可诱导表达AML1-ETO白血病细胞模型的建立及其对白血病细胞脂肪酸代谢的影响  

Establishment of leukemia cell model with inducible AML1-ETO expression and its effect on fatty acid metabolism in leukemia cells

作　　者：谢婉清 杨雪 顾闰夏 田征 邢海燕 唐克晶 饶青 邱少伟 王敏 王建祥 Xie Wanqing;Yang Xue;Gu Runxia;Tian Zheng;Xing Haiyan;Tang Kejing;Rao Qing;QiuShaowei;Wang Min;Wang Jianxiang(State Key Laboratory of Experimental Hematology,National Clinical Research Center for Blood Diseases,Haihe Laboratory of Cell Ecosystem,Institute of Hematology&Blood Diseases Hospital,Chinese Academy of Medical Sciences&Peking Union Medical College,Tianjin 300020,China;Tianjin Institutes of Health Science,Tianjin 301600,China)

机构地区：[1]中国医学科学院血液病医院(中国医学科学院血液学研究所),实验血液学国家重点实验室,国家血液系统疾病临床医学研究中心,细胞生态海河实验室,天津300020 [2]天津医学健康研究院,天津301600

出　　处：《中华血液学杂志》2023年第5期366-372,共7页Chinese Journal of Hematology

基　　金：国家科技部重点研发计划(2021YFC2500300);国家自然科学基金(81830005、82000131);天津市细胞生态海河实验室创新基金(22HHXBSS00032);中国医学科学院创新工程(2021-I2M-1-041);国家血液系统疾病临床医学研究中心临床研究基金(2023NCRCA0101)。

摘　　要：目的通过建立可诱导表达AML1-ETO(AE)融合基因的白血病细胞模型,研究AE融合基因对U937白血病细胞生物学功能的影响。方法利用慢病毒载体系统,构建强力霉素(Dox)依赖的可诱导表达AE融合基因的U937细胞系(U937-AE)。在AE融合基因表达前后,分别采用CCK-8法、流式细胞术进行细胞增殖、周期、诱导分化检测,并进行转录组学测序和代谢组学测序分析,初步探讨AE融合基因对白血病细胞生物学功能的影响。结果①成功构建Dox依赖的Tet-on调控系统,调控AE融合基因在U937-AE细胞中稳定表达。②诱导AE融合基因表达24 h后,表达AE的U937-AE细胞增殖倍率为3.47±0.07,低于AE阴性组的3.86±0.05(P<0.05);处于G0/G1期的细胞比例为(63.45±3.10)%,明显高于AE阴性组的(41.36±9.56)%(P<0.05);表达CD13、CD14的细胞比例较AE阴性组明显下降(P<0.05)。③转录组学测序进行基因集富集分析显示,与静息相关、NF-κB和干扰素α/γ应答相关的炎症反应和免疫调节的基因集被明显富集在表达AE的U937-AE细胞。④U937-AE细胞表达AE融合基因后脂肪酸代谢发生紊乱,AE阳性组细胞的部分中、短链脂肪酸酰基肉碱的代谢物浓度降低[丙酰基-L-肉碱:AE阳性组0.46±0.13,AE阴性组1.00±0.27(P<0.05)];部分长链脂肪酸酰基肉碱的代谢物浓度升高[十四烷酰肉碱:AE阳性组1.26±0.01,AE阴性组1.00±0.05(P<0.05)]。结论成功建立可诱导表达AE融合基因的白血病细胞模型。AE融合基因表达使U937-AE细胞增殖变慢、周期阻滞、分化受抑,炎症反应和免疫调节的相关基因集被明显富集,细胞的脂肪酸代谢发生紊乱。Objective To investigate the effect of the AML1-ETO(AE)fusion gene on the biological function of U937 leukemia cells by establishing a leukemia cell model that induces AE fusion gene expression.Methods The doxycycline(Dox)-dependent expression of the AE fusion gene in the U937 cell line(U937-AE)were established using a lentivirus vector system.The Cell Counting Kit 8 methods,including the PI and sidanilide induction,were used to detect cell proliferation,cell cycle-induced differentiation assays,respectively.The effect of the AE fusion gene on the biological function of U937-AE cells was preliminarily explored using transcriptome sequencing and metabonomic sequencing.Results①The Dox-dependent Tet-on regulatory system was successfully constructed to regulate the stable AE fusion gene expression in U937-AE cells.②Cell proliferation slowed down and the cell proliferation rate with AE expression(3.47±0.07)was lower than AE non-expression(3.86±0.05)after inducing the AE fusion gene expression for 24 h(P<0.05).The proportion of cells in the G0/G1 phase in the cell cycle increased,with AE expression[(63.45±3.10)%)]was higher than AE non-expression[(41.36±9.56)%](P<0.05).The proportion of cells expressing CD13 and CD14 decreased with the expression of AE.The AE negative group is significantly higher than the AE positive group(P<0.05).③The enrichment analysis of the transcriptome sequencing gene set revealed significantly enriched quiescence,nuclear factor kappa-light-chain-enhancer of activated B cells,interferon-α/γ,and other inflammatory response and immune regulation signals after AE expression.④Disorder of fatty acid metabolism of U937-AE cells occurred under the influence of AE.The concentration of the medium and short-chain fatty acid acylcarnitine metabolites decreased in cells with AE expressing,propionyl L-carnitine,wherein those with AE expression(0.46±0.13)were lower than those with AE non-expression(1.00±0.27)(P<0.05).The metabolite concentration of some long-chain fatty acid acylcarnitine inc

关 键 词：白血病细胞 脂肪酸代谢 免疫调节 短链脂肪酸 长链脂肪酸 转录组学 代谢组学 细胞增殖 

分 类 号：R733.7[医药卫生—肿瘤]