靶向CXC趋化因子受体1/2联合Ara-C对急性髓系白血病U937细胞恶性生物学行为的影响与调控机制  

The Effects and Regulatory Mechanism of Targeting CXC Chemokine Receptor 1/2 Combined with Ara-C on the Malignant Biological Behaviors of U937 Cells of Acute Myeloid Leukemia

作　　者：刘彦权 沈建箴[2] 殷悦[2] 陈玉婷 杨慧 唐焕文 LIU Yan-Quan;SHEN Jian-Zhen;YIN Yue;CHEN Yu-Ting;YANG Hui;TANG Huan-Wen(Department of Hematology,The First Clinical Medical College of Guangdong Medical University,Dongguan Key Laboratory of Environmental Medicine,Dongguan 523808,Guangdong Province,China;Institute of Hematology,Fujian Medical University Union Hospital,Fuzhou 350001,Fujian Province,China)

机构地区：[1]广东医科大学第一临床医学院血液内科,东莞市环境医学重点实验室,广东东莞523808 [2]福建医科大学附属协和医院血液病研究所,福建福州350001

出　　处：《中国实验血液学杂志》2023年第2期364-376,共13页Journal of Experimental Hematology

基　　金：国家自然科学基金面上项目(82073582);广东省教育厅高校特色创新项目(2020KTSCX048);广东医科大学学科建设项目(4SG22003G、4SG21209G);广东医科大学科技创新专项计划项目(4SG22046G)。

摘　　要：目的:探讨CXC趋化因子受体1/2(CXCR1/2)靶向抑制剂Reparixin联合Ara-C对急性髓系白血病(AML)细胞恶性生物学行为的作用及对CXCR家族表达的影响,并探究其间存在的分子机制,为AML新型分子标记物和靶向治疗提供科学依据与参考。方法:不同浓度Reparixin、Ara-C单药或联合干预AML U937细胞,倒置显微镜下观察细胞形态;Wright-Giemsa法检测细胞形态学改变;CCK-8法检测细胞增殖;Transwell小室法检测细胞侵袭能力;集落形成实验检测细胞克隆形成能力;Hoechst 33258法、Annexin V/PI双染流式细胞术检测细胞凋亡;单丹黄酰尸胺(MDC)荧光示踪染色法检测细胞自噬;Western blot检测凋亡、自噬及相关信号通路蛋白的表达情况;实时荧光定量聚合酶链式反应(qRT-PCR)检测CXCR家族的表达变化。结果:Reparixin可抑制U937细胞增殖、侵袭、迁移及克隆形成能力并促发其凋亡。与单药组相比,Reparixin联合Ara-C干预U937细胞时,增殖、侵袭、集落形成等恶性生物学行为能力显著下降,凋亡和自噬水平显著升高(P<0.01)。Reparixin联合Ara-C干预U937细胞后,可上调促凋亡蛋白Bax表达并显著下调抗凋亡蛋白Bcl-2的表达,同时亦使Caspase-3水解活化,继而诱发细胞凋亡。Reparixin联合Ara-C干预U937细胞后可上调LC3Ⅱ、Beclin-1蛋白的表达,且细胞中LC3Ⅱ/LC3Ⅰ比值较单药或对照组明显上调(P<0.01),MDC荧光示踪法亦示细胞中酸性囊泡绿色颗粒显著增多,且出现大量破碎细胞(P<0.01)。Reparixin联合Ara-C可显著抑制PI3K、AKT及NF-кB信号分子的磷酸化水平,通过抑制PI3K/AKT/NF-кB通路激活从而扼制白血病细胞恶性生物学行为,并诱导细胞发生程序性死亡。Ara-C干预U937细胞对CXCR家族受体表达基本无影响(P>0.05),利用Reparixin单药干预U937细胞后可下调CXCR1、CXCR2、CXCR4 mRNA的表达(P<0.05),其中CXCR2表达相较对照组及其他CXCR家族受体表达下调水平更为显著(P<0.01);当ReparObjective:To investigate and analyze the effect of CXC chemokine receptor 1/2(CXCR1/2)targeting inhibitor Reparixin combined with cytarabine(Ara-C)on the malignant biological behaviors of acute myeloid leukemia cells and its effect on the expression of the CXCR family,while exploring the accompanying molecular mechanism,providing scientific basis and reference for new molecular markers and targeted therapy for AML.Methods:Acute myeloid leukemia U937 cells were treated with different concentrations of Reparixin,Ara-C alone or in combination,and the cell morphology was observed under an inverted microscope;Wright-Giemsa staining was used to detect cell morphological changes;CCK-8 method was used to detect cell proliferation;the ability of cell invasion was detected by Transwell chamber method;the ability of colony formation was detected by colony formation assay;cell apoptosis was detected by Hoechst 33258 fluorescent staining and Annexin V/PI double-staining flow cytometry;monodansylcadaverine(MDC)staining was used to detect cell autophagy;the expression of apoptosis,autophagy and related signaling pathway proteins was detected by Western blot and the expression changes of CXCR family were detected by real-time quantitative polymerase chain reaction(qRT-PCR).Results:Reparixin could inhibit the proliferation,invasion,migration and clone formation ability of U937 cells.Compared with the single drug group,when U937 cells were intervened by Reparixin combined with Ara-C,the malignant biological behaviors such as proliferation,invasion and colony formation were significantly decreased,and the levels of apoptosis and autophagy were significantly increased(P<0.01).After Reparixin combined with Ara-C intervenes in U937 cells,it can up-regulate the expression of the pro-apoptotic protein Bax and significantly down-regulate the expression of the anti-apoptotic protein Bcl-2,and also hydrolyze and activate Caspase-3,thereby inducing cell apoptosis.Reparixin combined with Ara-C could up-regulate the expressions of LC3Ⅱand B

关 键 词：急性髓系白血病 CXC趋化因子受体 恶性生物学行为 调控机制 

分 类 号：R733.[医药卫生—肿瘤]