二烯丙基二硫启动人白血病HL-60细胞凋亡模型的建立

Establishment of the model of apoptosis initiation phase in human leukemia HL-60 cells induced by DADS

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。

Aim To investigate the apoptosis initiation phase in human leukemia HL-60 cells induced by DADS,and establish the apoptosis initiation model.Methods After incubation of HL-60 cells with 3.6 mg·kg^-1 DADS,the inhibitory effects of DADS on HL-60 cell growth were observed with growth curve.A flow cytometry method was used to determine the induction of apoptosis and the expression of activated caspase-3.DNA agarose electrophoresis was used to determine the induction of apoptosis.Protein activity of caspase-3,Bcl-2 was tested by Western blot analysis.Results The growth curve show 3.6 mg·kg^-1 DADS could not inhibit the growth of HL-60 cells at day 1,but remarkably reduced the growth during day 2~6,cell number was reduced by 24.1%,36.5%,44.2%,52%,and 53.6% respectively compared with control group.Flow cytometry analysis showed that the apoptotic rate of DADS treated cells at day 1 and day 2 was 3.1% and 4.3%respectively,with no difference from control cells（3%）.While from day 3 to day 5,the apoptotic rate was 8.5%,15.2% and 27.4% respectively,which had great difference from control cells.In the day 2~5,the apoptotic rates were 7.9%,12.4%,16.5% and 18.8% in the DADS-withdrawal groups respectively,which were significantly increased compared with the control group and the 1 day with DADS withdrawal（P〈0.05）.The expression of activated caspase-3 from day 2 to day 5 were 6.3%,10%,10.4%,14.9% and 17.3% respectively,which were higher than control group and DADS treated 1 day（P〈0.05）.DNA agarose electrophoresis showed that the DNA ladder appeared both in the DADS treated groups from day 4,and in the DADS-withdrawal groups from day 2 Western-blotting showed that caspase-3 was upregulated and Bcl-2 was downregulated from day 2.Conclusion Diallyl disulfide could significantly induce apoptosis of human Leukemia HL-60 cells,and the initiation phase of apoptosis triggered by 3.6mg · L-1 DADS is at day 2.