E.coli DH5a.Positive clones were indentified by double restriction enzyme digestion and sequence analysis. The eukaryotic expression vectors of Rab5a and Rab5aN133I were transfected into RAW264.7cells by liposome, and screened with G418. The G418-resistant colonies were obtained and amplified. The transformed cell lines were identified by RT-PCR, Real-Time PCR and Western Blot. The production of cytokines were measured after transformed cell lines of Rab5a、Rab5aN133I、Rab7and Rab7T22N stimulation with CpG after8hr. Results:Sequence analysis showed that the Rab5a-pcDNA plasmids were100%homologous with the sequence of Rab5a in Genbank, Rab9-pcDNA plasmids were99%homologous with the sequence of Rab9in Genbank, no mutations were found. Rab5a and Rab7expression in transfected cells was significantly higher than the control cell group. Overexpression of Rab5a significantly promoted the production of TNF-α, IL-1β and IFN-β in CpG stimulated macrophages. The production of cytokines was restored in overexpression of Rab5aN133I cell line. Overexpression of Rab7inhibited CpG induced expression of IL-6, IL-1β and IFN-β in RAW264.7cells. However, overexpression of Rab7T22N significantly promoted the expression of the above cytokines in RAW264.7macrophages induced by CpG Conclusion:Rab5a and Rab9eukaryotic expression vectors were successfully constructed. Rab5a may positively regulates TLR9signaling pathway, However, Rab7can negatively regulate TLR9signaling. The study may lay a foundation for our further study the role of Rab proteins in TLRs signaling pathways.