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

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（10 μmol·L^-1）单用组和氯喹（50 μmol·L^-1）联用地西他滨组（联用组）。联用组细胞使用氯喹孵育6 h后再与其他组细胞同时开始实验。孵育24及48 h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48 h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24 h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。

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

Objective:To study the influence of chloroquine combined with decitabine in the apoptosis of leukemia K562 cells and KG-1a1Aor1a cells,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-1a1Aor1a cells were cultivated in vitro and divided into blank control group,decitabine group (10 μmol · L 1) and chloroquine (50 μmol · L 1) combined with decitabine group (combined group).The leukemia cells in combined group were pre-treated with chloroquine for 6 h before experiment.After treatment with drugs for 24 and 48 h,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 48 h,the number of K562 and KG-1a1Aor1a cells 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-1a1Aor1a in 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-1a1Aor1a cells 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-1a1Aor1a cells 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.