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目的细胞高通量筛选发现苯并噻唑衍生物BD960具有免疫抑制活性,文中探讨BD960抑制T细胞增殖的作用机制。方法免疫磁珠纯化人外周血T细胞,Anti-CD3/CD28m Abs或同种异型抗原活化T细胞。流式细胞术检测BD960对活化T细胞的增殖抑制作用、静息T细胞的细胞毒性作用、CD25表达及细胞周期。酶联免疫吸附法测定BD960对活化T细胞分泌IL-2、IL-4、IL-6、IL-10、IL-17A及IFN-γ等细胞因子的影响。结果 BD960抑制Anti-CD3/CD28 m Abs或同种异型抗原刺激的T细胞增殖,IC50分别为(2.3±0.3)μmol/L和(2.5±0.3)μmol/L,且浓度达100μmol/L也不影响静息T细胞和PBMC的存活。Anti-CD3/CD28 m Abs刺激T细胞72 h表达的CD25比为69.7%,BD960在(0.625、2.5、10)μmol/L均不抑制活化T细胞表达CD25,而0.1μmol/L FK506可抑制CD25表达低至9.4%。活化96 h的T细胞,G0/G1期细胞比为58.5%。BD960能阻滞细胞周期于G0/G1期,并随着浓度的提高G0/G1比例增大。BD960在(0.625、2.5、10)μmol/L能抑制活化T细胞分泌IFN-γ、IL-6和IL-17并呈剂量依赖效应,但不影响IL-2、IL-4和IL-10。结论 BD960抑制作用在T细胞增殖为细胞活化的后期,同时抑制IL-6、IL-17A和IFN-γ等促炎细胞因子的分泌,抑制作用不同于FK506。BD960有望作为先导化合物开发新型免疫抑制剂。
The target cell high-throughput screening found that benzothiazole derivative BD960 has immunosuppressive activity, BD960 in the paper to explore the mechanism of T cell proliferation. Methods Immunomagnetic beads were used to purify human peripheral blood T cells, and anti-CD3 / CD28m Abs or alloantigen-activated T cells. The inhibitory effect of BD960 on the proliferation of activated T cells, cytotoxicity of resting T cells, CD25 expression and cell cycle were detected by flow cytometry. The effect of BD960 on cytokines such as IL-2, IL-4, IL-6, IL-10, IL-17A and IFN-γ secreted by activated T cells was detected by enzyme-linked immunosorbent assay. Results BD960 inhibited the proliferation of T cells stimulated by Anti-CD3 / CD28 m Abs or alloantigen with the IC50 values of (2.3 ± 0.3) μmol / L and (2.5 ± 0.3) μmol / L, respectively. Affects the survival of resting T cells and PBMCs. Anti-CD3 / CD28 m Abs stimulated T cells 72h expression CD25 ratio was 69.7%, BD960 (0.625,2.5,10) μmol / L did not inhibit activated T cells express CD25, while 0.1μmol / L FK506 can inhibit CD25 The expression is as low as 9.4%. Activation of 96 h T cells, G0 / G1 phase cell ratio was 58.5%. BD960 can block the cell cycle in G0 / G1 phase, and with the increase of G0 / G1 ratio increased. BD960 inhibited the secretion of IFN-γ, IL-6 and IL-17 by activated T cells in a dose-dependent manner at (0.625,2.5,10) μmol / L but did not affect IL-2, IL-4 and IL-10. Conclusion BD960 inhibits the secretion of pro-inflammatory cytokines, such as IL-6, IL-17A and IFN-γ, at different stages of T cell proliferation and late stage of cell activation. The inhibitory effect is different from that of FK506. BD960 is expected as a leading compound to develop new immunosuppressive agents.