DBO对人慢性髓系白血病细胞K562增殖和凋亡以及自噬影响  

Effect of DBO on the proliferation,apoptosis and autophagy of human chronic myeloid leukemia K562 cells

作　　者：廖琼 孙玉梅[1] 姜夏薇[2] 李珊 王希 白艳梅 任霞[3] 王大本[1] 姜国胜[3] LIAO Qiong;SUN Yu-mei;J IANG Xia-wei;LI Shah;WANG Xi;BAI Yan-mei;REN Xia;WANG Da-ben;J IANG Guo-sheng(Department of Hematology and Oncology , The 5th People ＇ s Hospital of Jinan , Jinan 250022, P. R. China 2. Department of Laboratory, The 4th People ＇s Hospital of J inan ,Jinan 250031 ,P. R. China;3. Institute of Basic Medicine, Shandong Academy of Medical Sciences , Key Laboratory for Modern Medicine and Technology of Shandong Provinc;Key Medical Laboratory for Tumor Immunology , Chinese Medicine Immunology of Shandong Province, J inan 250062, P. R. Chin)

机构地区：[1]济南市第五人民医院血液肿瘤科,山东济南250022 [2]济南市第四人民医院检验科,山东济南250031 [3]山东省医学科学院基础医学研究所.山东省现代医用药物与技术重点实验室.山东省医药卫生肿瘤免疫与中药免疫重点实验室,山东济南250062

出　　处：《中华肿瘤防治杂志》2017年第19期1342-1348,共7页Chinese Journal of Cancer Prevention and Treatment

基　　金：国家自然科学基金(81573467);山东省自主创新及成果转化专项(2014CGZH1313;2015ZDJS04003);山东省自然科学基金(ZR2015HM014;ZR2015YL028;ZR2014HM085);泰山学者基金(ts201511075)

摘　　要：目的 DBO(6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine)作为雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)抑制剂,可诱导人脐静脉内皮细胞发生自噬,促进其凋亡,但是DBO对白血病细胞是否亦具有同样的作用却不明确。本研究旨在探讨DBO对人慢性髓系白血病K562细胞的增殖、自噬与凋亡的影响。方法常规方法复苏、传代培养K562细胞,设置对照组(DMSO)、DBO处理组。处理24、48和72h后收集细胞,采用蛋白质印迹法检测LC3蛋白的表达。细胞免疫荧光实验检测LC3斑点的表达;透射电镜观察细胞自噬现象。CCK-8比色法检测K562细胞的活性和增殖能力。细胞周期实验检测K562细胞分裂能力。AnnexinⅤFITC/PI双染流式细胞术检测细胞凋亡。结果蛋白质印迹结果显示,K562细胞经雷帕霉素处理后LC3-Ⅱ蛋白水平表达升高,p62蛋白水平表达降低;经50μmol/L DBO处理24、48和72h后,LC3-Ⅱ和p62蛋白水平表达与雷帕霉素处理后结果相似,并呈时间依赖性。K562细胞经10、25和50μmol/L处理后,LC3-Ⅱ蛋白水平表达升高,p62蛋白水平表达降低,呈剂量依赖性,而使用自噬抑制剂3-MA后LC3-Ⅱ蛋白表达降低,P62蛋白表达升高。细胞免疫荧光实验显示,DBO作用72h后,与对照组相比,DBO处理组LC3斑点的表达量增加。透射电镜观察结果显示,与对照组相比,DBO处理组细胞胞质内可见较多自噬小体和自噬溶酶体,细胞核不规则,染色质边缘化。CCK8检测结果显示,DBO对K562细胞的增殖具有明显的抑制作用,并呈现时间-剂量依赖性。DBO经10、25、50和100μmol/L处理24h的细胞增殖抑制率分别为(0.68±0.05)%、(2.76±0.35)%、(12.64±3.90)%和(22.58±2.41)%,F=67.389,P<0.001,处理组与对照组之间差异有统计学意义;48h的增殖抑制率分别为(3.83±1.06)%、(6.23±1.27)%、(15.90±1.10)%和(24.50±2.51)%,F=145.738,P<0.001,处理组与对照组之间差异有统计学意义;72h的增殖抑制率分别为(8.78�OBJECTIVE DBO（6,8-dichloro-2,3-dihydro-3-hydroxymethyl-1,4-benzoxazine）is one of rapamycin target protein（mTOR）inhibitors that is able to induce autophagy and apoptosis in human umbilical vein endothelial cells.But it is not clear whether DBO has the same effect on leukemia cells.This study was to investigate the effect of DBO on proliferation,autophagy and apoptosis of human chronic myeloid leukemia K562 cells.METHODS K562 cells were revived and cultured by conventional method.The control group（DMSO）and DBO treatment group were set up.Induced by DBO for 24,48 and 72 h,the protein expression of LC3 was detected by Western blotting.The expression of LC3 spots was detected by cell immunofluorescence assay.Autophagy was observed by transmission electron microscopy.Cell viability and proliferation were analyzed using CCK-8 colorimetry.The ability of K562 cell division was detected by cell cycle assay.The cell apoptosis was tested by Annexin ⅤFITC/PI double staining flow cytometry.RESULTS Western blot showed that after treated with rapamycin,the protein expression of LC3-Ⅱ in K562 cells increased and that of p62 reduced.After treated with 50μmol/L DBO for 24,48 and 72 h,the protein expression of LC3-Ⅱ and p62 in K562 cells is time-dependently similar to that of rapamycin treatment.Treated with different concentrations of DBO（10,25,50μmol/L）,the protein expression of LC3-Ⅱ in K562 cells increased dose-dependently,and that of p62 decreased dose-dependently.While the autophagy inhibitor 3-MA is used,LC3-Ⅱ protein expression is reduced and p62 expression is increased.Cell immunofluorescence experiments showed that compared with the control group,the expression of LC3 spots in the DBO-treated group was significantly higher after treated with DBO for 72 h.Transmission electron microscopy showed that many autophagosome and autolysosome,irregular nucleus,marginalized chromatin appeared in the DBO-treated cells cytoplasm.CCK8 results showed that DBO inhibited the proliferation of K562 cells in a dos

关 键 词：DBO K562细胞 凋亡 自噬 白血病 

分 类 号：R733.72[医药卫生—肿瘤]