活化T细胞表面Tn抗原动态变化与Cosmc之间的关系研究  

Study on the relationship between the Tn antigen and Cosmc in activated CD3^+ T cells

作　　者：石传芹 徐雪[1] 于晓锋[2] 刘栋[3] 于泽顺[3] 河恩玲 杜镇镇[1] 胡涛[1] SHI Chuan-qin XU Xue YU Xiao-feng LIU Dong YU Ze-shun HE En-ling DU Zhen- zhen HU Tao(Taishan Scholars Program, Department of Immunology, BinzhouMedical University, Yantai 264003, Shandong, China Linzi People＇s Hospital Binzhou Medieal University Hospital)

机构地区：[1]滨州医学院免疫学教研室,泰山学者团队,山东烟台264003 [2]临淄区人民医院,山东淄博255400 [3]滨州医学院附属医院,山东滨州256600

出　　处：《中国病原生物学杂志》2017年第5期428-433,共6页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.30972778);山东省自然科学基金项目(No.Y2007C143,ZR2014HM020)

摘　　要：目的探讨T细胞活化过程中表面Tn抗原的表达状况与Cosmc转录及蛋白表达水平之间的关系。方法30例健康志愿者,男15例,女15例,平均年龄(34±8.9)。抽取志愿者外周静脉血,肝素抗凝,采用Ficoll密度梯度离心法分离单个核细胞(peripheral blood mononuclear cell,PBMC),免疫磁珠法分选纯化的CD3+T细胞,分别采用T细胞活化剂Cocktail、CD3/CD28Dynabeads刺激12、24、36、48、60、72h。流式细胞术检测两种活化剂刺激活化前后不同时间点CD3+T细胞中Tn+细胞的百分率及Tn抗原平均荧光强度,采用RT-PCR和Western blot检测两种活化剂刺激活化前后不同时间点T细胞中Cosmc转录及蛋白表达状况,采用荧光分析法检测T-synthase活性,应用SPASS13.0软件进行统计学分析,阐明活化T细胞表面Tn抗原动态变化与Cosmc之间的关系。结果 Cocktail、CD3/CD28Dynabeads均能促使活化T细胞表面表达Tn抗原,以及Tn抗原表达的平均荧光强度随活化时间的延长先升高后下降。CD3/CD28Dynabeads活化组Tn+细胞百分率的峰值出现在活化后48h,而Cocktail活化组峰值出现在活化后60h,差异具有统计学意义(P<0.05)。Cosmc转录及蛋白水平、T-synthase活性随活化时间延长先降低后升高,变化趋势与Tn抗原表达水平相反,差异具有统计学意义(P<0.05)。结论 T细胞活化过程中Tn抗原表达呈动态变化,表达水平与Cosmc转录及蛋白水平下降、T-synthase活性降低密切相关,为以Tn抗原为靶点治疗由T细胞活化引起的免疫性疾病提供了重要依据。Objective To explore the relationship between Tn antigen expression in T cells and Cosmc （Core 1 β3-galactosyltransferase specific molecular chaperone） during T cell activation. Methods Peripheral blood samples from 30 healthy volunteers （15 males and 15 females, average age： 34±8.9 years） were selected from the central blood bank in the City of Yantai and anticoagulated with heparin. Human peripheral blood mononuclear cells （PBMCs） were isolated using Ficoll density gradient centrifugation. CD3＋ T cells were isolated from PBMCs via negative selection using a CD3＋ T cell MACS isolation kit. CD3＋ T cells were then stimulated with activators, i.e. CD3/CD28 Dynabeads and Cocktail, for 12 h, 24 h, 36 h, 48 h, 60 h, and 72 h. The percentage of Tn-positive cells and mean fluorescence intensity of activated T cells were measured using flow cytometry （FCM）. RT-PCR and Western blotting were used to detect Cosmc transcription and the level of protein expression in CD3＋ T cells at different time points before and after activation. T-synthase ac- tivity in these T cells was measured using a fluorescent assay. Data were analyzed using SPSS13.0 to ascertain the rela- tionship between dynamic changes in Tn antigen expression on the surface of activated T cells and Cosine. Results Both Cocktail and CD3/CD28 Dynabeads resulted in Tn antigen expression in activated T cells. The percentage of Tn- positive cells and the mean fluorescence intensity of Tn antigen expression first increased and then decreased within 72 h of stimu- lation with either activator. The peak activation of Tn＋ cells after stimulation with CD3/CD28 Dynabeads occurred 48 h after activation, but the peak activation of Tn＋ cells after stimulation with Cocktail occurred 60 h after activation. The percentage of Tn＋ ceils and the mean fluorescence intensity of Tn antigen expression in T cells activated with CD3/CD28 Dynabeads were higher than the percentage of Tn＋ cells and the mean fluorescence intensity of Tn antigen expression

关 键 词：T淋巴细胞 Tn抗原 分子伴侣Cosmc T细胞活化剂(CD3CD28 Dynabeads Cocktail) 

分 类 号：R378.91[医药卫生—病原生物学]