EZH2负性调控CDKN2A介导SLE自身抗体产生的机制研究

Systemic Lupus Erythematosus(SLE) is an autoimmune disease characterized by abundant autoantibody production and multisystem damage. However, the mechanism underlying production of autoantibodies in SLE is still unclear. EZH2 is a histone methyltransferase. Our previous studies demonstrated that EZH2 mediated terminal differentiation from naïve B cells to plasma cells and secretion of antibodies. We also identified EZH2 protein levels in B cells and plasma cells from patients with SLE were elevated and in positive correlation with SLEDAI score. Meanwhile, CDKN2A, an EZH2 target gene, was down-regulated in patients with SLE. Since CDKN2A participates in the repression of cell proliferation and promotion of cell apoptosis, we hypothesize that EZH2 may contribute to the terminal differentiation of autoreactive plasma cells and production of autoantibodies resulting from impaired B cell peripheral immune tolerance by inhibition of CDKN2A, promotion of proliferation and suppression of apoptosis. Based on preliminary work, we will further explore the molecular and cellular mechanisms of autoantibody production in SLE through inducing ex vivo and in vivo plasma cells terminal differentiation and through Ezh2-inducible knockout mouse model, adoptive transfer mouse model and CHIP experiment. Our expected findings may facilitate the identification of new target for the treatment of SLE.

系统性红斑狼疮（SLE）是一种以大量病理性自身抗体产生为特征并累及多器官系统的自身免疫病，但SLE自身抗体产生的机制尚不明确。EZH2是一种组蛋白甲基化酶，申请者的前期研究显示，EZH2促进小鼠B细胞向浆细胞终分化及抗体分泌，SLE患者B细胞及浆细胞内EZH2表达增高且与疾病活动度呈正相关，同时SLE患者EZH2靶基因CDKN2A表达下降，该基因可抑制细胞分裂，促进细胞凋亡。因此申请者提出“EZH2通过抑制CDKN2A表达，促进B细胞增殖、抑制B细胞凋亡，导致SLE患者B细胞外周免疫耐受缺陷，并终分化为自身反应性浆细胞，进而产生自身抗体”的假说。本项目拟通过在细胞及动物模型中诱导B细胞向浆细胞终分化，并通过Ezh2基因敲除、过继转移模型，CHIP等实验方法探索EZH2影响SLE自身抗体产生的分子及细胞机制，同时探索EZH2抑制剂对SLE的治疗作用，有望为SLE的治疗提供新靶点。

目的:探讨zeste基因增强子同源物2 (EZH2)在系统性红斑狼疮(SLE)患者B细胞免疫耐受破坏和自身抗体产生中的作用，以及EZH2抑制剂对狼疮小鼠模型的治疗作用。.方法:收集初发SLE患者外周血单个核细胞(PBMCs)进行流式细胞术检测。构建pristane诱导的狼疮小鼠模型，腹腔注射EZH2抑制剂GSK343 (2.5mg/kg)治疗狼疮小鼠。收集狼疮小鼠血液和尿液检测自身抗体和蛋白尿，并进行肾脏病理评分。磁珠分选小鼠脾脏B细胞，流式细胞术检测细胞凋亡，实时荧光定量PCR (RT-qPCR)和蛋白质印迹法(WB)检测细胞凋亡。.结果:EZH2在SLE患者和pristane诱导的狼疮小鼠的多种B细胞亚群中均升高。EZH2抑制剂可减轻pristane诱导的狼疮小鼠的狼疮样症状并抑制自身抗体的产生。抑制EZH2也减少了狼疮患者浆细胞自身抗体的分泌。机制上，EZH2可通过抑制细胞周期蛋白依赖性激酶抑制剂(CKI)基因的表达，抑制B细胞凋亡，促进B细胞增殖，从而介导外周免疫耐受的破坏和自身抗体的产生。.结论:EZH2通过抑制CKI基因介导B细胞外周免疫耐受的破坏，参与SLE自身抗体的产生。EZH2抑制剂可作为一种有前景的SLE治疗药物。