构建雄黄诱导维甲酸耐药急性早幼粒细胞白血病NB4-R1细胞凋亡的蛋白质双向电泳图谱

Establishment of Two-dimensional Electrophoresis Proteomic Profiles of Retinoid Acid Resistant Human Acute Promyelocytic Leukemia NB4-R1 Cells with Apoptosis Induced by Realgar

目的构建雄黄(四硫化四砷,tetra-arsenic tetra-sulfide,As4S4)诱导急性早幼粒细胞白血病(APL)维甲酸耐药细胞株NB4-R1细胞凋亡前后的差异蛋白质组表达谱。方法 首先应用MTT法、透射电镜、AnnexinV/PI双染法和激光共聚焦显微镜的一系列体外实验定性化和定量化雄黄诱导NB4-R1细胞凋亡的作用;然后应用高分辨二维双向电泳技术构建雄黄诱导维甲酸耐药细胞株NB4-R1凋亡前后的差异蛋白质组表达谱。结果 雄黄对NB4-R1细胞的生长抑制效应呈剂量和时间依赖性,雄黄作用24h半数抑制浓度(IC50)为(24.06±0.19)μmol/L,48h IC50为(9.50±0.13)μmol/L,72h IC50为(6.55±0.03)μmol/L;确定了25μmol/L雄黄作用24h和48h时分别为NB4-R1细胞凋亡主群的早期和凋亡晚期阶段;成功构建了雄黄诱导维甲酸耐药细胞株NB4-R1凋亡前后的差异蛋白质组表达谱,ImageMasterTM2D Platinum图像分析软件分析显示,未处理组(R0)、处理24h组(R24)和处理48h组(R48)的蛋白质组表达图谱分别平均含1069、975和893个点;R24与R0的匹配率为79.94%,R48与R0的匹配率为69.33%,R24与R48的匹配率为71.91%。结论 成功构建雄黄诱导维甲酸耐药细胞株NB4-R1凋亡前后的差异蛋白质组表达谱,为今后从整体上更好地理解雄黄诱导耐药APL细胞凋亡的蛋白质组传导网络以及筛选恶性血液肿瘤新型生物标志物和药物靶标方面奠定了基础。

Objective To establish the comparative proteomic profiles of retinoid acid（RA） resistant human acute promyelocytic leukemia（APL） NB4-R1 cells before and after apoptosis induced by realgar（tetra-arsenic tetra-sulfide,As4S4）.Methods First a serial of assays were performed using MTT,transmission electron microscopy,Annexin V FITC/PI double-stain,flow cytometry and confocal laser scanning microscopy to qualitatively and quantitatively observe the in vitro apoptosis inducing effect of realgar on RA-resistant cells.Then the comparative proteomic profile before and after NB4-R1 apoptosis was established using high-resolution two-dimensional electrophoresis system.Results The inhibition effect of realgar on NB4-R1 cell growth was dose and time dependent.The 24-h 50% inhibiting concentration（IC50） was 24.06±0.19 μmol/L,and the 48-h IC50 9.50±0.13 μmol/L,and 72-h IC50 6.55±0.03 μmol/L,respectively.24 h and 48 h were the early and late phase of major NB4-R1 apoptotic cell populations induced by 25 μmol/L realgar respectively.Differential proteomic profiles before and after realgar induced NB4-R1 apoptosis were successfully established.Averagely 1 069,975 and 893 spots could be detected of the untreated group（R0）,the 24-h treatment group（R24）,and the 48-h treatment group（R48）,respectively by ImageMasterTM 2D Platinum Software.The matching rate between R24 and R0 was 79.94% and that between R48 and R0 69.33%,and that between R24 and R48 71.91%.Conclusion Differential proteomic profiles of realgar induced NB4-R1 apoptosis were successfully established for the first time,which provided a basis for comprehensively understanding the signal transduction of realgar induced apoptosis in RA-resistant APL cells,also for screening new bio-markers and drug targets of hematopoietic malignant tumor.