NZW型IL-10RA基因导入引发小鼠巨噬细胞系RAW264.7功能亢进

发布时间：2018-09-05 11:12

【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。
[Abstract]:NZB 脳 NZW/F1 mice are (SLE) model mice of systemic lupus erythematosus. Our previous study found that there is a SLE susceptibility gene locus to IL-10RA gene on chromosome 9 of NZW mice. There were several changes in the coding region of IL-10RA gene. G1066A codon change led to the change of encoding amino acid from glycine to glutamate, resulting in the deletion of tyrosine kinase JAK1 and TYK2 phosphorylation binding motif, which is responsible for the negative regulation of IL-10 signal transduction. So it can not inhibit cellular immunity and inflammatory response, causing a series of pathological immune process leading to the pathogenesis of SLE. The effect of NZW type IL-10R on the biological function and signal transduction of mouse macrophages was studied in order to elucidate its role in the pathogenesis of SLE. Methods NZW vector [pcDNA3.1 () -NZW-IL-10R] and wild-type vector [pcDNA3.1 () -WILD-IL-10R] constructed in our laboratory were transfected into mouse macrophage cell line RAW264.7, by liposome transfection. And confirmed by flow cytometry. The expression of signal transduction protein was detected by Western blot and the secretion of inflammatory cytokines was measured by real-time quantitative PCR. Results 1 mIL-10 could not inhibit the proliferation of murine macrophages expressing NZW type IL-10R. 2. Similarly, mIL-10 showed a dose-dependent increase in IL-1a,TNF-a secretion from RAW264.7 cells of NZW type IL-10R. 路3 the signal transduction protein showed decreased phosphorylation in NZW macrophages. Conclusion NZW type IL-10R can not play an immunosuppressive effect on mouse mononuclear macrophages due to the sequence changes of its multiple coding regions, especially the deletion of JAK1 and TYK2 phosphorylation binding motifs. Thus, the inflammatory response can not be limited to cause excessive inflammatory damage, so it may play an important role in the pathogenesis of autoimmune diseases.
【学位授予单位】：中国医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392.2

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