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目的 :用反义 bcr/abl寡核苷酸片段诱导 K562细胞凋亡 ,观察 Caspase3表达及活性变化 ,以阐明 Caspase3在慢性髓性白血病 ( CML)发病机制中的作用。方法 :用反义 bcr/abl寡核苷酸片段 AS和对照无义片段 NS处理 CML急变细胞系 K562 ,检测细胞凋亡及 Caspase3表达和活性的变化。结果 :K562细胞经 AS处理 72 h,流式细胞仪检测未见亚二倍体细胞增多。原位末端标记法显示 AS组 2 4 h始出现细胞凋亡增多 ,72 h达高峰 ,为 ( 78.50± 6.60 ) % ,较对照组 ( 1 8.2 1± 3.32 ) %及 NS组 ( 2 4 .1 3± 4.1 7) %明显增多 ( P<0 .0 5)。Western blot检测显示 ,培养 72 h AS组 Caspase3原酶及 p17活性亚单位的表达增强。荧光分光光度法测定 Caspase3活性示 ,AS处理 1 2 h Caspase3活性即已升高 ,48h达高峰 ,为处理前的 4.71倍。对照组及NS组上升幅度较小 ,72 h最高 ,分别为处理前的 2 .78倍和 2 .30倍。结论 :Caspase3参与了 CML的 bcr/abl基因抑制细胞凋亡的作用。
Objective : To induce apoptosis of K562 cells with antisense bcr/abl oligonucleotide fragment and observe the changes of Caspase3 expression and activity to elucidate the role of Caspase3 in the pathogenesis of chronic myelogenous leukemia (CML). METHODS: The CML blast cell line K562 was treated with antisense bcr/abl oligonucleotide fragment AS and control nonsense fragment NS to detect the changes of apoptosis and Caspase3 expression and activity. RESULTS: K562 cells were treated with AS for 72 h. There was no subdiploid cell count detected by flow cytometry. In situ end-labeling showed that the apoptosis of AS group began to increase at 24 h and peaked at 72 h (78.50± 6.60) % compared with the control group (18.2 1± 3.32) % and NS group (2 4.1 3±4.1 7) % increased significantly (P<0.05). Western blot analysis showed that the expression of caspase 3 pro-enzyme and p17 activity subunit was increased in AS group after 72 hours culture. The fluorescence spectrophotometric determination of Caspase3 activity showed that the activity of Caspase3 was increased after AS treatment for 12 h and peaked at 48 h, 4.71 times before treatment. The increase in the control group and the NS group was smaller and highest at 72 h, which was 2.78-fold and 2.30-fold before treatment respectively. Conclusion: Caspase3 is involved in the inhibition of apoptosis by bcr/abl gene in CML.