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目的寻找二烯丙基二硫(diallyl disulfide,DADS)诱导人白血病HL-60细胞凋亡的启动点,建立DADS启动HL-60细胞凋亡模型。方法实验设未处理组、处理组和撤药组,分别采用细胞计数、流式细胞术、DNA凝胶电泳、Western blot等方法,绘制生长曲线,检测凋亡率、活化的caspase-3表达率以及DNALadder、凋亡相关蛋白的检测。结果3.6mg.L-1DADS作用HL-60细胞1d,与对照组相比,细胞数没有明显变化,而作用2～6d,细胞数分别减少24.1%、36.5%、44.2%、52%、53.6%。DADS作用1、2d,凋亡率分别为3.1%、4.3%,与未处理组(3.0%)差异无显著性,而作用3～5d的凋亡率分别达到8.5%,15.2%,27.4%,均高于未处理组(P<0.05)。DADS作用2～5d撤药后再培养至d6,凋亡率分别为7.9%,12.4%,16.5%,18.8%,高于未处理组的3.3%(P<0.05)。处理2～5d后,活化的caspase-3的表达率分别为10.0%、10.4%、14.9%、17.3%,均高于未处理组5.4%(P<0.05)。处理组中DADS处理4d后DNA凝胶电泳出现梯状条带,而DADS作用2～5d撤药后再培养至d6均出现明显梯状条带。Westernblot结果表明,从作用2d起,caspase-3表达开始上调,而Bcl-2表达开始下调。结论3.6mg.L-1DADS诱导人白血病HL-60细胞凋亡的启动点为处理2d。
OBJECTIVE: To investigate the initiation point of diallyl disulfide (DADS) -induced apoptosis in human leukemia HL-60 cells and to establish a model of apoptosis induced by DADS in HL-60 cells. Methods The untreated group, treatment group and withdrawal group were set up. The growth curve was drawn by cell counting, flow cytometry, DNA gel electrophoresis and Western blot, respectively. The apoptosis rate, the expression of activated caspase-3 As well as DNALadder, apoptosis-related protein detection. Results The number of cells in HL-60 cells treated with 3.6 mg.L-1DADS for 1 day was 24.1%, 36.5%, 44.2%, 52%, 53.6% . The apoptotic rates of DADS treated with DADS for 1 and 2 d were 3.1% and 4.3%, respectively. There was no significant difference between untreated group and untreated group (3.0%), while the apoptotic rates reached 8.5%, 15.2% and 27.4% Were higher than the untreated group (P <0.05). After DADS treatment for 2 to 5 days, the apoptosis rate was 7.9%, 12.4%, 16.5% and 18.8%, respectively, which was higher than that of untreated group (P <0.05). After 2 to 5 days of treatment, the expression rates of activated caspase-3 were 10.0%, 10.4%, 14.9% and 17.3%, respectively, which were all higher than those of the untreated group (5.4%, P <0.05). After treated with DADS for 4 days, DNA ladder electrophoresis showed a ladder-shaped band, while DADS treated for 2 ~ 5 days and then cultured to d6 showed obvious ladder-like bands. The result of Western blot showed that the expression of caspase-3 began to up-regulate and the expression of Bcl-2 began to decrease from the 2nd day. Conclusion The initiation point of 3.6mg.L-1DADS-induced apoptosis in human leukemia HL-60 cells was treated for 2 days.