摘要
目的探讨三氧化二砷(As 2O 3)单用或联合雷帕霉素(Rapa)对急性髓系白血病THP-1细胞增殖、凋亡和自噬的影响及其可能的分子机制。方法将对数生长期急性髓系白血病THP-1细胞分为对照组、As 2O 3单药组、Rapa单药组及As 2O 3+Rapa组。As 2O 3、Rapa单药组分别应用终浓度0.75、1.50、3.00、6.00、12.00μmol·L^-1 As 2O 3以及10、20、40、80、160 nmol·L^-1 Rapa干预细胞,As 2O 3+Rapa组细胞给予3 mol·L^-1 As 2O 3和40 mmol·L^-1 Rapa干预,对照组细胞采用正常培养基培养。应用细胞计数试剂盒-8(CCK-8)检测3μmol·L^-1 As 2O 3+40 nmol·L^-1 Rapa联合用药对THP-1细胞的增殖抑制率;流式细胞术检测各组THP-1细胞的凋亡率;实时荧光定量聚合酶链反应检测各组THP-1细胞中自噬相关基因Beclin1、LC3和p62 mRNA的表达;Western blotting检测各组THP-1细胞中自噬相关蛋白微管相关蛋白1轻链3(LC3)-II/LC3-I的表达。结果As 2O 3单独用药和Rapa单独用药时,随着药物浓度增加对急性髓系白血病THP-1细胞的增殖抑制作用逐渐增强,呈剂量依赖关系(P<0.05)。As 2O 3和Rapa对急性髓系白血病THP-1细胞的IC 50分别为5.794μmol·L^-1和77.274 nmol·L^-1;As 2O 3+Rapa组急性髓系白血病THP-1细胞增殖抑制率显著高于As 2O 3、Rapa单药组(P<0.05);与As 2O 3、Rapa单药组比较,As 2O 3+Rapa组THP-1细胞中自噬相关基因Beclin-1和LC3 mRNA表达水平显著升高(P<0.05),p62 mRNA表达水平显著降低(P<0.05)。与As 2O 3和Rapa单药组比较,As 2O 3+Rapa组THP-1细胞中LC3-II/LC3-I比值显著增高(P<0.05)。结论As 2O 3联合Rapa可协同抑制THP-1细胞增殖,诱导THP-1细胞凋亡,其效应可能通过引起THP-1细胞过度自噬实现。
Objective To observe the effects of arsenic trioxide(As 2O 3)combined with rapamycin(Rapa)on the proli-feration,apoptosis and autophagy of acute myeloid leukemia THP-1 cells,and investigate the underlying mechanisms.Methods The THP-1 cells in the logarithmic growth stage were divided into the control group,the As 2O 3 treatment group,the Rapa treatment group and the As 2O 2+Rapa group.The As 2O 3 treatment group and the Rapa treatment group were treated with 0.75,1.50,3.00,6.00,12.00μmol·L^-1 As 2O 3 and 10,20,40,80 and 160 nmol·L^-1 Rapa,respectively;the cells in As 2O 2+Rapa grouup were treated with 3μmol·L^-1 As 2O 2 and 40 nmol·L^-1 Rapa;the cells in blank control group were cultured in normal medium.The inhibitory effects of As 2O 3 and Rapa alone,as well as in a combination of As 2O 3 and Rapa on THP-1 cell proliferation were detected by using cell counting kit-8 method;the apoptosis of THP-1 cells in the blank control group,As 2O 3 treatment group,Rapa treatment group and As 2O 3+Rapa group was detected by using flow cytometry;the expressions of autophagy associated gene Beclin1,microtublule-associated 1 light chain 3(LC3)and p62 mRNA and protein in THP-1 cells in the four groups were measured by using real-time quantitative polymerase chain reaction and Western blotting,respectively.Results The growth inhibition rate of As 2O 3 or Rapa alone on the cell proliferation increased with the augment of drug concentration in a concentration-dependent manner(P<0.05).The IC 50 of As 2O 3 and Rapa on THP-1 cells were 5.794μmol·L^-1 and 77.274 nmol·L^-1,respectively.The cell proliferation inhibition rate and apoptosis ratio in the As 2O 3+Rapa group were significantly higher than those in the As 2O 3 treatment group and the Rapa treatment group(P<0.05).The expression of autophagy related genes Beclin-1,LC-3 mRNA in the THP-1 cells in the As 2O 3+Rapa group were significantly higher than those in the As 2O 3 treatment group and the Rapa treatment group(P<0.05),the expression of p62 mRNA was significantly low
作者
肖蓬莉
郭淑利
王双琳
刘思哲
彭靓
王万里
王松云
王慧睿
XIAO Pengli;GUO Shuli;WANG Shuanglin;LIU Sizhe;PENG Liang;WANG Wanli;WANG Songyun;WANG Huirui(Department of Hematology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471009,Henan Province,China;Graduate School,Xinxiang Medical University,Xinxiang 453003,Henan Province,China)
出处
《新乡医学院学报》
CAS
2020年第9期818-823,共6页
Journal of Xinxiang Medical University
基金
河南省医学科技攻关计划项目(编号:2018020905,SB201903035)
洛阳市科技计划医疗卫生项目(编号:1910014A)。
