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中文摘要：
目的研究体外实验中不同细胞刺激剂对小鼠调节性T细胞（Treg）表面T淋巴细胞毒性相关抗原-4（CTLA-4）及叉头翼状螺旋转录因子（Foxp3）表达的影响，并对不同细胞刺激剂在Treg抑制活性发挥中的作用进行比较。方法免疫磁珠法分离健康清洁级BALB／c小鼠脾脏CD4＋CD25＋Treg。采用植物凝集素（PHA，20mg／L）、刀豆蛋白A（ConA,5g／L）及固相包被抗CD3（1mg／L）刺激Treg，观察刺激后24、48和72hCTLA-4及Foxp3表达的时间一效应关系。结果PHA刺激对小鼠Treg细胞表面CTLA-4及Foxp3表达均无显著影响（P均〉0．05）；而ConA表现出一过性的Treg活化作用，24hCTLA-4表达明显上调（P〈0.01），但随刺激时间延长作用减弱，48h和72h后其表达已接近未刺激前的范围，同时ConA未能明显诱导Foxp3表达（P〉0．05）；抗CD3可有效刺激Treg活化，在24、48和72hCTLA-4及Foxp3表达均明显上调（P〈0．05或P〈0．01），其中以作用24h和48h表达上调尤为显著（P均〈0.01）。结论抗CD3在体外实验中能明显刺激Treg活化并诱导其抑制性相关分子CTLA-4及Foxp3的表达，为进一步探讨CD4＋CD25＋Treg的免疫调节效应提供了可能。
英文摘要：
Objective To investigate the expression of cytotoxic T lymphocyte - associated antigen 4 （CTLA - 4） and intranuclear forkhead/winged helix transcription factor p3 （Foxp3） of splenic regulatory T cells （Tregs） in mice, and compare the effects of different stimuli on the immunological activity of Tregs. Methods CD4＋ CD25＋ Tregs isolated from the spleens of male BALB/c mice by magnetic beads were seeded on 96- well （1 × 10^3 cells/well） cell culture plates supplemented with phytohemagglutin （PHA, 20 mg/L）, concanavalin A （ConA, 5 g/L） or coated with anti -CD3 （1 mg/L）. After being stimulated with different stimuli for various length of time, the expressions of CTLA - 4 and Foxp3 molecules of Tregs were determined. The time - dependent responses between different stimuli and CTLA - 4 as well as Foxp3 were analyzed by means of flow cytometry. Results After stimulation with PHA, the CTLA - 4 expressions on surface of mice splenic Tregs and intranuclear Foxp3 molecules were unchanged （both P〉 0.05）. In ConA- treated group, the expression of CTLA- 4 was significantly up- regulated at 24 hours （P〈0. 01）, while the CTLA -4 expression returned to the control range at 48 and 72 hours. The expression of Foxp3 was not markedly different between ConA - treated group and controls. When treated with plate - bound anti- CD3, both CTLA -4 and Foxp3 expressions were significantly up -regulated at 24, 48 and 72 hours （P〈0.05 or P〈0. 01）, especially at 24 and 48 hours （all P〈0.01）. Conclusion These data indicated that various stimuli can result in different modulatory responses of the CTLA - 4 and Foxp3 expressions in mice splenic Tregs. Anti -CD3 appears to be an effective immunoregulatory signal that influences the activation of CD4＋ CD25＋- Tregs.
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