HL-60细胞向单核系和粒系分化的比较蛋白质组学研究

Comparative proteomics analysis on differentiation of human promyelocytic leukemia HL-60 cells into granulocyte and monocyte lineages

背景与目的:已有研究发现NSC67657和全反式维甲酸可以诱导HL-60细胞分别向单核系和粒系分化。本研究比较分析不同诱导剂诱导HL-60细胞向单核系和粒系分化前后以及分化中的蛋白表达差异,发现与单核系和粒系分化有关的关键蛋白。方法:分别采用粒系分化诱导剂全反式维甲酸和单核系分化诱导剂NSC67657诱导HL-60细胞分化,MTT法检测细胞增殖情况;流式细胞技术分析细胞表面特异性分化抗原(CD11b/CD14)的表达,根据CD14表达情况计算细胞分化百分率;细胞化学染色对HL-60的两系分化进行验证。改良双向电泳分离技术对HL-60细胞全蛋白进行分离,PDQuest软件分析寻找差异蛋白点,基质辅助激光解吸-飞行时间质谱(MALDI-TOF MS)对蛋白点进行分析,RT-PCR和免疫印迹对差异蛋白ICAT进行验证。结果:全反式维甲酸和NSC67657均抑制HL-60细胞增殖,并分别诱导HL-60细胞向粒系和单核系分化。在2μmol/L ATRA和10μmol/L NSC67657作用5d后,CD11b和CD14的表达均在90%以上,细胞化学染色支持细胞两系分化的结论。蛋白质组学分析表明,HL-60细胞分别向粒系和单核系分化后,有25个差异蛋白点的变化趋势在两系分化中是相同的,有10个差异蛋白点在单核系分化后表达有差异。有15个差异蛋白点在粒系分化后表达有差异;ICAT是其中差异蛋白之一,通过基因和蛋白水平验证,发现ICAT在NSC67657诱导HL-60细胞单核系分化过程中表达升高,而在粒系和非处理细胞中表达无显著性差异。结论:双向电泳/MOLDI-TOF MS对HL-60细胞分化前后细胞全蛋白进行分析,发现了一批与两系分化有关的蛋白分子,为关键蛋白的筛选及其功能研究提供了重要的启示。

Background and Objective： Human promyelocytic leukemia cell line HL-60 has potential to differentiate into granulocytes and monocytes by different inducers, such as NSC67657 and all-trans retinoic acid （ATRA）. However, the mechanism is unclear yet. This study was to induce differentiation of HL-60 cells using ATRA and NSC67657, and compare the protein expression patterns using two-dimensionar electrophoresis （2-DE）. Methods： HL-60 cells were cultured with ATRA and NSC67657 respectively. Cell proliferation was analyzed by MTT assay. Cellular surface specific antigen CD11b/CD14 was detected using flow cytometry （FCM） to assess cell differentiation. The alterations of cell morphology were observed with cellular chemical staining under electron microscope. Total protein was separated by modified 2-DE. The differentially expressed proteins were identified using PD-Quest software and analyzed by MOLDI-TOF MS. ICAT protein with differential expression was verified by reverse transcription-polymerase chain reaction （RT-PCR） and Western blot. Results： The granulocytic and monocytic differentiation of HL-60 cells was induced by ATRA and NSC67657, respectively. The positive rates of both CD11b and CD14 in HL-60 cells were over 90% after 5-day treatment （2 μmol/L ATRA or 10 μmol/L NSC67657）; cell morphology also represented characteristics of differentiation. Proteomic analysis showed that 25 proteins were differentially expressed with the same pattern in both differentiation groups, ten were differentially expressed only in monocytic differentiation group and 15 only in granulocytic differentiation group. Among them, ICAT expression was up-regulated during NSC67657-induced differentiation of HL-60 cells. Conclusion： A batch of differentially expressed proteins has be found by 2-DE in HL-60 cells with granulocytic and monocytic differentiation, which would contribute to the following functional research on related proteins.