论文部分内容阅读
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 investigate the relationship between Tn antigen expression and Cosmc transcription and protein expression during T cell activation. Methods Thirty healthy volunteers, 15 males and 15 females, average age (34 ± 8.9). Peripheral venous blood was collected from patients and anticoagulated with heparin. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Purified CD3 + T cells were sorted by immunomagnetic beads method. T cell activators Cocktail , CD3 / CD28 Dynabeads stimulated 12, 24, 36, 48, 60, 72h. The percentage of Tn + cells and the average fluorescence intensity of Tn antigen in CD3 + T cells at different time points before and after stimulation were detected by flow cytometry. The levels of Tn antigen and Tn antigen were measured by RT-PCR and Western blot, respectively The mRNA and protein expression of Cosmc in the cells were detected by fluorescence analysis. The activity of T-synthase was detected by fluorescence analysis. The statistical analysis was performed by using SPASS13.0 software to elucidate the relationship between the dynamic changes of Tn antigen and Cosmc on activated T cells. Results Both Cocktail and CD3 / CD28 Dynabeads promoted the expression of Tn antigen on activated T cells, and the mean fluorescence intensity of Tn antigen increased first and then decreased with the prolongation of activation time. The peak of percentage of Tn + cells in CD3 / CD28 Dynabeads activation group appeared 48h after activation, while the peak value of Cocktail activation group appeared 60h after activation, the difference was statistically significant (P <0.05). Cosmc transcription and protein levels, T-synthase activity first decreased and then increased with the prolongation of activation time, and the trend of change was opposite to that of Tn antigen (P <0.05). Conclusions Tn antigen expression changes dynamically during T cell activation. The expression of Tn antigen is closely related to the decline of Cosmc transcription and protein level and the decrease of T-synthase activity. It provides a basis for Tn antigen therapy for immune diseases caused by T cell activation Important reference.