TNFAIP3调控SLE自身免疫炎症形成的分子缺陷及其机制

The gene of tumor necrosis factor alpha-induced protein3(TNFAIF3),known as one of important endogenous negative regulators of TNFα mediated NF-κB inflammatory singnaling, has been found to be associated with the susceptibility of systemic lupus erythematosus(SLE) patients with genome-wide analyses from various global areas. However, the molecule evidences of deficient functions of TNFAIF3 to regulate the inflammation of SLE are still unclear.In present project, the expression levels of TNFAIF3 mRNA and protein on periphery blood T subgroup cells from patients with SLE will be detected by real-time PCR and immuno-blotting respectively and the mutations of TNFAIF3 gene will be analysed to be associated with clinical phenotypes of SLE patients by single nucleotide polymorphisms(SNPs).From these bases on,co-expression vehicles containing different genotypes will be constructed and transferred into Jurkat cells. Then, the diversify pattens of inflammatory signaling activation in transferred Jurkat cells will be observed by Western blotting following the stimulations of tumor necrosis factor alpha or lipopolysaccharide（LPS）.The anti-inflammatory effects with different mutants will be explored in C57/6-lpr mice. The aims of this project are to explore the molecule deficient basis and mechanism of function failure of TNFAIF3 to negatively regulate the activation of NF-κB inflammatory signaling pathways which induce auto-inflammatory processes of SLE and to provide some novel evidences about both the elucidation of illness diversification and the findings of new molecular targets to treat SLE.

全球多个地区报道用全基因扫描技术从SLE患者中都筛选到相关的易感基因TNFAIP3，而TNFAIP3又是TNFα介导的NF-κB信号途径异常活化的重要负性调控分子。本课题选择TNFAIP3为切入点，以SLE患者为研究对象，采用SNP分析、定量PCR等技术，观察患者外周血T细胞亚群中TNFAIP3mRNA和蛋白表达水平及基因多态性，分析其与临床关联性。在此基础上，体外构建不同基因型别的共表达载体，作用于Jurkat细胞，观察TNF和脂多糖作用后细胞炎症信号分子的变化，同时观察不同基因型别表达的蛋白对狼疮鼠体内炎症反应的影响，以明确TNFAIP3调控SLE自身免疫炎症形成的分子缺陷及其信号变化特征。本研究可望为解释SLE患者病情多样化和重症化提供全新的依据，为建立选择性关闭SLE炎症信号的个体化疗法提供新的分子靶位。

系统性红斑狼疮（SLE）是一种自身免疫性炎症性疾病，临床表现多样。发病机制至今未完全阐明，既往研究表面异常活化的单核细胞和核因子-κB（NF-κB）介导的炎症反应通路是机制之一。其中肿瘤坏死因子-α（TNF-α）是介导自身免疫反应炎症形成的重要效应因子,长期成为SLE等自身免疫性疾病研究的热点。TNF-α诱导蛋白3/与A20结合的核因子-κB抑制蛋白1 （TNFAIP3/ABIN1）是影响TNF-α介导的炎症反应形成的内源性调控基因。锌指蛋白A20是TNFAIP3编码的蛋白，越来越多的研究证实A20是NF-κB活化的负反馈调控机制中的重要一环。我们的研究发现来自SLE患者的单核细胞在TNF-α刺激下会产生显著的NF-κB介导的炎症应答，表现为IL-1β和NF-κB表达增加、IκBα表达增加及p-IκBα表达减少，然而TNF-α诱导的A20表达未显著增加。Daphnetin是瑞香科属植物的自然提取物，它能显著提高小鼠体内A20的表达。我们的研究显示，通过daphnetin的干预能够缓解NZB/WF1狼疮鼠肾炎，表现为daphnetin治疗组血中尿素氮水平显著下降，dahpnetin降低了血清IL-6,TNF-α水平,狼疮鼠经过dahpnetin干预后高表达A20并能抑制NF-κB活性。进一步的研究显示在小鼠腹腔巨噬细胞中通过腺病毒介导的A20过表达可以减低pristane狼疮模型小鼠的血清中细胞因子IL-6、IL-1β水平、降低自身抗体anti-dsDNA和anti-nRNP 的IgG水平；病理切片显示过表达A20可以缓解pristane诱导的狼疮肾炎，表现为肾小球和肾小管间质的活动性评分下降和24小时尿蛋白减少；机制研究显示过表达A20抑制了NF-κB活性。综上，SLE中存在TNFAIP3相关分子的调控缺陷，A20作为TNFAIP3编码的蛋白具有潜在的治疗价值。