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目的:探讨超抗原金黄色葡萄球菌肠毒素(SEB)体外诱导外周T细胞免疫耐受的作用机制。方法:采用SEB体外刺激C57BL/6J(B6)小鼠的脾细胞后,以MTT比色法检测脾细胞的增殖,并用PI染色后以流式细胞术(FCM)分析不同时间段处于S期、G0～G1期的细胞及无能T细胞的凋亡,测定T细胞亚群及MHC-I(H一2Kb)表达的变化。用琼脂糖凝胶电泳,观察不同时问段凋亡T细胞的DNA特征。结果:部分去除CD8+T细胞后,SEB可刺激B6小鼠脾细胞中CD4+T细胞大量增殖。在SEB刺激后第3天,CD4+T细胞中处于S期的比率最大,此后开始下降;而处于G0～G1期的CD4+T细胞变化则相反。在初次刺激后第3天,增殖的CD4+T细胞出现无能。FCM检测及用琼脂糖凝胶电泳检查DNA ladder证实,在第7天,无能CD4+ T细胞出现凋亡,且凋亡细胞的比率逐渐增多,不因加入抗CD3抗体或ConA而逆转。在SEB刺激后,CD4+ T细胞表面MHC-I类分子(H-2Kb)的表达,随细胞无能的出现而明显下调。结论:SEB诱导的T细胞免疫耐受,可能与CD4+ T细胞的无能、凋亡及细胞表面分子MHC-I的表达下凋有关。
Objective: To investigate the mechanism of superantigen staphylococcal enterotoxin (SEB) inducing peripheral T cell immune tolerance in vitro. Methods: The splenocytes of C57BL / 6J (B6) mice were stimulated with SEB in vitro and the proliferation of splenocytes was detected by MTT colorimetric assay. Flow cytometry (FCM) G0 ~ G1 phase cells and impaired T cell apoptosis, measured T cell subsets and MHC-I (H-2Kb) expression changes. The DNA characteristics of apoptotic T cells at different time points were observed by agarose gel electrophoresis. Results: After partial removal of CD8 + T cells, SEB stimulated the proliferation of CD4 + T cells in splenocytes of B6 mice. On the third day after SEB stimulation, the percentage of CD4 + T cells in S phase was the highest and then decreased. The changes of CD4 + T cells in G0-G1 phase were opposite. On the third day after the initial stimulation, proliferating CD4 + T cells became impotence. FCM and DNA ladder with agarose gel electrophoresis confirmed that on day 7, the apoptosis of incompetent CD4 + T cells was observed, and the percentage of apoptotic cells gradually increased, not reversed by the addition of anti-CD3 antibody or ConA. After SEB stimulation, the expression of MHC class I molecules (H-2Kb) on the surface of CD4 + T cells was significantly down-regulated with the onset of cell inactivity. Conclusion: The immune tolerance of T cells induced by SEB may be related to the impotence and apoptosis of CD4 + T cells and the expression of MHC-I on cell surface.