茴香霉素在小鼠淋巴细胞增殖和活化中的作用

Effect of anisomycin on the proliferation and activation of lymphocytes in mice

目的研究茴香霉素在小鼠淋巴细胞增殖和活化中的作用。方法以活体染料羧基荧光素乙酰乙酸琥珀酰业胺酯染色,建立了在多克隆刺激剂刀豆蛋白A(Con A)刺激下评价小鼠淋巴细胞增殖的模型,通过流式细胞术及MTT法分析茴香霉素在不同剂量下对淋巴细胞增殖的作用;采用碘化丙锭染色分析茴香霉素对Con A或佛波醇酯(PDB)加离子霉素(Ion)刺激的小鼠淋巴细胞周期变化的作用;利用荧光标记的单克隆抗体双染技术结合流式细胞仪检测茴香霉素对小鼠CD3+淋巴细胞早期及中期活化标志分子CD69和CD25表达的影响。结果随着茴香霉素浓度从1.0 ng/ml逐渐增至25.0 ng/ml,Con A对淋巴细胞的促增殖作用逐渐减弱,以10.0 ng/ml和25.0 ng/ml茴香霉素的抑制作用最为明显,呈剂量依赖关系(r=-0.96,P<0.01)。进一步发现,1.0～25.0 ng/ml茴香霉素能够使Con A或PDB加Ion诱导的淋巴细胞停滞于G0/G1期,阻止其进入S期,且阻止作用随上述浓度的增加而增强,呈明显剂量依赖关系(r=-0.97,P<0.01和r=-0.98,P<0.01)。据此选用最佳剂量10.0 ng/ml,茴香霉素能够明显抑制淋巴细胞表面分子CD69和CD25的表达(P均<0.01)。结论茴香霉素能明显抑制小鼠淋巴细胞的增殖和活化。

Objective To investigate the effect of anisomycin on proliferation and activation of lymphocytes in the mice. Methods A model to evaluate lymphocyte proliferation stimulated with a polyclonal T cell activator, concanavalin A （Con A）, was established by a vital dye carboxyl fluorescin diacetate succinimidyl ester （CFSE） labeling technique. Effects of anisomycin on the lymphocyte proliferation at different doses were estimated by flow cytometry and MTr method. The propidium iodide labeling technique was applied to assay the effect of different doses of anisomycin on lymphocyte cell-cycle. The expression level of CD69 and CD25 on the surface of activated lymphocytes was evaluated by a fluorescin-conjugated monoclonal antibody double labeling technique. Results The promoting effect of Con A on lymphocyte proliferation was suppressed in the presence of anisomycin ranging from 1.0 ng/ml to 25.0 ng/ml. At the range of 10.0 ng/ml to 25.0 ng/ml, the inhibitory effect was the most significant. Anisomycin induced G0/G1 arrest and blocked S phase entry sfimulated by Con A or by PDB plus Ion, and the inhibitory effect of anisomycin was enhanced by the increase of its concentration in a dose-dependent manner （ r = -0.97, P 〈 0.01 and r = -0.98, P 〈 0.01）. At the dose of 10.0 ng/ml, anisomycin down-regulated the expression level of CD69 and CIY25. Conclusion Anisomycin may inhibit the proliferation and activation of lymphocytes.