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目的分析Forskolin对佛波醇酯多克隆刺激剂(phorbol12,13-dibutyrate,PDB)联合离子霉素(ionomycin,Ion)刺激的小鼠CD3+T淋巴细胞体外增殖和周期的影响,在此基础上进一步研究Forskolin对单纯PDB刺激的小鼠CD3+T淋巴细胞早期活化标志CD69分子和中期活化标志CD25分子的影响,从而阐明其作用机制。方法无菌分离小鼠淋巴结并制备单个淋巴细胞悬液,与不同浓度的Forskolin孵育48h后,运用羧基荧光素已酰已酸(CFDA-SE)染色技术和碘化丙锭(PI)染色技术结合流式细胞术、CELLQuest和ModFitLT软件,分别检测Forskolin对PDB+Ion刺激下小鼠CD3+T淋巴细胞增殖和周期的影响;与不同浓度的Forskolin孵育2h、10h,运用流式细胞术结合双色荧光抗体染色技术检测Forskolin对PDB诱导下的小鼠CD3+T淋巴细胞CD69分子和CD25分子表达的影响。结果Forskolin(10-7、10-6、10-5mol.L-1)均能明显抑制PDB+Ion刺激的小鼠CD3+T淋巴细胞的增殖并将细胞阻滞在G0/G1期;同时也能明显抑制单纯PDB刺激下的CD3+T细胞早期和中期活化,且呈明显的量效关系。结论提示Forskolin在CD3+T细胞活化过程中信号转导的某个环节上有抑制作用,从而通过抑制T淋巴细胞的活化,发挥免疫抑制作用,是一种潜在的免疫抑制剂。
Objective To analyze the effect of Forskolin on proliferation and cell cycle of murine CD3 + T lymphocytes stimulated by phorbol 12,13-dibutyrate (PDB) combined with ionomycin (Ion) To further investigate the effect of Forskolin on CD69 molecule and CD25 molecule, a marker of early activation of mouse CD3 + T lymphocytes stimulated by PDB alone, and to elucidate its mechanism of action. Methods The mouse lymph nodes were aseptically isolated and the single lymphocyte suspension was prepared. After incubation with different concentrations of Forskolin for 48 hours, the cells were stained with carboxyfluorescein hexanoate (CFDA-SE) and propidium iodide (PI) staining Flow cytometry, CELLQuest and ModFitLT software were used to detect the effect of Forskolin on proliferation and cycle of CD3 + T lymphocytes stimulated by PDB + Ion in mice. Forskolin was incubated with different concentrations of Forskolin for 2h and 10h respectively. Flow cytometry combined with two-color fluorescence Antibody staining technique was used to detect the effect of Forskolin on PDCD-induced CD69 and CD25 expression in mouse CD3 + T lymphocytes. Results Forskolin (10-7, 10-6, 10-5mol.L-1) significantly inhibited PDB + Ion-stimulated proliferation of mouse CD3 + T lymphocytes and arrested cells in G0 / G1 phase Can significantly inhibit the early and mid-term activation of CD3 + T cells stimulated by PDB, and showed a significant dose-response relationship. The results suggest that Forskolin may inhibit some aspects of signal transduction during the activation of CD3 + T cells and thus may be an immunosuppressant by inhibiting the activation of T lymphocytes and exerting immunosuppressive effects.