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目的初步探讨肿瘤坏死因子凋亡诱导配体(TRAIL)诱导白血病细胞发生凋亡过程中可能存在的抵抗机制。方法用流式细胞仪检测经 TRAIL 处理后,白血病细胞系 K562、CEM 凋亡情况及线粒体跨膜电位的改变;采用 Western blot 检测 TRAIL 处理后细胞凋亡相关蛋白 Bcl-xL、Bax 及内源性半胱天冬酶(caspase)-8表达情况;并用 ELISA 法检测核转录因子(NF)-kB 活性变化。结果经TRAIL 处理后,K562、CEM 细胞出现不同程度的凋亡,其凋亡指数分别为29.98%、14.1%,后者显著低于前者(P<0.001);线粒体跨膜电位下降,分别为73.25%、25.4%(P<0.01);CEM 细胞中内源性caspase-8的表达水平低于 K562细胞,并且两者均表现出 Bcl-xL 表达上升、Bax 表达下降,在 CEM 细胞中 Bcl-xL/Bax 比例为18.8,显著高于 K562细胞 Bcl-xL/Bax 的比值(5.1);并且在 TRAIL 处理 CEM细胞后早期(2 h)即表现出 NF-kB 活性增加(0.48 μmol·L~(-1)·mg~(-1)蛋白),高于 K562细胞(0.326μmol·L~(-1)·mg~(-1)蛋白(P<0.001)。结论 TRAIL 诱导白血病细胞凋亡过程中,CEM 细胞对药物抵抗原因可能与 CEM 细胞内源性 caspase-8表达水平较低、线粒体内膜敏感程度降低、NF-kB 活性早期增加以及 Bcl-2家族蛋白的表达变化有关。
Objective To investigate the possible mechanism of resistance in apoptosis induced by tumor necrosis factor Apoptosis inducing ligand (TRAIL) in leukemia cells. Methods The apoptosis of leukemic cell lines K562 and CEM after transfection with TRAIL and the changes of mitochondrial transmembrane potential were detected by flow cytometry. The apoptosis-related proteins Bcl-xL, Bax and endogenous Caspase -8 expression; And activity of nuclear transcription factor (NF) -kB was detected by ELISA. Results After TRAIL treatment, K562 and CEM cells showed different degrees of apoptosis with apoptotic index of 29.98% and 14.1% respectively, the latter being significantly lower than the former (P <0.001). The mitochondrial transmembrane potential decreased to 73.25 %, 25.4% (P <0.01). The expression of endogenous caspase-8 in CEM cells was lower than that in K562 cells, and both of them showed the increase of Bcl-xL expression and the decrease of Bax expression in CEM cells. / Bax ratio was 18.8, which was significantly higher than that of K562 cells (5.1); and the activity of NF-κB was increased in the early stage of TRAIL treatment (2 h) (0.48 μmol·L ~ (- 1) · mg -1 protein), which was higher than that of K562 cells (0.326μmol·L -1 · mg -1) (P <0.001) .Conclusion During TRAIL-induced leukemia cell apoptosis, The reason of resistance of CEM cells to drug may be related to the low level of endogenous caspase-8 expression, the decrease of mitochondrial inner membrane sensitivity, the early increase of NF-κB activity and the change of Bcl-2 family protein in CEM cells.