Analysis Of Protein Expression In Retinoic Acid-Induced HL60 Cells By Modified Two-Dimensional Electrophoresis

ObjectivesTo establish the differentiation model that HL60 cells are induced to granulocyte cells treating with All-trans retinoic acid (ATRA), the modified two-dimensional electrophoresis conditions was used to separate the proteins between treated and untreated HL60 cells. The mass spectrometry was used to analyze the differentially expressed proteins, and then search them in database. To provide new experimental evidence for the study of granulocyte differentiation of the HL60 cells in molecular mechanisms.Methods1. HL60 cells were induced treating with All-trans retinoic acid (ATRA). In order to select the appropriate drug concentration and induction time, MTT and flow cytometry are used to detect the HL60cell proliferation and the expression of differentiation antigens CD11b respectively. Cellular chemical staining, transmission electron microscope, Cell cycle and apoptosis detection is used to verify the differentiation of the treated HL60 cells.2. To improve the key conditions which affect two-dimensional gel electrophoresis separation. Primarily through the comparison of different protein extraction methods and the comparison of traditional focusing conditions (50 V 5 h,100 V 2 h,500 V 2 h,1000 V 2 h, 1000～8500 Vlinear voltage rise4 h,8500 V 70000 Vh)and improved focusing conditions(450V 2h,4380V 2h,4380V 1h33min),to select the optimum protein extraction methods and improved focusing conditions.3. the protein of HL60 cell lines could be separated by modified two-dimensional electrophoresis (2-DE),then we use PDQuest software to analyze the differentially expressed protein between treated and untreated HL60 cells, finally the proteins could be identified by matrix-assisted laser desorption - time of flight mass spectrometry (MALDI-TOF).4. Mascot software was used to search for the differentially expressed protein proteins in Swissprot database.Results1. ATRA could inhibit HL60 cell proliferation, and with the increase in drug concentration, the effect of inhibiting the more significant. Treated with 2μM ATRA for five days, there are more than 90% of HL60 cells express antigenCD11b. Cellular chemical staining and transmission electron microscope results also show that ATRA could induce HL60 cells to granulocyte cells. Cell cycle arrest at G0 ~ G1 phase,and there is no obvious apoptosis phenomenon.2. Using the typical protein extraction method and the new isoelectrofocusing method, the number of protein spots increase to 1102±7.40 and the result of protein spots'repeatability is (74.8±6.44)%,which meet the requirement of the study.3. By the analysis of modified 2-DE and PDQuest software, 25 protein spots are detected in untreated cells, while 15 protein spots in treated cells. Some of them are oncogene protein and suppressor gene protein, while some of them are involved in apoptosis, etc.ConclusionsThe model of ATRA-induced HL60 cells to granulocyte differentiation was successfully established. Using the modified two-dimensional electrophoresis conditions, the protein separation was significantly improved, and it has good reproducibility. The increased and reduced expressed proteins of the HL60 cells after induced by ATRA, were involved in the process that HL60 cells was induced to granulocyte differentiation.These proteins help us to understand the molecular mechanism of differentiation.