Gene Expression Assay In LPS-stimulated Macrophages

Objective:To investigate the changes of gene expression profile in lipopolysaccharide(LPS)-stimulated primary Kupffer cells(KCs),and study the function of the target gene.Methods:Rat KCs were isolated and purified by means of in situ perfusion and density gradient centrifugation,KCs were stimulated with LPS.Gene expression profile were studied using gene microarrays.We studied the function of target gene in Raw 264.7 cells and the expression of Iraklbpl in LPS-stimulated Raw 264.7 cells during inflammatory response.IRF3 shRNA adenovirus was used to knock out the IRF3 gene in order to study the effect of IRF3 on the target gene.The expression of gene at mRNA level was detected by real-time fluorescence quantitative PCR.The secretion of inflammatory factors in cell supernatant was analyzed by Elisa.The expression of nuclear and cytoplasmic proteins was detected by Western blot.Results:27 genes were upregulated including Ceslf?Sicl7a3?Slc21a4?Hsd17b2?Sorbs2?Ccdc116?Mgam?Myo5b?Etl4?Fabp1?Kif4b?Fos11?Cyp4a1?Penk?Tmem221?Rp15?Nr2f1?Hoxb1?Gpr165?Fam90a13p?Kpna6?Irak1bp1?Kcnh1 and 4 unnamed genes and 4 downregulated including Oc90?Tagln?Arxes2 and Olr830 in LPS-stimulated KCs.The secretion of IL-6 mRNA and protein was significantly higher than the control group.The expression of Irak1bp1 mRNA and total Irak1bp1 protein was significantly higher than the control group,and the expression of Iraklbpl protein in nucleus was higher than that in normal control group.The expression of Iraklbpl protein in cytoplasm was not significantly changed compared with normal.The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 were significantly higher than the control group(p<0.01).The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 with adenovirus strains carrying IRF3 shRNA(p<0.01).However,the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA.The nuclear constitutive and the LPS-induced expression of Irak1bp1 protein were not affected by IRP3 shRNA.Conclusion:There is a significant difference between LPS-stimulated and normal control cells in gene expression profile by microarray analysis.LPS can induce the expression of nuclear Irak1bp1 protein in Raw 264.7,IRF3 shRNA adenovirus can effectively inhibit the expression of nuclear IRF3 protein induced by LPS stimulation,but does not affect the expression of nuclear Irak1bp1 protein.