关键词: 骨髓间充质干细胞, 组蛋白乙酰化酶GCN5, 生物学特性, 免疫共沉淀, 串联质谱

Abstract: Objective The purpose of the experiment is explaining the role of histone acetyltransferase GCN5 in the process of mesenchymal stem cells induced by 5-azacytidine and the regulation mechanism of GCN5 complex. Methods Cell proliferation was evaluated by MTT assay, cell cycle by flow cytometry and expression of P21 by real-time PCR. Then we used co-immunoprecipitation strategy to separate interaction proteins with GCN5 and tandem mass spectrometry to identify interacting proteins. CHIP strategy was used for analyzing binding ability between P21 promoter and GCN5 together with acetylated histones at the GCN5 site on the P21 gene. Results The proportion of G0/G1 phase MSCs and expression of P21 gene reached the highest at 3 days after 5-azaC-induced, then gradually decreasing. Cell proliferation index PI value and the G2/S phase cell ratio were the opposite to the above. GCN5 physically interacted with the following proteins which were categorized as：ATP-dependent chromatin remodeling complex、transcription initiation complex、transcription factor and zinc finger protein. Binding-abilities and levels of acetylation of 5-azaC treated group were higher than that of untreated group. Conclusion High concentration of 5-azaC increases G0/G1 phase arrest for MSCs. G0/G1 phase arrest decreased and cell proliferation increased as the concentration of 5-azaC decreasing. 5-azaC raised the combination of GCN5-protein-complexes and the P21 gene promoter in vitro differentiation of MSCs through regulating G0/G1 phase of cell cycle and cell proliferation.

Key words: Mesenchymal Stem Cells, histone acetyltransferase GCN5, Biological Characteristics, Co-IP, MS/MS