脾脏调节性树突状细胞腹腔回输对实验性自身免疫性脑脊髓炎小鼠视神经的保护作用及其机制  

Spleen regulatory dendritic cells protect the optic nerve from optic neuritis in mice with experimental autoimmune encephalomyelitis

作　　者：侯艳丽[1] 杜然[1] 刘庆阳 王艳玲[1] HOU Yanli;DU Ran;LIU Qingyang;WANG Yanling(Beijing Friendship Hospital,Capital Medical University,Beijing 100050,China;不详)

机构地区：[1]首都医科大学附属北京友谊医院,北京100050 [2]应急总医院

出　　处：《山东医药》2020年第22期51-55,共5页Shandong Medical Journal

基　　金：国家自然科学基金资助项目面上项目(81870686,81272140)。

摘　　要：目的探讨脾脏调节性树突状细胞(CD11c^-CD45RB^high DC)腹腔回输对实验性自身免疫性脑脊髓炎(EAE)小鼠视神经的保护作用及其机制。方法无菌条件下制备小鼠脾脏DC,经抗CD45RB磁珠阳选获得CD45RBhigh DC,剪切掉细胞上的磁珠,再加入抗小鼠CD11c磁珠,MS磁性分离柱阴选部分即为CD11c^-CD45RB^high DC,经标染的抗CD11c单克隆抗体-别藻蓝蛋白和抗CD45RB单克隆抗体-藻红蛋白染色,上流式细胞仪鉴定。取C57BL/6小鼠50只适应性饲养7 d,随机分为对照组、模型组及低、中、高剂量组,每组10只。对照组常规饲养,不建模,模型组及低、中、高剂量组采用MOG35-55诱导EAE模型。低、中、高剂量组建模当日分别腹腔回输脾脏CD11c^-CD45RB^high DC 1×10^5、5×10^5、10×10^5个/只,对照组与模型组同期腹腔注射等量PBS。模型组及低、中、高剂量组建模当日开始,对照组同期,每日观察其神经功能并进行神经症状评分。模型组及低、中、高剂量组建模第1~3、7、15天,对照组同期,尾静脉取血,采用ELISA法检测血浆IL-6、TNF-α、IL-4,采用流式细胞仪计数CD4^+T细胞。各组末次尾静脉取血后,心脏灌注处死,自筛板后0.5 mm处取球后视神经组织,分别行HE染色和LFB染色,观察视神经组织病理学变化。结果通过磁珠分选仪获取脾脏CD11c^-CD45RB^high DC,经流式细胞仪检测,CD11c^-CD45RB^high DC纯度>85%。连续观察15 d,对照组未出现明显神经功能异常,神经症状评分均为0分。模型组及低、中、高剂量组建模第1~9天均未出现明显神经功能异常,神经症状评分均为0分;模型组建模第10、15天神经症状评分均高于低、中、高剂量组(P均<0.05),而低、中、高剂量组两两比较P均>0.05。模型组及低、中、高剂量组建模第1~3天血浆IL-6、TNF-α水平均高于对照组同期(P均<0.05);低、中、高剂量组建模第1~3天血浆IL-6、TNF-α水平均低于模型组同期,建模第2、3、7、15天Objective To investigate the protective effects of regulatory dendrite cells(CD11c^-CD45RB^high DC)on optic nerve in experimental autoimmune encephalomyelitis(EAE)mice model.Methods Mice spleen dendrite cells(DC)were prepared under sterile conditions.Magnetic cell sorting(MACS)method was employed for collection of CD11c^-CD45RB^high DC,with anti-CD45RB tagged beads for positive selection and anti-CD11c-for negative selection.The purity of the selected CD11c^-CD45RB^high DC was identified with monoclonal Abs(mAbs)to R-phycoerythrin(PE)-conjugated CD45RB and allophycocyanin(APC)-conjugated CD11c for flow cytometry.A total of 50 male C57BL/6 mice were fed adaptively for 7 days,which were then randomly divided into five groups:the normal group(n=10),the model group(n=10),and the intervention group(n=30)(which was further divided into the low-dose,medium-dose and high-dose subgroups,with 10 in each).An EAE model was induced with Myelin Oligodendrocyte Glycoprotein(MOG35-55)in the model group and intervention group but not in normal control group.Mice in the intervention group were infused with different dose of CD11c^-CD45RB^high DC(1×10^5/mouse in low-dose subgroup,5×10^5/mouse in medium-dose subgroup and 10×10^5/mouse in high-dose subgroup)on day 0.All the mice were evaluated for neurological functions on a daily basis.Serum samples of mice were collected on day 1-3,7,15 after treatment,and the levels of interleukin 6(IL-6),tumor necrosis factor-α(TNF-α),and interleukin 4(IL-4)were analyzed by ELISA.The number of CD4^+T cells was evaluated by flow cytometry.On day 15,all mice were sacrificed by heart perfusion.The optic nerve was taken from 0.5 mm after the ethmoid plate and stained with HE and LFB staining,respectively,and then we observed the histopathological changes of the optic nerve.Results The purity of spleen CD11c^-CD45RB^high DC collected with MACS method was over 85%as determined with fluorescence-activated cell sorting(FACS).The neurological function of the control group did not show obvious abnorma

关 键 词：多发性硬化相关性视神经炎 实验性自身免疫性脑脊髓炎 调节性树突状细胞 炎症细胞因子 CD4^+T细胞 小鼠 

分 类 号：R774.6[医药卫生—眼科]