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背景与目的:甲异靛可有效治疗慢性粒细胞白血病,但其抗白血病的机制尚不清楚。本研究拟观察甲异靛诱导急性白血病细胞株HL-60细胞凋亡的作用及促凋亡机制。方法:用台盼蓝拒染实验观察甲异靛对HL-60细胞增殖抑制作用;琼脂糖凝胶电泳检测DNA条带;荧光显微镜观察细胞形态学的变化;流式细胞术检测细胞凋亡率和Fas蛋白的表达;免疫蛋白印迹法分析caspase-9、caspase-8、caspase-3、PARP、bcl-2、bax和胞浆细胞色素c的含量。结果:甲异靛可抑制HL-60细胞增殖和诱导其凋亡。20μmol/L甲异靛处理HL-60细胞12～48h可明显抑制HL-60细胞增殖;20μmol/L甲异靛作用于HL-60细胞1h,凋亡百分比为(3.70±0.56)%,当延长至3h、6h和12h凋亡细胞比例分别上升至(19.80±1.13)%、(29.20±2.69)%和(47.05±7.70)%,较阴性对照组[(2.65±0.78)%]明显增高(P<0.05)。HL-60细胞经甲异靛处理3h后出现细胞核染色质浓集和DNA梯形条带。甲异靛处理HL-60细胞1h后死亡受体Fas阳性率由(9.35±0.21)%升高到(21.30±1.27)%(P<0.05)。甲异靛可活化HL-60细胞的caspase-8、caspase-9、caspase-3和PARP,降低抗凋亡蛋白bcl-2的表达和上调促凋亡蛋白bax,并升高细胞浆中细胞色素c的浓度。caspase-3抑制剂(z-DEVD-fmk)可以减少甲异靛对HL-60细胞的增殖抑制,降低细胞凋亡率。100μmol/Lz-DEVD-fmk预处理HL-60细胞后再加入20μmol/L甲异靛,5h后凋亡率为(16.5±5.5)%,甲异靛组为(29.8±5.4)%(P<0.05);12h后计数,抑制剂加甲异靛组活细胞高达(3.57±0.18)×105/ml,甲异靛组活细胞仅为(1.80±0.14)×105/ml(P<0.05)。结论:甲异靛可诱导HL-60细胞凋亡,其机制与caspases和bcl-2家族蛋白的调节有关。
BACKGROUND & OBJECTIVE: Methyldiotecan can effectively treat chronic myeloid leukemia, but the mechanism of anti-leukemia is not clear. This study was intended to observe the apoptosis and the mechanism of apoptosis in HL-60 cells induced by methohepatic-induced acute leukemia. Methods: The inhibitory effect of meisoindigo on the proliferation of HL-60 cells was observed by trypan blue exclusion test. The DNA bands were detected by agarose gel electrophoresis. The morphological changes of cells were observed by fluorescence microscopy. The apoptosis rate was detected by flow cytometry The expressions of caspase-9, caspase-8, caspase-3, PARP, bcl-2, bax and cytoplasmic cytochrome c were detected by Western blotting. Results: Methyindigo can inhibit the proliferation and induce the apoptosis of HL-60 cells. The proliferation of HL-60 cells was inhibited by 20μmol / L methylisoindigo for 12-48h, while the apoptosis rate was 3.70 ± 0.56% when HL-60 cells were exposed to 20μmol / The percentage of apoptotic cells increased to (19.80 ± 1.13)%, (29.20 ± 2.69)% and (47.05 ± 7.70)% at 3h, 6h and 12h respectively, which was significantly higher than that in the negative control group [(2.65 ± 0.78)%] <0.05). HL-60 cells were treated with methylisoindigo 3h nuclear chromatin condensation and DNA trapezoidal bands. The Fas positive rate of death receptor in HL-60 cells treated with methytoindigo increased from (9.35 ± 0.21)% to (21.30 ± 1.27)% (P <0.05). Methylisothiocyanate activated caspase-8, caspase-9, caspase-3 and PARP in HL-60 cells, decreased the expression of anti-apoptotic protein bcl-2 and up-regulated the pro-apoptotic protein bax, and increased cytochrome Concentration of c. Caspase-3 inhibitor (z-DEVD-fmk) can reduce the proliferation inhibition of HL-60 cells and decrease the apoptosis rate of HL-60 cells. After being pretreated with 100μmol / Lz-DEVD-fmk, HL-60 cells were treated with 20μmol / L mechlorethamine and the apoptosis rate was (16.5 ± 5.5)% after 5h and 29.8 ± 5.4% 0.05). After 12 hours, the viable cells in the inhibitor plus methylisothiocyanate group reached as high as (3.57 ± 0.18) × 105 / ml, while the live cells in the methylisothiocyanate group were only (1.80 ± 0.14) × 105 / ml (P <0.05). Conclusion: Methylindigo can induce the apoptosis of HL-60 cells. The mechanism is related to the regulation of caspases and bcl-2 family proteins.