摘要
前言
NZB×NZW/F1小鼠是系统性红斑狼疮(SLE)模型小鼠,我们的前期研究发现在NZW小鼠的第9号染色体有一SLE易感基因位点与IL-10RA基因连锁,其IL-10RA基因编码区序列有多处改变。G1066A密码子改变导致编码氨基酸由甘氨酸变为谷氨酸,造成承担IL-10信号转导负向调节的酪氨酸激酶JAK1和TYK2磷酸化结合模序缺失,从而不能抑制细胞免疫和炎症应答,引起一系列病理免疫过程导致SLE发病。本实验通过研究NZW型IL-10R对小鼠巨噬细胞生物功能及信号转导的影响,从而阐明其在SLE发病过程中的作用。
实验方法
本实验室前期构建的NZW型载体[pcDNA3.1(+)—NZW—IL-10R]和野生型载体[pcDNA3.1(+)—WILD—IL-10R]通过脂质体转染技术转染到小鼠巨噬细胞株RAW264.7,利用G418筛选高表达IL-10R的克隆,并通过流式细胞术确证。通过MTT法测定mIL-10刺激的高表达克隆增殖情况,免疫印迹方法检测信号转导蛋白表达,实时定量PCR技术测定炎性细胞因子分泌。
研究结果
1、mIL-10不能抑制表达NZW型IL-10R的小鼠巨噬细胞增殖。
2、同样mIL-10呈现剂量依赖性促进NZW型IL-10R的RAW264.7细胞炎性细胞因子IL-1a,TNF-a分泌。
3、信号转导蛋白在NZW型巨噬细胞表现为磷酸化程度降低。
结论
NZW型IL-10R由于其多处的基因编码区域序列改变,尤其是JAK1和TYK2磷酸化结合模序的缺失,导致IL-10与其结合后不能在小鼠的单核巨噬细胞发挥免疫抑制效应,从而不能限制炎症应答造成过度的炎症损伤,因此可能在自身免疫性疾病的发病过程中发挥重要作用。
Preface
NZB╳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.
Methods
Employing 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.
Conclusion
Because 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.
引文
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