c-Myc抑制剂10058-F4对实验性自身免疫性葡萄膜炎小鼠模型Th17细胞比例及细胞因子表达的影响  

Effects of c-Myc inhibitor 10058-F4 on T helper 17 cell proportion and cytokine expression in mouse model of experimental autoimmune uveitis

作　　者：刘玉玲 赵璐 杨超 陈思思 魏瑞华 粘红 LIU Yuling;ZHAO Lu;YANG Chao;CHEN Sisi;WEI Ruihua;NIAN Hong(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、眼视光学院、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,天津市300384

出　　处：《眼科新进展》2023年第6期429-433,共5页Recent Advances in Ophthalmology

基　　金：国家自然科学基金项目(编号:81970793,82070929);天津市医学重点学科(专科)建设项目(编号:TJYXZDXK-037A)。

摘　　要：目的探讨c-Myc抑制剂10058-F4对实验性自身免疫性葡萄膜炎(EAU)小鼠模型光感受器间维生素A类结合蛋白1-20(IRBP 1-20)特异性辅助性T细胞17(Th17)细胞比例及细胞因子表达的影响。方法取健康C57BL/6J小鼠(8~10周龄)6只,采用随机数字表法将小鼠分为正常对照组、EAU组,每组3只。EAU组小鼠腹腔内注射350 ng百日咳毒素,单后足和脊柱两侧皮下注射含150μg IRBP 1-20、0.8 mg结核分枝杆菌H37RA和弗氏不完全佐剂的乳化剂以诱导EAU模型;正常对照组小鼠不给予特殊干预。免疫后13 d,行间接检眼镜检查并通过小动物视网膜成像系统可视化检测小鼠眼底炎性病灶,HE染色观察视网膜组织病理学改变,实时荧光定量PCR(qRT-PCR)检测T细胞和眼球组织中c-Myc mRNA的表达。将EAU模型小鼠T细胞分为对照组和10058-F4组,10058-F4组T细胞加入50μmol·L^(-1)10058-F4,对照组T细胞不给予药物干预。培养6 h后,洗去药物,将两组T细胞与IRBP 1-20(10 mg·L^(-1))及骨髓来源树突细胞共培养,同时加入20μg·L^(-1)白细胞介素(IL)-23(Th17细胞诱导条件)。采用qRT-PCR检测c-Myc、维甲酸相关核孤儿受体γt(RORγt)、IL-17A、IL-17F和粒细胞-巨噬细胞集落刺激因子(GM-CSF)mRNA相对表达量,ELISA检测共培养细胞上清液中IL-17含量,流式细胞仪检测共培养细胞中Th17细胞比例。结果本研究EAU组小鼠模型建立成功。qRT-PCR检测结果显示,EAU组小鼠T细胞、眼球组织中c-Myc mRNA相对表达量分别为1.85±0.37、2.31±0.47,较正常对照组(1.01±0.02、0.99±0.05)均明显升高,差异均有统计学意义(P=0.017、0.008);10058-F4组T细胞c-Myc mRNA相对表达量为0.57±0.03,明显低于对照组(1.04±0.02),差异有统计学意义(P<0.001)。ELISA检测结果显示,10058-F4组共培养细胞上清液中IL-17含量明显低于对照组,差异有统计学意义(P=0.025)。流式细胞仪检测结果显示,10058-F4组共培养细胞中Th17细胞比例为(17.97±0.91)%,明�Objective To investigate the effects of c-Myc inhibitor 10058-F4 on the proportion of interphotoreceptor retinoid-binding protein 1-20(IRBP 1-20)-specific T helper 17(Th17)cells and expression of cytokines in the mouse model of experimental autoimmune uveitis(EAU).Methods Six healthy C57BL/6J mice(8-10 weeks old)were included and randomly divided into the normal control(NC)group and the EAU group using the random number table method,3 for each group.Mice in the EAU group were injected intraperitoneally with 350 ng pertussis toxin and injected subcutaneously with an emulsion containing 150μg IRBP 1-20,0.8 mg mycobacterium tuberculosis H37RA and Freund’s incomplete adjuvant on a hind leg and flank to induce the EAU model,while mice in the NC group were not given any special interventions.At 13 d after the immunization,fundus inflammatory lesions in EAU mice were evaluated by indirect ophthalmoscopy and visualized via retinal imaging system for small animals,hematoxylin-eosin staining was performed for histopathological examination of the retina,and quantitative real-time PCR(qRT-PCR)was used to detect the messenger ribonucleic acid(mRNA)expression of c-Myc in T cells and eyeball tissues.T cells isolated from EAU mice were divided into the control group and the 10058-F4 group,T cells in the 10058-F4 group were added with 50μmol·L^(-1)10058-F4,and T cells in the control group were not given drug treatment.After cultivation for 6 h,T cells in the two groups were washed and cultured with IRBP 1-20(10 mg·L^(-1)),bone marrow-derived dendritic cells and 20μg·L^(-1) interleukin(IL)-23(Th17 cell inducing conditions).Relative mRNA expressions of c-Myc,retinoic acid-related nuclear orphan receptorγt(RORγt),IL-17A,IL-17F and granulocyte-macrophage colony-stimulating factor(GM-CSF)were detected by qRT-PCR,the content of IL-17 in the supernatant of co-cultured cells was detected by enzyme-linked immunosorbent assay(ELISA),and the proportion of Th17 cells in the co-cultured cells was analyzed by flow cytometry.Results M

关 键 词：辅助性T细胞17 实验性自身免疫性葡萄膜炎 c-Myc抑制剂 10058-F4 

分 类 号：R773[医药卫生—眼科]