CD80双价抗体对降植烷诱导的小鼠狼疮样肾炎模型的免疫干预效应研究

Immune intervention with anti-CD80 bivalent antibody in pristane-induced mouse model of lupus nephritis

目的 运用CD80双价抗体抑制CD80/CD28信号通路,研究观察双价抗体对小鼠狼疮样肾炎的逆转效应并探究其分子机制.方法 运用化学法建立小鼠狼疮样肾炎模型,一次性腹腔注射0.5 ml降植烷（Pristane）,4个月时小鼠出现蛋白尿,外周ANA、抗dsDNA抗体水平也明显升高,模型建立成功.取成模小鼠分为2组,CD80双价抗体干预组：每只小鼠分别于第1、3、5、8、15天尾静脉注射200μg CD80双价抗体,随后3个月每隔1个月注射1次;模型组注射同型蛋白,给药方式及剂量与双价抗体干预组相同.同时设立正常对照组.Albustix试纸法监测小鼠尿蛋白动态变化,流式细胞术分析脾脏中免疫相关细胞的活化程度,间接免疫荧光法检测小鼠血清中自身抗体（ANA和抗dsDNA抗体）的表达水平及IFN-γ、IL-4的含量,取肾脏组织进行HE染色分析、免疫复合物（IC）检测.结果 小鼠尿蛋白显示双价抗体干预组的尿蛋白程度显著低于建模组,差异有统计学意义（P＆lt;0.05）;流式细胞术分析双价抗体干预组脾脏中巨噬细胞（CD11b＋）、树突状细胞（CD11c＋）、中性粒细胞（Gr1＋）及B细胞（CD21＋）活化程度显著低于模型组（P〈0.05）,并且T细胞中CD4＋、CD154＋细胞数量干预组明显少于模型组（P〈0.05）;血清中ANA、dsDNA的阳性率和滴度干预组低于模型组,差异有统计学意义（P〈0.05）;干预后小鼠血清中IFN-γ及IL-4的含量与建模组相比出现下降,差异具有统计学意义（P〈0.05）;肾脏HE染色和免疫荧光结果显示,肾小球炎症损伤和坏死程度减轻,肾脏免疫复合物沉积情况减少.结论 CD80双价抗体高效特异地结合抗原提呈细胞表面的CD80分子,阻断了CD80/CD28协同刺激信号,下调机体的免疫应答,对小鼠狼疮样肾炎病理损伤具有缓解和逆转作用.

Objective To study the therapeutic effect of anti-CD80 bivalent antibody on mouse lu-pus nephritis and to explore the possible molecular mechanism. Methods A mouse model of lupus nephritis was established through intraperitoneal injection of 0. 5 ml of pristine in female C57BL/6J mice. Appearance of urinary protein and significantly increased levels of peripheral antinuclear antibody （ ANA） and anti-doub-le-stranded DNA （ anti-dsDNA） antibody in the fourth month after injection indicated that the mouse model was established successfully. Then the mice were divided into two groups including anti-CD80 bivalent anti-body intervention group （injected with 200μg of anti-CD80 bivalent antibody at day 1, 3, 5, 8 and 15, fol-lowed by three times of injection with an interval of one month） and model group （ injected with the same protein using the same strategy）. A normal control group was set up accordingly. Albustix test paper was used to monitor the dynamic changes in mouse urinary protein. Flow cytometry was used to analyze the acti-vation of immune-related cells in spleen. Levels of autoantibodies （ ANA and anti-dsDNA） and levels of IFN-γ and IL-4 in serum were detected by indirect immunofluorescence assay. Renal tissue samples were an-alyzed with hematoxylin and eosin （ HE） staining and immunocomplex （ IC） assay. Results Urinary pro-tein level of the anti-CD80 bivalent antibody intervention group was significantly lower than that of the model group （P〈0. 05）. Activated macrophages, dendritic cells, neutrophils and B cells in spleen tissues of the anti-CD80 bivalent antibody intervention group were significantly less than those of the model group （ P〈0. 05）, and the numbers of CD4＋ and CD154＋ T cells were significantly less than those of the model group （P〈0. 05）. Positive rates and titers of ANA and dsDNA in serum samples of the intervention group were lower than those of the model group （P〈0. 05）. Levels of IFN-γand IL-4 in serum samples of the interven-tion group were decreased as compared with those of the model group （P〈0. 05）. HE staining and immuno-fluorescence assay showed that glomerular inflammatory injury and necrosis were alleviated and kidney im-mune complex deposition was reduced after anti-CD80 bivalent antibody intervention. Conclusion Anti-CD80 bivalent antibody specifically binds to the CD80 molecule on antigen presenting cell surface, blocks the CD80/CD28 co-stimulatory signaling pathway and down-regulates the body′s immune response, which al-leviates and reverses the lupus-like nephritis-induced pathological damages in mice.