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目的:探讨利用流式细胞术(FCM)检测细胞增殖(BrdU掺入)与T细胞的活化及细胞因子表达的关系。方法:正常人PBMC经PMA+Ionomycin刺激不同时间,在培养结束前1 h加入BrdU,利用抗BrdU以及多种抗细胞表面和抗细胞因子抗体标记,FCM检测。结果:体外刺激PBMC 48 h后,可见T细胞有BrdU掺入,但随着时间的延长,掺入率没有明显增加。对比分析BrdU掺入与活化分子表达的关系表明,CD69在刺激后8 h达高峰,而CD25则需要24 h后达到高峰。另外,BrdU掺入与细胞因子表达没有明显关系,当PBMC刺激后8 h,即有大量IFN-γ的表达,而当培养时间延长到24、48和72 h后,IFN-γ的表达没有明显改变。OKT3+antiCD28刺激T细胞后BrdU的阳性率高于PMA+Ionomycin刺激的T细胞,CD8+T细胞掺入率高于CD4+T细胞。结论:利用FCM检测BrdU+细胞可以用于评价T细胞的增殖反应,但在多克隆刺激的条件下,只有少数细胞发生增殖,并且BrdU掺入率受刺激剂种类和时间的影响,BrdU掺入与活化分子和细胞因子的表达均没有明显关系。
Objective: To investigate the relationship between cell proliferation (BrdU incorporation) and T cell activation and cytokine expression by flow cytometry (FCM). Methods: Normal human PBMCs were stimulated with PMA + Ionomycin for different time. BrdU was added 1 h before the end of culture. Anti-BrdU and various anti-cell surface and anti-cytokine antibody markers were used to detect FCM. RESULTS: PBMCs were stimulated with BrdU for 48 h after in vitro stimulation, but the incorporation of BrdU did not increase significantly with time. Comparative analysis of BrdU incorporation and activation of molecular expression showed that CD69 peaked at 8 h after stimulation, whereas CD25 peaked at 24 h. In addition, there was no significant relationship between BrdU incorporation and cytokine expression. When PBMC was stimulated for 8 h, a large amount of IFN-γ was expressed. However, when cultured for 24, 48 and 72 h, the expression of IFN-γ was not significantly change. The positive rate of BrdU after T cells was stimulated by OKT3 + antiCD28 was higher than that of PMA + Ionomycin-stimulated T cells, and the incorporation rate of CD8 + T cells was higher than that of CD4 + T cells. CONCLUSION: The detection of BrdU + cells by FCM can be used to evaluate the proliferative response of T cells. However, under the conditions of polyclonal stimulation, only a few cells proliferated and the BrdU incorporation rate was affected by the type and duration of stimulants. BrdU incorporation There was no significant relationship between the expression of activated molecules and cytokines.