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目的银杏内酯B(GB)是已知的天然而强效的血小板激活因子(PAF)受体(PAFR)拮抗剂,本文研究GB对小鼠T淋巴细胞活化、增殖及凋亡3大体外行为的影响,初步探讨其潜在的免疫调节作用与机制,从而为临床应用提供可靠的实验依据。方法分离小鼠淋巴细胞,培养前以不同浓度的GB预孵;以刀豆蛋白A(Con A)诱导细胞的活化与增殖,以荧光抗体双色标记(anti-CD3 mAb-PE/anti-CD69 mAb-FITC、anti-CD3 mAb-PE/anti-CD25 mAb-FITC)结合流式细胞术分别检测T淋巴细胞早、中期活化标志--表面抗原CD69与CD25的表达,以活体荧光染料CFDA-SE标记结合流式细胞术检测细胞增殖并用MTT法佐证;以地塞米松(Dex)诱导细胞的凋亡,以荧光染料双色标记[DiOC6(3)/PI]结合流式细胞术区别凋亡、死亡与活细胞群。结果 Con A刺激后6 h和24 h,T细胞早期活化标志CD69和中期活化标志CD25分别大量表达,Con A刺激后48 h活化的T细胞增殖至第4代,而经终浓度为5、10、20μmol/L GB在Con A刺激前对细胞进行4 h预孵处理可以显著下调T细胞表面抗原CD69、CD25的表达并能有效抑制细胞增殖。Dex诱导后12 h细胞凋亡和死亡群显著增大,而经同样的GB预孵处理可以在一定程度上抑制细胞的凋亡进程,对细胞凋亡显示出一定的保护作用。结论 GB能有效抑制小鼠T淋巴细胞的活化与增殖,并且对细胞凋亡起到一定的保护作用,凭借GB对T细胞此3大行为出色的调节作用,理应将其作为天然的免疫抑制剂候选者进行更深入的研究。
Objective Ginkgolide B (GB) is a known natural and potent PAF receptor antagonist (PAFR) antagonist. In this paper, we investigated the general behavior of GB on mouse T lymphocyte activation, proliferation and apoptosis Of the impact of preliminary study of its potential immune regulatory role and mechanism, so as to provide a reliable experimental basis for clinical application. Methods Lymphocytes from mice were isolated and preincubated with different concentrations of GB before incubation. The cells were activated and proliferated with Con A, and labeled with anti-CD3 mAb-PE / anti-CD69 mAb FITC, anti-CD3 mAb-PE / anti-CD25 mAb-FITC) combined with flow cytometry were used to detect the expression of CD69 and CD25, the early and mid-stage activation markers of T lymphocytes. Flow cytometry was used to detect cell proliferation and confirmed by MTT assay. Dexamethasone (Dex) was used to induce cell apoptosis. Fluorescent dye labeled DiOC6 (3) / PI combined with flow cytometry was used to distinguish apoptosis, Live cell population. Results At 6 and 24 h after Con A stimulation, CD69 and CD25, the early activation markers of T cells, were expressed in large quantities. T cells activated at 48 h after Con A stimulation proliferated to the 4th generation, whereas the final concentrations of 5 and 10 , Pretreatment with 20 μmol / L GB for 4 h before Con A stimulation significantly down-regulated the expression of T cell surface antigens CD69 and CD25 and effectively inhibited cell proliferation. After 12 h of Dex induction, the apoptotic and death groups were significantly increased. However, the same GB preincubation could inhibit the apoptosis process to a certain extent and showed a protective effect on apoptosis. Conclusion GB can effectively inhibit the activation and proliferation of T lymphocytes in mice, and play a protective role in apoptosis. By virtue of GB’s excellent regulation of T cell’s 3 major behaviors, it should be used as a natural immunosuppressant Candidates for more in-depth study.