氯喹抑制的自噬对地西他滨促进髓性白血病细胞凋亡的影响

Influence of autophagy inhibited by chloroquine in apoptosis of myelogenous leukemia cells promoted by decitabine

目的:研究氯喹与地西他滨联合应用对白血病K562和KG-1a1Aor1a细胞凋亡的影响,探讨自噬对地西他滨诱导白血病细胞凋亡的作用,阐明其作用机制。方法:体外培养髓性白血病K562和KG-1a1Aor1a细胞,分为空白对照组、地西他滨(10μmol·L-1)单用组和氯喹(50μmol·L-1)联用地西他滨组(联用组)。联用组细胞使用氯喹孵育6h后再与其他组细胞同时开始实验。孵育24及48h后,CCK-8法检测细胞数量并计算增殖抑制率,流式细胞术检测细胞凋亡率和线粒体膜电位。Q-PCR法检测Atg7及Atg12基因表达水平,Western blotting法检测LC3蛋白表达。结果:孵育24及48h后,与空白对照组比较,地西他滨和联用组K562和KG-1a1Aor1a细胞数量数量明显减少(P<0.05或P<0.01);凋亡率明显升高(P<0.05或P<0.01),线粒体膜电势明显增加(P<0.05或P<0.01);与地西他滨组比较,联用组K562和KG-1a1Aor1a细胞明显减少,细胞数量凋亡率明显升高(P<0.05)。孵育24h后,与空白对照组比较,地西他滨组K562和KG-1a1Aor1a细胞Atg7、Atg12和LC3-Ⅱ/LC3-Ⅰ相对表达水平明显升高(P<0.05或P<0.01);与地西他滨组比较,联用组K562和KG-1a1Aor1a细胞Atg7、Atg12和LC3-Ⅱ/LC3-Ⅰ相对表达水平明显降低(P<0.05或P<0.01)。结论:地西他滨具有促进白血病细胞凋亡的作用,而联用氯喹可以抑制自噬从而增强地西他滨诱导细胞凋亡的作用。

Objective：To study the influence of chloroquine combined with decitabine in the apoptosis of leukemia K562 cells and KG-1a1Aor1 acells,to explore the effect of autophagy on the leukemia cell apoptosis induced by decitabine,and to clarify its mechanism.Methods：The leukemia K562 and KG-1a1Aor1 acells were cultivated in vitro and divided into blank control group,decitabine group（10 μmol· L^-1）and chloroquine（50μmol·L^-1）combined with decitabine group（combined group）.The leukemia cells in combined group were pre-treated with chloroquine for 6h before experiment.After treatment with drugs for 24 and 48h,the number of cells was detected and by CCK-8 method the inhibitory rates of proliferation cells were calculated;the apoptotic rates and mitochondrial membrane potential were detected by flow cytometry.Q-PCR method was carried out to determine the gene expression levels of Atg7 and Atg12,and Western blotting was used to test the protein expression of LC3.Results：After treatment for 24 and 48h,the number of K562 and KG-1a1Aor1 acells in decitabine group and combined group were decreased compared with blank control group（P〈0.05 or P〈0.01）;the apoptotic rates and mitochondrial membrane potential were remarkably increased（P〈0.05 or P〈0.01）.Compared with decitabine group,the number of K562 and KG-1a1Aor1 ain combined group was significantly decreased,and the apoptotic rates were remarkably increased（P〈0.05）.After treatment for 24 h,the expression levels of Atg7,Atg12 and LC3-Ⅱ/LC3-Ⅰ in the leukemia K562 and KG-1a1Aor1 acells in decitabine group were significantly increased compared with blank control group（P〈0.05 or P〈0.01）;the expression levels of Atg7,Atg12 and LC3-Ⅱ/LC3-Ⅰ in the leukemia K562 and KG-1a1Aor1 acells in combined group were significantly decreased compared with decitabine group（P〈0.05 or P〈0.01）.Conclusion：Decitabine could promote the apoptosis of leukemia cells,and the inhibition of autophagy by chloroquine can promote the apoptosis induced by decitabine.