双氢青蒿素对小鼠T细胞的免疫抑制作用

Immunosuppressive effect of dihydroartemisinin on murine T lymphocytes

目的:研究双氢青蒿素(DHA)在体外对ConA诱导的小鼠T细胞增殖的影响,探讨其可能的免疫作用机制。方法:加入不同浓度DHA,以多克隆刺激剂刀豆蛋白A(ConA)诱导T细胞活化增殖,用羧基荧光素双醋酸盐琥珀酰亚胺酯(CFDA-SE)染色法分析T细胞增殖情况;利用流式细胞术(FCM)结合双色免疫荧光染色技术检测CD3+T细胞早、中、晚期活化标志CD69、CD25、CD71表达情况;用Fluo-4/AM荧光钙离子探针技术检测细胞内钙离子([Ca2+]i)浓度的变化;以碘化丙锭(PI)染色分析细胞周期分布;运用流式细胞术(FCM)结合三色荧光染色技术检测CD4+CD25highTreg早期活化抗原CD69表达情况。结果:CFDA-SE染色结果显示,DHA能有效抑制ConA诱导的T细胞增殖,并呈时间-剂量依赖性;DHA对ConA刺激的CD3+T细胞CD69、CD25的表达有促进作用,而对CD71的表达有抑制作用,均呈剂量依赖性;DHA单独作用不能引起T细胞[Ca2+]i的升高,在ConA的刺激下能引起[Ca2+]i浓度升高;PI染色流式分析结果显示,DHA阻滞细胞于G0/G1期,阻止细胞进入S期和G2/M期;DHA能够下调CD4+CD25highTreg细胞CD69的表达。结论:DHA对小鼠淋巴细胞的增殖有明显的抑制作用,是一种潜在的免疫抑制剂。

AIM： To investigate the effect of dihydroartemisinin （DHA） on the proliferation of murine T lymphocytes stimulated by Con A in vitro and its related immunosuppressive mechanism. METHODS： Murine T lymphocytes were stimulated by Con A and treated with different concentrations of DHA. Cell proliferation was measured by carboxyl fluoresce in diacetate succinmidyl ester （CFDA-SE） staining. The expression of CD69, CD25 and CD71,which was the marker of early, middle, later activation of CD3＋ T lymphocytes, was measured by flow cytometry （FCM） combined with two-color immunofluorescent staining of cell surface antigen. Fluorescence calcium indicator fluo-4/AM was used to measure the change of the intracellular calcium concentration （[Ca2＋]i） of murine T lymphocytes. The distribution of the cell cycle was analyzed by PI staining. The expression of CD69, the early activation antigen on CD4＋CD25high Treg was also measured by FCM combined with three-color immunofluorescent staining. RESULTS： The result of CFDA-SE staining showed that DHA efficiently inhibited the Con A-induced proliferation of T-lymphocytes in a time-and dose-dependent manners. DHA showed modestly increased proportions of CD69 and CD25 on Con A-stimulated CD3＋T cells, but inhibited the expression of CD25 in a dose dependent manner. DHA with Con A, but not DHA alone, caused an increase in intracellular calcium concentration of T cells. The results of FCM analysis with PI staining showed that DHA imposed a total cell cycle arrest in G0/G1 and prevented cells entering S phase and G2/M phase. Furthermore, DHA reduced the expression of CD69 on CD4＋CD25high Treg. CONCLUSION： DHA, which exhibits immunosuppressive effect on the proliferation of murine T-lymphocytes, is promising to be developed as an immunosuppressive reagent.