lncRNA 5033413D16Rik对实验性自身免疫性葡萄膜炎Th17细胞活性的抑制作用  

Inhibitory effect of lncRNA 5033413D16Rik on the activity of T helper 17 cells in experimental autoimmune uveitis

作　　者：李学佳 张开朗 陈思思 魏瑞华[1] 粘红 Li Xuejia;Zhang Kailang;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、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》2024年第11期983-990,共8页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金(81970793、82070929);天津市医学重点学科(专科)建设项目(TJYXZDXK-037A);天津市视网膜功能与疾病重点实验室自主与开放课题(2020tjswmm001)。

摘　　要：目的探讨长链非编码RNA 5033413D16Rik(lncRNA 5033413D16Rik)对实验性自身免疫性葡萄膜炎(EAU)中光感受器间维生素A类结合蛋白1-20(IRBP_(1-20))特异性辅助性T17(Th17)细胞活性的影响。方法选取SPF级8~10周龄C57BL/6雌性小鼠12只,采用随机数字表法分为EAU组和正常对照组,每组各6只。正常对照组小鼠不接受任何处理,将IRBP_(1-20)和完全弗氏佐剂充分乳化后主动免疫EAU组小鼠。免疫后13 d,行眼底检查并进行炎症评分,苏木精-伊红染色观察视网膜组织病理学改变;分离EAU小鼠脾脏及淋巴结中T细胞,实时荧光定量PCR检测lncRNA 5033413D16Rik相对表达量。将分离培养的T细胞分为短发夹RNA(shRNA)-5033413D16Rik转染组和shRNA-阴性对照(NC)转染组,分别转染相应shRNA,实时荧光定量PCR验证shRNA-5033413D16Ri的敲低效率。将2个组细胞分别与骨髓源性树突状细胞在Th17极化条件下共培养,实时荧光定量PCR检测2个组共培养细胞中转录因子维甲酸相关核孤儿受体γt(RORγt)、白细胞介素17(IL-17)、IL-23受体(IL-23R)和粒细胞-巨噬细胞集落刺激因子(GM-CSF)mRNA相对表达量;ELISA试剂盒检测2个组共培养细胞上清中IL-17质量浓度;流式细胞仪检测2个组共培养细胞中Th17细胞百分比。结果成功建立小鼠EAU模型。实时荧光定量PCR检测结果显示,与正常对照组相比,EAU组小鼠T细胞中lncRNA 5033413D16Rik相对表达量显著降低,差异有统计学意义(t=-13.332,P<0.001)。与shRNA-NC转染组相比,shRNA-5033413D16Rik转染组T细胞中lncRNA 5033413D16Rik相对表达量显著降低,差异有统计学意义(t=-6.338,P<0.01)。shRNA-5033413D16Rik转染组共培养细胞中RORγt、IL-17、IL-23R和GM-CSF mRNA相对表达量分别为1.61±0.13、1.51±0.13、1.85±0.33和1.45±0.11,明显高于shRNA-NC转染组的1.00±0.01、1.00±0.01、1.00±0.01和1.00±0.01,差异均有统计学意义(t=-6.839、-8.221、-4.538、-4.189,均P<0.05);ELISA检测结果显示,shRNA-50334Objective To investigate the effect of long noncoding RNA(lncRNA)5033413D16Rik(lncRNA 5033413D16Rik)on the activity of interphotoreceptor retinoid-binding protein_(1-20)(IRBP)1-20-specific T helper 17(Th17)cells in experimental autoimmune uveitis(EAU).Methods Twelve SPF grade C57BL/6 female mice aged 8 to 10 weeks were selected and divided into EAU group and normal control group using the random number table method,with 6 mice in each group.Mice in the normal control group received no treatment.Mice in the EAU group were immunized with IRBP_(1-20)emulsified in complete Freund's adjuvant to induce EAU.On day 13 after immunization,fundus examination and inflammation scoring were performed,and retinal histopathological changes were observed with hematoxylin and eosin staining.T cells were isolated from the spleen and lymph nodes of EAU mice,and the relative expression of lncRNA 5033413D16Rik was detected by real-time fluorescence quantitative PCR(qRT-PCR).In addition,the isolated T cells were divided into short hairpin RNA(shRNA)-5033413D16Rik transfected group and shRNA-negative control(NC)transfected group,and transfected with corresponding shRNAs,respectively.The knockdown efficiency of shRNA-5033413D16Rik was verified by qRT-PCR.T cells in the two groups were co-cultured with bone marrow-derived dendritic cells(BMDCs)under Th17 polarizing conditions.The relative expression of retinoic acid-related orphan receptor(RORγt),interleukin 17(IL-17),IL-23 receptor(IL-23R),and granulocyte-macrophage colony-stimulating factor(GM-CSF)mRNA in co-cultured cells were analyzed by qRT-PCR.The IL-17 concentration in co-culture supernatants was assayed by ELISA and percentages of Th17 cells were determined by flow cytometry.The use and care of experimental animals complied with the Regulations for the Administration of Affairs Concerning Experimental Animals promulgated by the State Science and Technology Commission.The study protocol was reviewed and approved by the Experimental Animal Ethics Committee of Tianjin Medical Univer

关 键 词：葡萄膜炎 自身免疫性疾病 实验性自身免疫性葡萄膜炎 辅助性T17细胞 lncRNA 5033413D16Rik 

分 类 号：R773.9[医药卫生—眼科]