初诊急性髓系白血病患者来源的骨髓间充质干细胞通过调节Caspase-3/survivin抑制DNR诱导的HL-60细胞凋亡  

Newly Diagnosed Acute Myeloid Leukemia Patients-Derived Bone Marrow Mesenchymal Stem Cells Suppress Daunorubicin Induced HL-60 Cell Apoptosis via Modulating Caspase-3/Survivin

作　　者：宁红梅 王军 苏永锋 徐晨 扈江伟 楼晓 李秀森 毛宁 陈虎 NING Hong-Mei;WANG Jun;SU Yong-Feng;XU Chen;HU Jiang-Wei;LOU Xiao;LI Xiu-Sen;MAO Ning;CHEN Hu(Department of Hematopoietic Stem Cell Transplantation,The Fifth Medical Center of Chinese PLA General Hospital,Beijing 100071,China;Institute of Military Cognition and Brain Sciences,Academy of Military Medical Sciences,Beijing 100850,China)

机构地区：[1]解放军总医院第五医学中心造血干细胞移植科,北京100071 [2]军事医学研究院军事认知与脑科学研究所,北京100850

出　　处：《中国实验血液学杂志》2019年第6期1736-1743,共8页Journal of Experimental Hematology

基　　金：国家自然科学基金项目(81070435)

摘　　要：目的:探讨急性髓系白血病(AML)患者的骨髓微环境在化疗耐药中的作用,观察骨髓间充质干细胞(MSC)对柔红霉素(DNR)诱导的AML细胞HL-60凋亡的影响,并探索其初步作用机制。方法:分别将健康供者和初诊AML患者来源的骨髓MSC与HL-60细胞共培养,不同的实验组添加或者不添加DNR,采用流式细胞术检测AnnexinⅤ/PI标记的HL-60细胞凋亡;通过瑞氏-吉姆萨染色观察各组HL-60细胞形态,统计原始和分化细胞所占的比例;采用Western blot检测凋亡相关蛋白Caspase-3、Caspase-8、Caspase-9和Survivin的表达情况。结果:流式细胞术显示,健康供者MSC以及初诊AML患者来源MSC分别与HL-60细胞共培养未见HL-60细胞的凋亡情况有显著变化。加入DNR后,HL-60细胞凋亡率为(49.57±7.44)%,健康供者MSC共培养加药组和初诊AML患者来源的MSC共培养加药组凋亡率显著降低,分别为(30.72±4.05)%(P<0.01)和(22.99±4.08)%(P<0.01),但健康供者MSC组与初诊AML患者来源MSC组对DNR诱导HL-60细胞凋亡的抑制作用无统计学差别(P>0.05)。瑞氏-吉姆萨染色结果显示,初诊AML患者来源MSC共培养的HL-60细胞绝大多数处于原始状态,极少见到细胞分化。初诊AML患者来源MSC共培养组中,DNR引起的细胞凋亡和分化明显减少,HL-60细胞大多数处于原始状态。Western blot检测结果表明,初诊AML患者来源MSC和健康供者MSC共培养组的HL-60细胞内Caspase-3活性剪切均较HL-60细胞单独培养组下降,且初诊AML患者来源MSC组显著降低。此外,初诊AML患者来源MSC和健康供者MSC共培养的HL-60细胞Survivin的表达较单独药物作用组表达更高,且初诊AML患者来源MSC组表达显著增高。结论:初诊的AML患者骨髓中存在的MSC与健康人骨髓MSC可以抑制DNR诱导的HL-60细胞凋亡,其作用机制可能与抑制了HL-60细胞内Caspase-3活性,提高了Survivin的表达有关。Objective:To explore the role of bone marrow niche in the chemotherapy resistance of patient with acute myeloid leukemia(AML),and to investigate the effects of the MSCs on the apoptosis of HL-60 cell and its underlying mechanisms.Methods:MSCs were derived from the bone marrow of newly diagnosed AML patients(AML-MSCs)and health donors(MSCs)were co-cultured with HL-60 cells respectively.The apoptosis of HL-60 cells in the presence/absence of MSCs and/or Daunorubicin were determined by flow cytometry with Annexin V/PI double staining.In addition,the morphological features of HL-60 cells were observed by Wright-Giemsa staining,and the ratio of blasts and differentiated cells were counted.Furthermore,the expressions of apo pto sis related factors including Caspase-3,Caspase-8,Caspase-9 and Survivin were detected by Western blot.Results:The flow cytometry showed that there was no significant change in apoptosis of HL-60 cells co-eultured with MSC derived from healthy donors or AML patients.After adding Daunorubicin into different cultural systems,the apoptotic rates of HL-60,HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs were(49.57±7.44)%,(30.72±4.05)%and(22.99±4.08)%,respectively,which showed that normal MSCs and AML-MSCs could remarkably supress Daunorubicin-induced HL-60 apoptosis,however,there was no statistically significant difference of apoptosis between HL-60 co-cultured with normal MSCs and HL-60 co-cultured with AML-MSCs.Wright-Giemsa staining showed that most of the HL-60 cells co-cultured with AML-MSCs were primitive,and cell differentiation was unusual.In AML-MSCs co-cultured group,the cell apoptosis and differentiation caused by DNR was significant decreased,and most of HL-60 cells were initial.Western blot showed that the cleavage activity of Caspase-3 of HL-60 in AML-MSCs and normal MSCs co-cultured group was decreased,compared with HL-60 in single cultured group,moreover,the decrease was significantly in AML-MSC group.Additionally,the expression of survivin in AML-MSCs and no

关 键 词：初诊急性髓系白血病 骨髓微环境 间充质干细胞 HL-60细胞 凋亡 

分 类 号：R733.71[医药卫生—肿瘤] R329.28[医药卫生—临床医学]