脂氧素A_4通过抑制Jurkat T细胞钙池操纵的钙通道电流而抑制其激活和增殖

Lipoxin A_4 inhibits Jurkat T cell activation and proliferation by blocking calcium entry current I_(SOC)

目的通过研究脂氧素A4（LXA4）对Jurkat T细胞内游离钙离子浓度变化、钙池操纵的钙通道电流（Isoc）及细胞激活和增殖的影响，初步探讨其对T细胞的调节作用及机制。方法钙离子荧光探针Fluo-3／AM负载细胞后，用激光共聚焦扫描显微镜技术动态检测活细胞内游离钙离子浓度的变化；全细胞膜片钳技术记录Isoc；ELISA测IL-2水平；3^H-TdR掺入法测细胞增殖情况，并结合流式细胞仪进行细胞周期分析。结果（1）用含钙细胞外液时，LXA4降低正常Jurkat T细胞内游离钙离子浓度，而用无钙细胞外液时则无显著差异；（2）LXA4呈剂量依赖性抑制钙池操纵的钙通道（SOC）激活剂thapsigargin（TG）引起Jurkat T细胞内游离钙浓度的增高，100nmol／L LXA4也能抑制anti-CD3或植物血凝素（PHA）引起的细胞内钙离子浓度的增高，SOC阻滞剂2-aminoethoxydiphenylborate（2-APB）能显著抑制TG引起的钙内流；（3）膜片钳结果显示，LXA4抑制正常Jurkat T细胞Isoc，TG能显著增大Isoc，而LXA4呈剂量依赖性抑制TG引起的Isoc的升高；（4）LXA4剂量依赖性抑制anti-CD3和anti-CD28诱导的Jurkat T细胞IL-2表达及细胞增殖，细胞周期分析发现LXA4阻止Jurkat T细胞由GI1期进入S期，降低S期细胞比例。结论LXA4可能通过抑制Jurkat T细胞Isoc引起的胞质内游离钙离子浓度的升高而抑制其激活和增殖。

Objective To study the effects of LXA4 on T cells and its mechanism through investigating the effects of lipoxin A4 （LXA4）on the changes of intracellular free calcium concentration, store-operated calcium channels current（Isoc ）, activation and proliferation in Jurkat T cells. Methods After incubated with Fluo-3/AM, laser confocal scanning microscopy was used to dynamically monitor the changes of intracellular calcium concentration. Whole-cell patch clamp technique was employed to investigate the influence of LXA4 on Isoc. The concentrations of IL-2 were determined by ELISA. 3^ H-TdR incorporation was used for detecting Jurkat T cell proliferation, and cell cycle progression was measured by flow cytometry. Results （1） LXA4 reduced the intracellular free calcium concentration of Jurkat T cells in the presence of external Ca^2＋ , but the effects weren＇t obvious in the ab sence of external Ca^2＋ . （2） LXA4 inhibited the rise of intracellular free calcium concentration of Jurkat T cells induced by thapsigargin （TG） which was an excitomotor of store-operated calcium channels （SOC） in a dose-dependent manner. LXA4, at concentration of 100 nmol/L, also inhibited the rise of intracellular free calcium concentration induced by anti-CD3 or PHA. SOC inhibitor 2-aminoethoxydiphenylborate（2-APB） could notably suppress the Ca^2＋ entry evoked by TG. （3） LXA4 blocked Isoc in Jurkat cells, and TG could markedly induce the increase of Isoc. LXA4, in a dose-dependent manner, inhibited the increase of Isoc caused by TG obviously. （4） LXA4 suppressed the production of IL-2 and the proliferation of Jurkat T cells activated by anti-CD3 and anti-CD28 antibodies in a dose-dependent manner. By the analysis of the cell cycle, we found LXA4 could suppress the entry of Jurkat T cells from G1 to S phase and decreased the radio of cells in the S phase. Conclusion LXA4 could inhibit Jurkat T cells activation and proliferation by blocking calcium entry current Isoc.