全反式维甲酸诱导HL60细胞分化前后蛋白表达差异分析

Analysis of protein expression in retinoic acid-induced HL60 cells by modified two--dimensional electrophoresis

目的:建立全反式维甲酸(Alltrans-retinoic acid,ATRA)诱导HL60细胞向粒系分化的模型,研究采用改良双向电泳条件对分化前后的细胞全蛋白进行分离分析,寻找差异表达蛋白。方法:采用全反式维甲酸对HL60细胞进行诱导分化。通过MTT、流式细胞技术分析细胞增殖情况和CD11b细胞表面分化抗原的表达,选择最适的药物浓度和诱导时间;再通过细胞化学染色对分化结果进行验证,构建分化模型。采用改良双向电泳分离技术对细胞全蛋白进行分离,运用PDQuest软件分析HL60细胞分化前后蛋白表达差异,并用基质辅助激光解吸-飞行时间质谱(MALDI-TOF)对差异蛋白点进行分析。结果:ATRA明显抑制HL60细胞增殖,并随药物浓度的增加,抑制效果也越显著;2μmol/L的ATRA连续作用HL60细胞5d即有90%以上的细胞有粒系分化标志抗原CD11b的表达。细胞化学染色结果亦表明诱导后的HL60细胞向粒系分化。将分化前后的HL60细胞全蛋白经过改良双向电泳技术分离,PDQuest软件比对,MAIDI-TOF分析,发现有25个蛋白点仅在未分化凝胶谱中找到,有15个蛋白点仅在分化细胞蛋白表达谱中找到。其中有的是癌基因表达蛋白,有的是抑癌基因表达蛋白,还有的则参与凋亡过程等。结论:在选定的药物浓度和作用时间下,ATRA可以诱导HL60细胞向粒系分化。改良双向电泳技术对药物作用前后细胞蛋白的分离,可发现不同于传统双向电泳分离所得的蛋白结果。

Objective： To establish the granulocyte-differentiation model of the HL60 cells which are treated with All-trans retinoic acid （ATRA）, and to use the modified two-dimensional electrophoresis conditions to analyze the differences of protein expression between treated and untreated HL60 cells. Methods： HL60 cells were induced through treatment with All-trans retinoic acid （ATRA）.For selection of the appropriate drug concentration and induction time, MTT and flow cytometry are used to detect the HL60cell proliferation and the expression of differentiation antigens CDllb respectively. Cellular chemical staining was used for the verification of the differentiation of the treated HL60 ceils. The protein of HL60 cell lines could be separated by modified two-dimensional electrophoresis （2-DE）. PDQuest software was used to analyze the different protein expression between treated and untreated HL60 cells.The protein was analyzed by matrix-assisted laser desorption - time of flight mass spectrometry （MALDI-TOF）. Results： ATRA could inhibit HL60 cell proliferation, and with the increase in drug concentration, the effect of inhibiting was more significant. Treated with 2 μ M ATRA for five days, there were more than 90% of HL60 cells expressing antigenCD11b. Cellular chemical staining also showed that ATRA could induce HL60 cells to granulocyte cells. By the analysis of modified 2-DE and PDQuest software, 25 protein spots was detected in untreated cells, while 15 protein spots was promoted Some of them were oncogene protein and suppressor gene protein, while some of others are involved in apoptosis. Conclusion： ATRA could induce HL60 cells to granulocyte cells in selected drug concentration and induction time. Using the modified two-dimensional electropboresis conditions, different protein expression can be found from the traditional two-dimensional electrophoresis.