摘要目的:研究Bcl-2 BH4选择性抑制剂BDA-366对NK/T细胞淋巴瘤（NK/TCL）的抑制作用和杀伤机制。方法:用0、0.05、0.10、0.20、0.30、0.40、0.50 μmol/L的BDA-366处理人NK白血病细胞株YT和人NK/TCL细胞株NK92，使用CCK-8法计算BDA-366对细胞的半抑制浓度（IC 50）值；流式细胞术检测对照组和IC 50浓度BDA-366处理组细胞凋亡水平；蛋白质印迹法检测对照组、1/2 IC 50、IC 50、2倍IC 50浓度BDA-366处理组细胞凋亡相关蛋白表达水平；TMRE和Fluo-3荧光探针法检测对照组和IC 50浓度BDA-366处理组细胞线粒体膜电位，以及对照组、IC 50、2倍IC 50浓度BDA-366处理组细胞内Ca 2+浓度；对对照组和10 mg/kg BDA-366腹腔注射组NOD-SCID小鼠进行称重和小鼠组织HE染色，以评估BDA-366是否具有体内毒性。 结果:BDA-366对于YT和NK92细胞株的IC 50分别为0.065、0.086 μmol/L。流式细胞术结果显示，对照组和0.065 μmol/L BDA-366组YT细胞凋亡率分别为（6.62±1.59）%、（34.60±3.06）%，对照组和0.086 μmol/L BDA-366组NK92细胞凋亡率分别为（5.57±0.88）%、（29.18±0.90）%，差异均具有统计学意义（ t=14.05， P<0.001； t=32.58， P<0.001）。对照组、0.043、0.086、0.172 μmol/L BDA-366组NK92细胞Bax相对表达量分别为0.85±0.00、1.26±0.04、1.51±0.18、1.15±0.10（ F=20.70， P<0.001），各BDA-366处理组Bax相对表达量均高于对照组（均 P<0.05）。对照组和0.065 μmol/L BDA-366组YT细胞TMRE荧光强度分别为8 372.00±330.47、6 419.67±311.34，对照组和0.086 μmol/L BDA-366组NK92细胞TMRE荧光强度分别为9 169.00±535.72、7 311.67±295.52，差异均具有统计学意义（ t=7.45， P=0.002； t=5.26， P=0.006）。在YT细胞中，0.065、0.130 μmol/L BDA-366组细胞内Ca 2+浓度均高于对照组（5 791.67±220.45、6 729.33±585.39、4 874.67±112.61， F=19.16， P=0.003）（ P=0.039； P=0.002）；在NK92细胞中，0.086、0.172 μmol/L BDA-366组细胞内Ca 2+浓度均高于对照组（4 553.67±17.62、4 740.33±254.50、4 185.67±17.67， F=10.96， P=0.010）（ P=0.039； P=0.007）。BDA-366组和对照组小鼠第12天相对于第0天体重变化差异无统计学意义[（3.18±0.01）g比（2.73±0.58）g， t=0.60， P=0.570]，HE染色结果未见BDA-366组小鼠心、肝、脾、肺、肾形态异常。 结论:BDA-366在体外可促进NK/TCL细胞发生凋亡，在体内不引起小鼠体重减轻和器官HE染色形态改变。BDA-366对NK/TCL细胞的抑制作用可能是通过提高Bax表达、诱导Ca 2+释放和降低线粒体膜电位实现的。

abstractsObjective:To investigate the inhibitory effect and killing mechanism of Bcl-2 BH4 selective inhibitor BDA-366 on NK/T cell lymphoma (NK/TCL) .Methods:Human NK cell leukemia cell line YT and human NK/TCL cell line NK92 cells were treated with 0, 0.05, 0.10, 0.20, 0.30, 0.40, 0.50 μmol/L BDA-366. CCK-8 assay was used to calculate the half inhibitory concentration (IC 50) value of BDA-366 on these cells. The apoptosis levels of cells in control group and IC 50 BDA-366 treated group were detected by flow cytometry. Western blotting was used to detect the expression levels of apoptosis-related proteins in cells of control group and 1/2 IC 50, IC 50, 2× IC 50 BDA-366 treated groups. TMRE and Fluo-3 fluorescent probe were used to detect mitochondrial membrane potential of control group and IC 50 BDA-366 treated group, and the intracellular Ca 2+ concentration of control group, IC 50, 2× IC 50 BDA-366 treated groups. NOD-SCID mice in control group and 10 mg/kg BDA-366 intraperitoneal injection group were weighed and HE staining was performed to evaluate the toxicity of BDA-366 in vivo. Results:The IC 50 of BDA-366 for YT and NK92 cells were 0.065 and 0.086 μmol/L respectively. The apoptosis rates of YT cells in the control group and 0.065 μmol/L BDA-366 group were (6.62±1.59) % and (34.60±3.06) % respectively. The apoptosis rates of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were (5.57±0.88) % and (29.18±0.90) % respectively, both with statistically significant differences ( t=14.05, P<0.001; t=32.58, P<0.001). The relative expression of Bax in NK92 cells of the control group, 0.043, 0.086 and 0.172 μmol/L BDA-366 groups were 0.85±0.00, 1.26±0.04, 1.51±0.18, 1.15±0.10 ( F=20.70, P<0.001), the relative expression of Bax in BDA-366 groups were higher than that in the control group (all P<0.05). The fluorescence intensity of TMRE of YT cells in the control group and 0.065 μmol/L BDA-366 group were 8 372.00±330.47 and 6 419.67±311.34, and that of NK92 cells in the control group and 0.086 μmol/L BDA-366 group were 9 169.00±535.72 and 7 311.67±295.52 respectively, and there were statistically significant differences ( t=7.45, P=0.002; t=5.26, P=0.006). In YT cells, the intracellular Ca 2+ concentrations of 0.065 and 0.130 μmol/L BDA-366 groups were significantly higher than that of the control group (5 791.67±220.45, 6 729.33±585.39, 4 874.67±112.61, F=19.16, P=0.003) ( P=0.039; P=0.002). In NK92 cells, the intracellular Ca 2+ concentrations of 0.086 and 0.172 μmol/L BDA-366 groups were significantly higher than that of the control group (4 553.67±17.62, 4 740.33±254.50, 4 185.67±17.67, F=10.96, P=0.010) ( P=0.039; P=0.007). There was no statistically significant difference in body weight change on day 12 compared with day 0 of NOD-SCID mice between BDA-366 group and control group [ (3.18±0.01) g vs. (2.73±0.58) g, t=0.60, P=0.570], and HE staining showed no abnormal morphology of heart, liver, spleen, lung and kidney in BDA-366 group. Conclusion:BDA-366 promotes NK/TCL cells apoptosis in vitro, but does not cause weight loss and morphological changes of organs by HE staining in vivo. The inhibitory effect of BDA-366 on NK/TCL cells may be achieved by increasing Bax expression, inducing Ca 2+ release and reducing mitochondrial membrane potential.