Anti-IL-12/IL-23 p40抗体对实验性自身免疫性葡萄膜炎的抑制作用及其机制  

Therapeutic effect of anti-IL-12/IL-23 p40 on experimental autoimmune uveitis and its mechanism

作　　者：崔雪雪 张智慧 吴凌子 栗勇涛 陈爽 陈努 张晓敏[1] Cui Xuexue;Zhang Zhihui;Wu Lingzi;Li Yongtao;Chen Shuang;Chen Nu;Zhang Xiaomin(Tianjin Key Laboratory of Retinal Functions and Diseases,Tianjin Branch of National Clinical Research Center for Ocular Disease,Eye Institute and School of Optometry,Tianjin Medical University Eye Hospital,Tianjin 300384,China)

机构地区：[1]天津医科大学眼科医院,天津医科大学眼视光学院,天津医科大学眼科研究所,国家眼耳鼻喉疾病临床医学研究中心,天津市分中心天津市视网膜功能与疾病重点实验室,天津300384

出　　处：《中华实验眼科杂志》2022年第8期707-715,共9页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目(81671642、82171042、81870651);天津市科技支撑重点项目(20YFZCSY00990);天津市自然科学基金重点项目(20JCZDJC00100);天津市医学重点学科(专科)建设项目(TJYXZDXK-037A)。

摘　　要：目的探讨Anti-白细胞介素(IL)-12/IL-23 p40抗体对实验性自身免疫性葡萄膜炎(EAU)的抑制作用及其机制。方法选取SPF级健康无眼疾6~8周龄雌性C57BL/6N小鼠66只,其中24只采用光感受器维生素A类结合蛋白(IRBP)651-670诱导小鼠EAU模型,分别在免疫前及免疫后第3、12、18天各取6只小鼠,流式细胞术检测各时间点小鼠脾脏、淋巴结和眼球中IL-17A^(+)γ干扰素(IFN-γ)^(+)CD4^(+)T细胞比例。取6只小鼠制作EAU模型,免疫后18 d采用小动物成像仪进行眼底拍照并行光相干断层扫描(OCT)检查。检查完成后处死小鼠,摘取眼球,采用苏木精-伊红染色法检测小鼠视网膜炎症反应和组织结构形态学改变;取淋巴结行流式细胞术检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例,按照表达数量不同分为IL-17A^(+)IFN-γ^(+)细胞高表达组和IL-17A^(+)IFN-γ^(+)细胞低表达组,比较2个组小鼠视网膜损伤情况。取36只小鼠制作EAU模型,采用随机数字表法分成Anti-IL-12/IL-23 p40组和IgG组,每组18只,分别尾静脉注射Anti-IL-12/IL-23 p40和IgG,每3天1次。免疫后第12天和第18天每组各取6只小鼠,分别取淋巴结和眼球组织,采用流式细胞仪检测T细胞亚群比例。免疫后第24天,每组各取6只小鼠,摘取眼球,采用苏木精-伊红染色法观察视网膜损害情况;采用流式细胞仪检测CD4^(+)T细胞体外诱导分化情况;采用酶联免疫吸附测定(ELISA)法检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞诱导分化后IL-17和IFN-γ表达情况;采用实时荧光定量PCR法检测IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞诱导分化后Th1细胞转录因子T-bet和Th17细胞转录因子维甲酸相关孤核受体γt(ROR-γt)相对表达量。结果免疫前和免疫后第3、12、18天,淋巴结、脾脏、眼球中IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞比例总体比较差异均有统计学意义(H=9.642、16.531、10.385,均P<0.05),其中与免疫前相比,EAU小鼠免疫后第12天淋巴结IL-17A^(+)IFN-γ^(+)CD4^(+)T细胞�Objective To explore the therapeutic effect of anti-interleukin(IL)-12/IL-23 p40 antibody on experimental autoimmune uveitis(EAU)and its mechanism.Methods Sixty-six SPF female C57BL/6N mice aged 6-8 weeks were selected.EAU model was established in 24 mice through immunization with the interphotoreceptor retinoid-binding protein(IRBP)651-670.The 24 mice were sacrificed before immunization,and on the 3rd,12th,and 18th day after immunization,with 6 at each time point.Flow cytometry was used to detect the proportion of IL-17A^(+)interferon-γ(IFN-γ)+CD4^(+)T cells in the spleen,lymph nodes and eyeballs.Another 6 mice were selected to establish EAU model,and fundus images of the mice were taken with a small animal imaging instrument and optical coherence tomography(OCT)18 days after immunization.The 6 mice were sacrificed after OCT examination and the eyeballs were collected.Hematoxylin-eosin staining was used to observe the retinal inflammation and morphological changes in tissue structure.Flow cytometry was employed to detect the proportion of IL-17A^(+)IFN-γ^(+)CD4^(+)T cells in lymph nodes.The 6 mice were divided into IL-17A^(+)IFN-γ^(+)highly expressed group and IL-17A^(+)IFN-γ^(+)lowly expressed group according to flow cytometry results,and the retinal injury was compared between the two groups.EAU model was established in another 36 mice,which were divided into anti-IL-12/IL-23 p40 group and IgG group by random number table method,with 18 mice in each group.Anti-IL-12/IL-23 p40 or IgG was injected by tail vein at a 3-day inteval according to grouping.On the 12th and 18th day after immunization,6 mice were selected from each group to collect lymph nodes and eyeballs,and the proportion of T cell subsets was detected by flow cytometry.Eyeballs of 6 mice in each group were extracted on the 24th day after immunization and retinal damage was observed by hematoxylin-eosin staining.The induced differentiation of CD4^(+)T cells in vitro was assayed by flow cytometry.The expressions of IL-17 and IFN-γwere detected b

关 键 词：葡萄膜炎 药物治疗 白细胞介素12亚基p40 单克隆抗体 光感受器间维生素A类结合蛋白 小鼠 

分 类 号：R773.9[医药卫生—眼科]