活化T细胞表面Tn抗原动态变化与Cosmc之间的关系研究

发布时间：2019-07-05 16:52

【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(P0.05)。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(P0.05)。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。
[Abstract]:Objective to investigate the relationship between the expression of Tn antigen and the transcription and protein expression of Cosmc during T cell activation. Methods 30 healthy volunteers, 15 males and 15 females, with an average age of 34 卤8.9, were enrolled in this study. Mononuclear cells (peripheral blood mononuclear cell,PBMC) were isolated from peripheral venous blood and heparin by Ficoll density gradient centrifugation. The purified CD3 T cells were isolated by immunomagnetizing beads and stimulated by T cell activating agent Cocktail,CD3/CD28Dynabeads for 12, 24, 36, 48, 60, 72 h, respectively. The percentage of Tn cells and the average fluorescence intensity of Tn antigen in CD3 T cells were detected by flow cytometry at different time points before and after activation. RT-PCR and Western blot were used to detect the transcription and protein expression of Cosmc in T cells at different time points before and after activation. T-synthase activity was detected by fluorescence analysis and statistically analyzed by SPASS13.0 software. To elucidate the relationship between the dynamic changes of Tn antigen on activated T cells and Cosmc. Results Cocktail,CD3/CD28Dynabeads could promote the expression of Tn antigen on the surface of activated T cells, and the average fluorescence intensity of Tn antigen expression increased at first and then decreased with the prolongation of activation time. The peak value of Tn cell percentage appeared at 48 h after activation, while that of Cocktail activation group appeared at 60 h after activation, the difference was statistically significant (P 0.05). The activity of T-synthase decreased at first and then increased with the prolongation of activation time, and the change trend was opposite to the expression level of Tn antigen, and the difference was statistically significant (P 0.05). Conclusion the expression of Tn antigen changes dynamically during T cell activation, and the expression level is closely related to the decrease of Cosmc transcription and protein level and the decrease of T-synthase activity, which provides an important basis for the treatment of immune diseases caused by T cell activation with Tn antigen as a target.
【作者单位】：滨州医学院免疫学教研室泰山学者团队;临淄区人民医院;滨州医学院附属医院;
【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)
【分类号】：R392

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