雷帕霉素对去甲氧柔红霉素诱导急性髓系白血病THP⁃1细胞凋亡的影响  

Effect of rapamycin on apoptosis of acute myeloid leukemia THP⁃1 cells induced by idarubicin

作　　者：郭淑利 肖蓬莉 王双琳 刘思哲 彭靓 王万里 王松云 王慧睿 Guo Shuli;Xiao Pengli;Wang Shuanglin;Liu Sizhe;Peng Liang;Wang Wanli;Wang Songyun;Wang Huirui(Department of Hematology,Luoyang Central Hospital Affiliated to Zhengzhou University,Luoyang 471009,China;Graduate School,Xinxiang Medical University,Xinxiang 453003,China)

机构地区：[1]郑州大学附属洛阳中心医院血液科,河南洛阳471009 [2]新乡医学院研究生院,河南新乡453003

出　　处：《白血病．淋巴瘤》2021年第5期267-271,共5页Journal of Leukemia & Lymphoma

基　　金：河南省医学科技攻关计划(2018020905、SB201903035);洛阳市科技计划医疗卫生项目(1910014A)。

摘　　要：目的探讨雷帕霉素(Rapa)对去甲氧柔红霉素(IDA)诱导急性髓系白血病THP⁃1细胞凋亡的影响及其分子机制。方法分别用10、20、40、80 nmol/L Rapa处理THP⁃1细胞1 h,另设未经Rapa处理的细胞。采用蛋白质印迹法检测THP⁃1细胞自噬标志物LC3蛋白的转换情况(LC3Ⅱ/LC3Ⅰ),采用流式细胞术检测细胞凋亡,确定Rapa处理浓度。用不同浓度IDA作用THP⁃1细胞24 h,采用CCK⁃8法检测IDA对THP⁃1细胞的增殖抑制率,计算半数抑制浓度(IC50)。以低于IC50的IDA作用Rapa处理或未处理的THP⁃1细胞24 h,CCK⁃8法检测细胞增殖抑制率,流式细胞术检测细胞凋亡情况,实时荧光定量聚合酶链反应检测自噬相关基因Beclin⁃1、LC3和p62的表达变化,蛋白质印迹法检测自噬标志物LC3蛋白的转换情况。结果20 nmol/L Rapa处理的THP⁃1细胞LC3Ⅱ/LC3Ⅰ高于未处理的细胞(P=0.0024);80 nmol/L Rapa处理的细胞凋亡率高于未处理的细胞(P=0.0073)。根据蛋白质印迹法和流式细胞术检测结果,选取20 nmol/L Rapa作为预处理浓度。IDA对THP⁃1细胞作用24 h的IC50为59.874 nmol/L。50 nmol/L IDA作用24 h后,Rapa预处理的THP⁃1细胞增殖抑制率[(69.67±5.03)%比(41.67±3.51)%]和细胞凋亡率[(74.35±4.83)%比(41.25±5.24)%]均高于未预处理的细胞(均P<0.05);Rapa预处理的THP⁃1细胞Beclin⁃1、LC3 mRNA表达水平及LC3Ⅱ/LC3Ⅰ均高于未预处理的细胞,p62 mRNA表达水平低于未预处理的细胞(均P<0.05)。结论Rapa能增强较低剂量IDA诱导的THP⁃1细胞凋亡,此效应可能是通过其引起THP⁃1细胞过度自噬实现的。Objective To investigate the effect of rapamycin(Rapa)on apoptosis of acute myeloid leukemia THP⁃1 cells induced by idarubicin(IDA)and its molecular mechanism.Methods The THP⁃1 cells were treated with 10,20,40 and 80 nmol/L Rapa for 1 h,and the cells without Rapa treatment were set up.Western blot was used to detect the conversion of autophagy marker LC3 protein in THP⁃1 cells(the ratio of LC3Ⅱ/LC3Ⅰ),flow cytometry was used to detect the apoptotic rate,and the pretreatment concentration of Rapa was determined.THP⁃1 cells were treated with different concentrations of IDA for 24 h,the cell proliferation inhibition rate of IDA for THP⁃1 cells was detected by CCK⁃8 method,and the half maximal inhibitory concentration(IC50)was calculated.THP⁃1 cells with or without Rapa treatment were treated by IDA with the concentration of lower than IC50 for 24 h,CCK⁃8 method was used to detect cell proliferation inhibition rate,flow cytometry was used to detect cell apoptosis,real⁃time fluorescent quantitative polymerase chain reaction was used to detect the expression changes of autophagy⁃related genes Beclin⁃1,LC3 and p62,and Western blot was used to detect the conversion of autophagy marker LC3 protein.Results The ratio of LC3Ⅱ/LC3Ⅰin THP⁃1 cells treated by 20 nmol/L Rapa was higher than that in the untreated cells(P=0.0024).The apoptotic rate in THP⁃1 cells treated by 80 nmol/L Rapa was higher than that in the untreated cells(P=0.0073).According to the results of Western blot and flow cytometry,20 nmol/L Rapa was selected as the pretreatment concentration.The IC50 of IDA for THP⁃1 cells treated with IDA for 24 h was 59.874 nmol/L.After treated with 50 nmol/L IDA for 24 h,the proliferation inhibitory[(69.67±5.03)%vs.(41.67±3.51)%]and apoptotic rates[(74.35±4.83)%vs.(41.25±5.24)%]in THP⁃1 cells pretreated by Rapa were higher than those in the unpretreated cells(both P<0.05);the Beclin⁃1 and LC3 mRNA expression levels and the ratio of LC3Ⅱ/LC3Ⅰin THP⁃1 cells pretreated by Rapa wer

关 键 词：白血病 髓样 急性 雷帕霉素 去甲氧柔红霉素 THP⁃1细胞 细胞凋亡 自噬 

分 类 号：R965[医药卫生—药理学]