Mechanism Of CD40of RAW264.7Cells Stimulated By Escherichia Coli Lipopolysaccharide

Cluster of differentiation antigen40(CD40) is an important co-stimulating molecules, Abnormal expression of CD40can trigger autoimmune inflammatory disease. Lipopolysaccharide (LPS) is a glycolipid that constitutes the major portion of outmost membrane of gram-negative bacteria, and it is capable of inducing inflammatory. LPS is also a kind of inducer of gene expression of CD40, it can up-regulate the expression of CD40in RAW264.7. The function study of CD40may provide a new idea for the treatment of these kind of diseases.RNA interference can lead to gene silencing specifically, and it’s a powerful tool to study specific gene function and gene therapy. In this study, we intend to use RNAi technology to inhibit the expression of CD40in LPS-activated RAW264.7cells, and use Western blot to detect the CD40experssion under LPS stimulation and pretreatment with siRNA. And then, following pretreatment with siRNA and stimulation of LPS, the secretion of cytokines in supernatant, the protein experssion of iNOS in cells and the NO production in cell supernatant was evaluated by ELISA, Westren blot and Greiss assay. The results indicated that down-regulation of the protein expression of CD40under LPS stimulation can effectively down-regulate interleukin-1β(IL-1β), tumor necrosis factor alpha (TNF-α) and macrophage inflammatory protein-2(MIP-2) release and the protein expression of iNOS as well as NO production. These results provide some important informations for the treatment of LPS related diseases.