高迁移率族蛋白1氧化还原失衡在系统性红斑狼疮发生发展中的作用机制研究

Antigen-antibody complex binds to C1q complement and starts the classical activation pathway to clear foreign antigens and/or senescent cells is an important mechanism to maintain immune balance. Literature and preliminary results indicate that HMGB1 is contained in systemic lupus erythematosus (SLE) patients' circulating immune complexes and cause abnormal autoimmune response, impede the normal clearance of senescent cells. Because HMGB1 protein cellular localization and immune function are determined by the redox station of its cysteine residues , we hypothesize that the HMGB1 protein is mildly oxidized ( 23,45 cysteine residues were oxidized to form a disulfide bond, while the 106 cysteine residue remain reduced) and then enter the cytoplasm and extracellular further bound to immune complexes, thus prevents the clearance of immune complexes, resulting in the deposition of immune complexes and autoimmune response , causing in the occurrence and development of SLE. This project intends to induce G →C mutation on exon 4 21st bp of HMGB1 in mice to construct a HMGB1C106S gene mutation mice to establish the SLE animal model, and further using confocal microscope, flow cytometry technologies to explore the relationship between HMGB1 protein oxidative modification status and its immune functions, as well as the role of HMGB1 in the development of anti-nuclear antibodies and the pathogenesis of SLE, and assess the potentiality of HMGB1 as a therapeutic target of SLE.

抗原抗体复合物结合补体C1q启动补体经典激活途径清除外来抗原和自身衰老细胞是保持机体免疫平衡的重要机制。文献和前期研究结果表明系统性红斑狼疮（SLE）患者体内循环复合物中HMGB1蛋白是引起异常自身免疫反应，妨碍衰老细胞正常清除的关键。而HMGB1蛋白细胞定位和免疫功能和其半胱氨酸残基氧化状态有关，据此提出HMGB1蛋白被轻度氧化（23，45位半胱氨酸残基被氧化形成二硫键，106位半胱氨酸残基仍为还原态）从而进入胞浆和胞外，进一步结合到免疫复合物上阻止了免疫复合物的清除，造成免疫复合物沉积和机体自身免疫反应，导致SLE的发生发展的假说。本项目拟通过构建HMGB1基因第四外显子21位碱基G→C点突变小鼠（C106S）建立SLE动物模型，并采用激光共聚焦、流式技术来探讨HMGB1蛋白氧化修饰状态和其免疫功能的关系，以及在抗核抗体和SLE发生发展中的作用，评估HMGB1作为SLE治疗靶点的潜力。

文献和我们的前期研究表明HMGB1蛋白在许多生理病理过程中发挥关键作用，并且其功能和其半胱氨酸残基的氧化还原状态有关。本项目通过CRISPR/Cas9技术构建HMGB1第106位氨基酸点突变的模式小鼠来研究106位氨基酸不同的氧化还原状态在系统性红斑狼疮发病中的作用机制，并探讨将HMGB1作为自身免疫病治疗靶点的潜在价值。首先研究了自身免疫性疾病（类风湿性关节炎和系统性红斑狼疮）患者外周血中HMGB1和抗HMGB1自身抗体的浓度和疾病严重程度的关系。采用CRISPR/Cas9构建了HMGB1敲除的小鼠黑色素瘤B16细胞株和HMGB1第106位氨基酸点突变小鼠模型（还原态：半胱氨酸突变为色氨酸；氧化态：半胱氨酸突变为丙氨酸），进一步构建系统性红斑狼疮疾病模型研究HMGB1第106位氨基酸氧化还原状态在系统性红斑狼疮发病中的机制。