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目的:研究Forskolin对刀豆蛋白A(Con A)刺激的小鼠CD3+T淋巴细胞活化、增殖和周期的影响,初步探讨其免疫调节作用机制。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,运用流式细胞术结合双色荧光抗体染色技术来检测CD3+T细胞早期活化标志CD69的表达水平;用羧基荧光素乙酰乙酸(CFDA-SE)染色技术结合流式细胞术和CELLQuest、Mod FitLT软件,分析T细胞增殖情况;用碘化丙锭(PI)染色技术分析细胞周期分布。结果:加入终浓度为10-7、10-6、10-5M的Forskolin,孵育8 h后,CD3+T细胞CD69的表达水平和平均荧光强度(MFI)与Con A组相比都有显著的降低,且呈梯度依赖性;对Con A刺激的小鼠CD3+T细胞的增殖指数(PI)也有梯度性抑制;对细胞周期的影响表现为:随着Forskolin浓度升高,处于G0/G1期的细胞增加,S期细胞减少,G2/M期的细胞也有少量减少。结论:Forskolin能有效抑制小鼠T淋巴细胞的体外活化和增殖,阻滞细胞从G0/G1期进入S、G2/M期。由此推断Forskolin在某种程度上是一种潜在的免疫抑制剂。
Objective: To study the effect of Forskolin on the activation, proliferation and cycle of mouse CD3+ T lymphocytes stimulated by Con A, and to explore the mechanism of immune regulation. METHODS: Mouse lymph nodes were isolated aseptically and single lymphocyte suspensions were prepared and incubated with different concentrations of Forskolin. Flow cytometry was used in combination with dual-color fluorescent antibody staining to detect the expression level of early activation marker CD69 in CD3+ T cells. Fluorescein acetoacetate (CFDA-SE) staining was combined with flow cytometry and CELLQuest and Mod FitLT software to analyze T cell proliferation. Propidium iodide (PI) staining was used to analyze cell cycle distribution. RESULTS: After adding Forskolin with final concentrations of 10-7, 10-6 and 10-5 M, the expression level of CD69 and the average fluorescence intensity (MFI) of CD3+ T cells were significantly higher than that of Con A group after 8 h of incubation. Decreased, gradient-dependent; ConA-stimulated mouse CD3+ T cell proliferation index (PI) also has a gradient inhibition; the effect on the cell cycle: As Forskolin concentration increases, in the G0/G1 phase The number of cells increased, cells in S phase decreased, and cells in G2/M phase also decreased slightly. CONCLUSION: Forskolin can effectively inhibit the activation and proliferation of mouse T lymphocytes in vitro, and block cells from G0/G1 phase to S, G2/M phase. It was concluded that Forskolin is to some extent a potential immunosuppressive agent.