The Study Of Gene Function On Mouse Membrane CD14

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram negative bacteria. Cluster of differentiation antigen14(CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule(siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14specifically, was identified, RAW264.7cells pretreated with siRNA-224were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-a), macrophage inflammatory protein-2(MIP-2) and interleukin-6(IL-6) and the NO production were evaluated. The results indicated that siRNA-224effectively inhibited TNF-a, MIP-2, and IL-6release and NO production from LPS-stimulated RAW264.7cells by down-regulating mRNA accumulation and protein expression of mCD14specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.