间充质干细胞对EAU大鼠抗原特异性T细胞和抗原递呈细胞功能的抑制作用  

Inhibitory effects of mesenchymal stem cells on antigen-specific T cells and antigen presenting cells in experimental autoimmune uveitis

作　　者：白伶伶 张灵君 郑慧 王梅艳 东莉洁[1] 李筱荣[1] 张晓敏[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 [2]北京市垂杨柳医院

出　　处：《中华实验眼科杂志》2015年第10期870-875,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：国家自然科学基金项目（81371005）;国家自然科学基金青年项目（31100991）

摘　　要：背景 我们前期研究发现,间充质干细胞（MSCs）可以有效治疗大鼠实验性自身免疫性葡萄膜炎（EAU）,减轻组织损害,但其具体作用机制仍在研究中.目的 研究MSCs对大鼠EAU模型中T细胞亚群和抗原递呈细胞（APCs）的影响.方法 收集6只清洁级4～6周龄Wistar雄性大鼠双侧股骨、胫骨骨髓,采用贴壁培养法纯化Wistar大鼠骨髓MSCs.采用随机数字表法将12只清洁级Lewis雌性大鼠分为MSCs组和PBS组,每组6只.于Lewis大鼠单后足及背部皮下注射200μl含30μg光感受器间维生素A类结合蛋白（IRBP） 1177-1191多肽片段R16及完全弗氏佐剂（CFA）的乳化液以建立EAU模型,造模后于裂隙灯显微镜下观察大鼠眼部炎症表现.造模后9～11d,MSCs组大鼠每日经尾静脉注射密度为5×106/ml的MSCs悬液1 ml;PBS组大鼠以同样的方法注射等容积的PBS.造模后15d,分离各组大鼠脾脏和引流淋巴结中的T细胞及APCs,采用流式细胞仪检测各组大鼠脾脏和引流淋巴结中γ干扰素（IFN-γ）阳性CD4＋T细胞、白细胞介素-17（IL-17）阳性CD4＋T细胞和叉头状螺旋转录因子p3（Foxp3）阳性CD4＋T细胞的比例,以评估辅助性T细胞1（Th1）、Th17和调节性T细胞（Treg）细胞亚群的作用;依据各组T细胞与APCs共培养的方式不同分为PBS共培养组、PBS-MSCs交叉培养组、MSCs-PBS培养组和MSCs共培养组,分别加入不同质量浓度（0.3、1.0、10.0 μg/ml）的R16抗原进行刺激,无R16抗原刺激的细胞作为空白对照,采用5-溴脱氧尿嘧啶核苷（BrdU）法测定各组大鼠T细胞吸光度（A）值,计算T细胞增生指数.结果 造模后11、12、13和14d,MSCs组大鼠眼前节炎症评分均明显低于PBS组,差异均有统计学意义（t=3.825、5.100、4.250、3.400,均P＜0.05）.与PBS组大鼠比较,MSCs组大鼠脾脏和淋巴结中IFN-γ＋ CD4＋T细胞比例均明显下降,差异均有统计学意义（t=5.651、4.376,均P＜0.05）;MSCs组大鼠脾脏和引流�Background Our previous studies found that mesenchymal stem cells （MSCs） can ameliorate experimental autoimmune uveitis （EAU） and reduce tissue impairment.Its mechanism is still pending.Objective This study was performed to investigate the effects of MSCs on T cell subsets and antigen presenting cells （APCs） in EAU rats.Methods MSCs were isolated from bone marrow of six male Wistar rats and cultured by plastic adherence method.Twelve female Lewis rats were assigned randomly into MSCs group and PBS group.EAU rat model was induced by immunization with 200 μl emulsion containing 30 μg interphotoreceptor retinoid-binding protein （IRBP） 1177-1191 polypeptide fragment R16 and complete Freund adjuvant （CFA）.The eye manifestations of the rats were observed and scored under the slit lamp microscope after modeling.The R16-immunized rats were treated intravenously with 5×106/ml MSCs for 3 consecutive days from day 9 to 11 after modeling in the MSCs group,and the equivalent volume of PBS was used with the same way in the PBS group.Fifteen days after modeling,the spleens and draining lymph nodes were collected to evaluate the proportion of interferon-γ （IFN-γ） positive CD4＋ T cells,interleukin-17 （IL-17）positive CD4＋ T cells and forkhead helix transcription factor p3 （Foxp3） positive CD4＋ T cells by flow cytometry.The T cells and APCs from the different groups were cocultured and divided into PBS cocultured group,MSCs cocultured group, PBS-MSCs cross-cultured group and MSCs-PBS cross-cultured group under the stimulation of R16 at the concentration of 0.3,1.0 or 10.0 μg/ml, and the proliferation indexes of the T cells in different groups were assayed by 5-bromodeoxyuridine （BrdU） Elisa kit.The use of experimental animals complied with the regulations on the management of experimental animals promulgated by the national science and technology commission.Results The ocular surface inflammatory scores of 11,12,13 and 14 days after modeling in the MSCs group were significantly lower

关 键 词：间充质干细胞 自身免疫性疾病/化学方法诱导 葡萄膜炎/预防和控制 辅助性T细胞1/ 免疫 辅助性T细胞17/免疫 调节性T淋巴细胞/免疫 抗原递呈细胞 动物模型 

分 类 号：R392.12[医药卫生—免疫学]