Abstract:Objective:To study the biological effects of silencing of Rel-A gene expression by RNA interference on the dendritic cells.
Methods :Mouse dendritic cells derived from bone marrow were amplificated through cytokine inducing system and purified with positive immunomicrobeads by flow cytometer.The mRNA and protein expression, aiming directly at Rel-A, were analyzed by semi-quantified RT-PCR and Western blot. A pair of the highest effective siRNA were synthesized selectively and packed by lentivirus transfer vector, then transfected to DC(RNA interference group), while control group was not transfected by RNAi; 12 hours later, lipipolysaccharide (LPS) at a concentration of 1mg/L was added to two groups and continued to culture. The cell microstructure was then observed under transmission electron microscopy. The cell surfacemarkers, such as MHC Ⅱ, CD86 and CD80, were detected by flow cytometer. The functional properties of DCs were detected by mixed lymphocyte reaction (MLR). The concentration of TNF-α, IFN-γ and IL-12 in the supernatant were detected by ELISA.
Results:mRNA and protein expression were reduced 90% in sequence 2 siRNA, the difference was significant statistically, in comparison with control group. Many phagocytic vacuoles were observed in the cytoplasm under transmission electron microscopy. The expression of CD80, CD86 and MHC Ⅱ in the cytomembrane of DCs,the proliferation of allogeneic T cells and the concentration of TNF-α, IFN-γ and IL-12 decreased more significantly in RNA interference group than in control group.
Conclusions:RNA interference can reduce Rel-A expression in murine myeloid dendritic cells and inhibit the maturation of DCs. The dendritic cells interfered with silencing of Rel-A gene are characterized as the biological features and immunological function of immature DC. This kind of RNAi Rel-A DCs may be used to induce immunotolerance as novel tolerogenic DCs.