维甲酸耐药细胞与敏感细胞的差异表达蛋白分析

Analysis of Differential Protein Expression Profile between Retinoic Acid Resistant and Sensitive Acute Promyelocytic Leukemia Cells

背景与目的:维甲酸耐药机制有待进一步的深入研究。蛋白质组学以所有蛋白质为研究对象,从细胞水平及整体水平研究蛋白质的组成及其变化规律。本实验应用蛋白组学技术整体比较维甲酸耐药细胞与敏感细胞蛋白表达谱,筛选差异表达蛋白,以期获得维甲酸耐药相关蛋白。方法:提取维甲酸耐药细胞株MR2及维甲酸敏感细胞株NB4的总蛋白,通过双向凝胶电泳分离,获得二者高质量的蛋白表达谱,经PDQuestv7.1软件比较分析,筛选出二者间差异表达的蛋白质点,通过质谱仪鉴定表达差异的蛋白。结果:获得了分辨率高、重复性好的APL细胞的双向电泳图谱。PDQuestv7.1软件分析结果表明,MR2细胞及NB4细胞的pH4～7双向凝胶电泳所展示的蛋白点分别有(890±45)个和(912±56)个。二者间有57个差异显著的蛋白点,其中23个在MR2细胞中上调,34个下调。经质谱鉴定了10个蛋白,鉴定成功率达70%,鉴定的蛋白涉及癌蛋白、细胞周期调控和信号转导相关蛋白质。结论:应用双向凝胶电泳技术整体展示了维甲酸耐药细胞株MR2及敏感细胞株NB4的蛋白表达谱,并分析、鉴定、筛选出与维甲酸耐药相关的差异表达蛋白,为进一步从多因素角度研究认识维甲酸耐药机制提供了新的线索。

BACKGROUND ＆ OBJECTIVE： This study was to compare the protein expression profiles between retinoic acid （RA） resistant and sensitive acute promyelocytic leukemia （APL） cells by proteomic research method. METHODS： Total cellular proteins extracted from a RA sensitive cell line NB4 and a RA resistant cell line MR2 were separated by two-dimensional （2D）polyacrylamide gel electrophoresis （PAGE）. High quality 2D-PAGE protein profiles were obtained and analyzed by PDQuest v7.1 analysis software to screen differentially expressed protein spots. Those sports were identified by mass spectrometry. RESULTS： 2-DE patterns of APL cell lines with high-resolution and reproducibility were obtained. The average spots for MR2 and NB4 cells were 890±45 and 912±56, respectively. 57 significantly differentially expressed protein spots were screened, among which 23 protein spots were identified to be up-regulated and 34 down-regulated in MR2 cells compared with NB4 cells. Ten proteins were identified by mass spectrometry, with a successful identification rate of 70%. The identified proteins could be classified into different catogories： oncogenes,cell cycle regulator and signal transducer. CONCLUSION- The utilization of 2D-PAGE is effective in identifying the protein expression profiles between RA resistant and sensitive APL cells, therefore this study may provide a novel clue to elucidate the drug resistant mechanisms of all-trans retinoic acid.