TLR7激动剂CL097对实验性自身免疫性葡萄膜炎中Th17细胞活性的促进作用  

Enhancement effect of TLR7 agonist CL097 on the activity of Th17 cells in experimental autoimmune uveitis

作　　者：陈琳 粘红 魏瑞华[1] 张红梅 张晓敏[1] 肖青 李雪 李筱荣[1] 

机构地区：[1]天津医科大学眼科医院天津医科大学眼科研究所天津医科大学眼视光学院,300384

出　　处：《中华实验眼科杂志》2015年第4期294-299,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81100646）;国家教育部留学回国人员科研启动基金项目（第48批）;天津市自然科学基金面上项目（13JCYBJC23300）;天津市科委自然科学基金项目（11JCYBJC26000）;天津医科大学校级基金项目（2013kyQ10）

摘　　要：背景 研究表明,Toll样受体7（TLR7）在自身免疫性疾病中发挥重要作用,但TLR7在实验性自身免疫性葡萄膜炎（EAU）中是否通过树突状细胞（DCs）调控辅助性T淋巴细胞17（Th17）的作用机制鲜见报道.目的 探讨TLR7激动剂CL097处理的小鼠骨髓树突状细胞（BMDCs）对光感受器间维生素A类结合蛋白（IRBP） 1-20-特异性Th17细胞的影响.方法 采用C57BL/6小鼠股骨和胫骨分离小鼠骨髓细胞,将重组小鼠粒细胞-巨噬细胞集落刺激因子（rmGM-CSF）及重组小鼠白细胞介素-4（rmIL-4）加入培养基在体外定向诱导BMDCs,于诱导后第6天在培养基中加入5μg/ml CL097作用8h,对照组加入PBS,采用实时荧光定量PCR技术检测CL097处理组与对照组BMDCs中IL-6、IL-23、IL-1β及转化生长因子-β（TGF-β） mRNA的相对表达量.小鼠腹腔内注射百日咳毒素1μg,注射后2h将IRBP1-20与等容积含有结核分枝杆菌HR37RA的完全弗氏佐剂充分乳化后主动免疫C57BL/6小鼠,眼底检查参照Caspi 0 ～4分的标准进行炎症评分.免疫后第13天取眼球制作病理切片,苏木精-伊红染色后行组织病理学检查,并分离小鼠脾脏及淋巴结中IRBP1.20-特异性T细胞,分别与CL097处理组及对照组的BMDCs共培养5d,收集细胞,采用实时荧光定量PCR技术检测2个组细胞中IL-17、γ干扰素（IFN-γ）、维甲酸相关核孤儿受体γt（RORγt）和T细胞中表达的T盒21转录因子（T-bet） mRNA的相对表达量;采用流式细胞仪检测并分析CL097处理组和对照组Th1与Th17细胞比例的变化.结果 EAU模型成功建立.实时荧光定量PCR检测结果显示,CL097处理组BMDCs中IL-6、IL-23、IL-1β mRNA的相对表达量明显高于对照组,差异均有统计学意义（t=4.560,P=0.045; t=5.476,P=0.032; t=17.240,P=0.003）,而TGF-β mRNA相对表达量明显低于对照组,差异有统计学意义（t=10.410,P=0.009）.CL097处理组BMDCs与IRBP1-20-特异性T细胞共培养后,RORγt和IL-17 mRNABackground Toll like receptor 7 （TLR7） has been implicated in the development of autoimmune diseases,but its role in the regulation of T helper cell 17 （Th17） response in experimental autoimmune uveitis （EAU） by dendritic cells （DCs） has not been revealed yet.Objective This study was to investigate the effect of bone marrow-derived dendritic cells （BMDCs） treated by TLR7 agonist CL097 on the regulation of interphotoreceptor retinoid-binding protein （IRBP）1-20-specific Th17 cells response in EAU.Methods BMDCs were isolated and collected from femur and tibia of naive C57BL/6 mice and cultured with recombinant mouse granulocyte-macrophage colony-stimulating factor （rmGM-CSF） and recombinant mouse interleukin-4 （rmIL-4）.After cultured for six days,5 μg/ml CL097 was added into the medium for 8 hours in the CL097 treated group,and PBS was used in the control group.Real-time fluorescence quantitative PCR （RT-qPCR） was employed to detect the relative expressions of IL-6,IL-23,IL-1β and transforming growth factor-β （TGF-β） mRNA in BMDCs.These mice received 1 μg of Bordetella pertussis toxin intra-peritoneally on the day of immunization,C57BL/6 mice were immunized with IRBP1-20 in complete Freund adjuvant containing heat-killed mycobacterium tuberculosis H37RA after two hours,EAU was evaluated by fundus examination on a scale of 0-4 reported by Caspi.Eyes harvested at 13 days after active immunization were processed for histopathological examination and stained with hemotoxylin and eosin.IRBP1-20-specific T cells were isolated from spleen cells and lymph node cells on the thirteenth day after immunization and co-cultured with CL097 treated or untreated BMDCs.After co-cultured for 5 days,cells were collected.Then,the relative expressions of IL-17,interferon-γ （IFN-γ）,retinoic acid-related orphan receptor-γt （RORγt）,T-box 21 transcription factor （T-bet） mRNA expression in T cells were tested by RT-qPCR.Percentages of Th1 cells and Th17 cells were assessed by flow c

关 键 词：Toll样受体7/激动剂 CL097 葡萄膜炎/免疫性 CD4+T淋巴细胞/分泌 辅助性T细胞17 细胞极性/免疫 炎性因子 树突状细胞/骨髓源性 C57BL/6小鼠 

分 类 号：R773.9[医药卫生—眼科]