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目的研究洋地黄毒苷(Digitoxin)在体外对人CD4+T细胞产生IL-17A和IFN-γ的影响及其机制。方法人PBMC用抗CD3和抗CD28抗体共刺激或在诱导Th17分化的条件下,同时加或不加Digitoxin,用酶联免疫吸附法检测培养上清中IL-17A和IFN-γ的产生。利用流式细胞术检测细胞因子IL-17A、IFN-γ以及转录因子RORγt的表达。利用Western blotting检测Digitoxin对RORγt表达的影响。结果 Digitoxin对人IL-17A有显著的剂量依赖性抑制作用,并且在刺激的早期(第0小时和第6小时)加入Digitoxin能显著地抑制IL-17A的产生,但不影响IFN-γ的产生,而晚期(第12小时和第24小时)加入Digitoxin对IL-17A的产生没有明显地抑制作用。进一步研究发现,Digitoxin抑制Th17细胞的分化,调控IL-17A的转录因子RORγt的表达。结论本实验表明,Digitoxin通过早期调控RORγt的表达来抑制IL-17A的产生,但不抑制IFN-γ的产生,提示Digitoxin除了对心血管作用外,也可以抑制炎症性疾病和自身免疫性疾病的发生发展。
Objective To investigate the effect and mechanism of Digitoxin on IL-17A and IFN-γ produced by human CD4 + T cells in vitro. Methods Human PBMCs were co-stimulated with anti-CD3 and anti-CD28 antibodies or treated with Digitoxin with or without induction of Th17 differentiation. The production of IL-17A and IFN-γ in culture supernatants was detected by enzyme-linked immunosorbent assay. Flow cytometry was used to detect the expression of cytokines IL-17A, IFN-γ and transcription factor RORγt. The effect of Digitoxin on the expression of RORγt was detected by Western blotting. Results Digitoxin had a significant dose-dependent inhibition of human IL-17A, and addition of Digitoxin early in the stimulation (0 and 6 hours) significantly inhibited the production of IL-17A but did not affect the production of IFN-γ , Whereas addition of Digitoxin at the late (12th and 24th hours) did not significantly inhibit IL-17A production. Further study found that Digitoxin inhibited the differentiation of Th17 cells and regulated the expression of RORγt, a transcription factor of IL-17A. Conclusion This experiment shows that Digitoxin can inhibit the production of IL-17A through the early regulation of RORγt expression but not the production of IFN-γ, suggesting that Digitoxin can inhibit inflammatory and autoimmune diseases in addition to cardiovascular effects Development took place.