TIM-3配体galectin-9诱导NK/T细胞淋巴瘤细胞的凋亡及其机制

Preliminary investigation of TIM-3 ligand galectin-9 inducing apoptosis of NK/T cell lymphoma cells

目的:观察TIM-3在NK/T细胞淋巴瘤(natural killer/T cell lymphoma,NK/TCL)细胞株中的表达及其配体galectin-9(GAL-9)诱导瘤细胞凋亡的作用及其部分机制。方法:Western blotting测定NK/TCL细胞株SNK-1、SNK-6、SNT-8和健康人外周血NK细胞中TIM-3的表达;采用不同质量浓度重组人GAL-9(rh GAL-9)作用于NK/TCL细胞株24 h后,通过CCK-8法检测细胞增殖活性;流式细胞术Annexin-V/PI双染法检测瘤细胞凋亡;Western blotting检测caspase-3、PARP及其剪接体和MAPK信号通路蛋白磷酸化表达水平变化。结果:NK/TCL细胞株SNK-1、SNK-6和SNT-8的TIM-3表达较健康人外周血NK细胞显著增高,其中SNK-1、SNK-6细胞株与对照组相比差异具有统计学意义(P<0.05);CCK-8检测结果显示,rh GAL-9对NK/TCL细胞增殖活力具有明显抑制作用,并有浓度依赖性;流式细胞术检测显示,rh GAL-9可诱导3种NK/TCL细胞株凋亡;Western blotting结果显示,cleaved-caspase-3、cleaved-PARP蛋白表达量增高,MAPK通路中JNK蛋白磷酸化水平升高。结论:TIM-3在NK/TCL细胞株中异常高表达,其配体GAL-9可诱导细胞凋亡,其机制可能与JNK磷酸化水平升高相关。

Objective： To identify the expression pattern of TIM-3 in natural killer/T-cell lymphoma （NK/TCL） cell lines, and to investigate the effect and mechanism of its ligand galectin-9 （GAL-9） inducing apoptosis of NK/TCL cell lines. Methods： Expression of TIM-3 in NK cell of peripheral blood from healthy donors and NK/TCL cell lines （SNK-1、SNK-6、SNT-8） was detected by Western blotting. After being treated with rhGAL-9 at various concentrations for 24h, the cell proliferation ability was analyzed with CCK-8 assay.Apoptosis ratio of the cells was determined by flow cytometry. Expressions of caspase-3, PARP and their cleavages were detected by Western blotting; moreover, phosphorylation levels of proteins in MAPK signaling pathway were also detected by Western blotting.Results： The expression of TIM-3 in SNK-1, SNK-6 and SNT-8 cell lines was significantly higher than that of NK cells from healthy donors （P〈0.05）. CCK-8 result showed that rhGAL-9 obviously inhibited the proliferation of NK/TCL cell lines in a concentration dependent manner. Flow cytometry showed that rhGAL-9 induced the apoptosis of NK/TCL cells; andWestern blotting proved that the expression of cleaved caspase-3, cleaved-PARP, and p-JNK in MAPK signaling pathway were significantly elevated. Conclusion： TIM-3 was over-expressed in NK/TCL cell lines, and its ligand galectin-9 induced cell apoptosis probably through the activation of JNK kinase pathways.