| PrefaceNZB╳NZW/F1 mice are the mouse model of Systemic lupus erythematosus (SLE),Our preliminary study found that a SLE susceptibility loci on the chromosome No.9 of the NZW mouse which linked with IL-10RA gene,which coding region sequences have changed.G1066A codon changes cause encoding amino acid change from glycine to glutamic acid,resulting in the deficiency of IL-10 signal transduction negative regulation of tyrosine kinase phosphorylation of JAK1 and TYK2,which lose to inhibit cell-mediated immunity and inflammatory response,causing a series of pathological processes which result in the immune pathogenesis of SLE.This experiment studied the NZW-type IL-10R on murine macrophage function and biology of signal transduction at the impact of SLE to clarify its role in pathogenesis.MethodsEmploying lipofection technology to transfect pre-built NZW vector[pcDNA3.1 (+)-NZW-IL-10R]and WILD vector[pcDNA3.1(+)-WILD-IL-10R]of our Laboratory into mouse macrophage cell line RAW264.7,using G418 to select the higher IL-10R expression cloning,and it is confirmed by flow cytometry.We used the MTT method to detect the proliferation of high-expression clone at the mIL-10 stimulating,the Western blot method to detect the signal transduction protein expression,and real-time quantitative PCR to measure secretion of inflammatory cytokines.Results 1,mlL-10 can not inhibit the proliferation of NZW-type IL-10R mouse macrophage cell.2,Similarly mIL-10 dose-dependent manner promote the inflammatory cytokines IL-1a,TNF-a secretion of NZW-type IL-10R in RAW264.7 cells.3,Signal transduction protein in the NZW-type macrophages showed reduced phosphorylation.ConclusionBecause many regions of the gene coding sequence on the NZW-type IL-10R changes,in particular phosphorylation of JAK1 and TYK2 deficiencies,IL-10 combination with the IL-10R can not exert immunosuppressive effects,which may play critical role in the process of autoimmune disease. |
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