芹菜素对小鼠T淋巴细胞体外活化和增殖的抑制作用

The inhibitory effects of apigenin in the in vitro activation and proliferation of mouse lymphocytes

研究芹菜素（apigenin,AP）对小鼠T细胞体外活化和增殖的影响,探讨其作用机制,以及将其开发为免疫抑制药物的可能。无菌分离小鼠淋巴结细胞,加入不同浓度（25μmol/L、50μmol/L、100μmol/L、150μmol/L和200μmol/L）的芹菜素预孵育4 h,用多克隆刺激剂刀豆蛋白A（concanavalin A,ConA）诱导T细胞活化和增殖,并用MTT法检测该药物浓度对T细胞的毒性作用。荧光标记抗体双染色结合流式细胞术,检测各浓度AP对ConA诱导的小鼠T细胞表达早期、中期和晚期活化抗原CD69、CD25和CD71的影响;以二乙酰羧基荧光素-琥珀酰亚胺酯（carboxyfluoresceindiacetate-succinim-idyl ester,CFDA-SE）染色结合流式细胞术,检测各浓度AP对ConA诱导的小鼠T细胞增殖的影响,并应用ModFit软件分析其增殖指数（proliferation index,PI）。结果显示,25-200μmol/L AP对ConA诱导的T细胞CD69、CD25的表达有显著抑制作用（P〈0.01）,且呈剂量依赖关系;25μmol/L和50μmol/L的AP抑制CD71的表达,但高浓度（大于100μmol/L）才有显著抑制作用（P〈0.01）。各浓度AP对ConA诱导的T细胞增殖均有显著抑制作用（P〈0.01）,且呈剂量依赖关系。表明在一定浓度范围内,AP可显著抑制ConA诱导的小鼠T细胞体外活化及增殖,有望通过进一步研究将其开发成免疫抑制药物。

To investigate the effects of apigenin on the activation and prolifrtation of mouse T lymphocytes in vitro, so as to elucidate its mechanism in order to develop a novel immunosuppressive drug, mouse lymphocytes were pre-treated with different concentration of apigenin（25, 50, 100 and 200μmol/L）for 4 hrs prior to stimulation with ConA. The cytotoxic effect of these concentrations of apigenin on T lymphocytes was assayed by MTT method, and those effects on the expressions of CD69, CD35 and CD71（the early, middle and late markers of the activated CD3^＋T cells）on T lymphocytes in response to ConA stimulation were measured by flow cytometry combined with two colored flow cytometry. While, the effect of apigenin on the proliferation of T lymphocytes in response to ConA stimulation was determined by flow cytometry combined with carboxyl fluorescein-diacetate-succinimidyl ester（CFD-SE）technology, and the proliferation index（PI）was analyzed by Modfit software. The experiment results showed that apigenin at concentration of 25～200 μmol/I, could inhibit the expressions of CD69 and CD25 on T lyrnphocytes in response to ConA stimulation obviously（P〈0.01 ） in a dose-dependent manner. Apigenin at concentration of 25 and 50 μmol/L inhibited the expression of CD71, but marked effect of inhibition could be demonstrated under higher con-centration of apigenin（more than 100 μmol/L） in a dose-dependent manner（P〈0.01）. It is evident from the above observation that apigenin can inhibit the activation and proliferation of mouse T lymphocytes in response to ConA stimulation in vitro, thus promising to be developed to immunosuppressive drug through further studies.