【摘要】 目的筛选并分析转染Islet-1慢病毒载体的C3H10T1/2细胞转化为心肌样细胞过程中与Islet-1相互作用的组蛋白乙酰化酶(HATs)和组蛋白去乙酰化酶(HDACs),明确Islet-1在C3H10T1/2细胞分化为心肌样细胞乙酰化调控网络中的关键枢纽作用。方法培养转染Islet-1慢病毒载体的C3H10T1/2细胞,观察细胞形态。免疫荧光和免疫印迹检测Islet-1的表达部位和最高表达时间点。免疫共沉淀与Islet-1结合的蛋白。免疫印迹验证Islet-1相互作用的HATs和HDACs。结果诱导组细胞形态出现心肌样细胞改变。各组Islet-1主要在胞质表达。诱导组Islet-1表达量在诱导后3周最高(0.782±0.015)。诱导组Islet-1表达量显著高于空白对照组和C3H10组(分别为0.819±0.026,0.127±0.006和0.126±0.001)(P<0.05),免疫共沉淀技术可行。与Islet-1相互作用的HATs和HDACs有GCN5、P300/CBP和HDAC4。结论 Islet-1与GCN5、P300/CBP和HDAC4相互作用特异性辅助C3H10T1/2细胞向心肌样细胞分化。更多还原

【Abstract】 Objective To find out which protein domains of histone acetylase(HATs) and histone deacetylase(HDACs) interact with Islet-1 during the differentiation of C3H10T1/2 cells transfected with Islet-1 lentiviral vector into cardiomyocyte-like cells,and to prove the pivotal role of Islet-1 in the network of histone acetylation.MethodsCulture and observe C3H10T1/2 cells transfected with Islet-1 lentiviral vector.The position of Islet-1 was detected by Immunofluorescence cytochemistry.The protein extraction time was determined by Western-blot according to expression levels of Islet-1-immunoprecipitation.The Islet-1-binding protein was precipitated by co-immunoprecipitation(Co-IP).The proteins of HATs and HDACs interacting with Islet-1 were verified by Western-blot.ResultsCells in induction group showed the appearance of cardiomyocyte-like cells.The location of Islet-1 was located in cytoplasm.The expression of protein in induction group was significant in the third week(0.782±0.015) compared with the first 2 weeks.The expression of Islet-1 in induction group was significantly higher than that in blank control group and C3H10 group(0.819±0.026,0.127±0.006 and 0.126±0.001 respectively,P<0.05).The proteins interacting with Islet-1 were GCN5、P300/CBP and HDAC4.Conclusions The synergism of Islet-1 and its interacting proteins such as GCN5、P300/CBP and HDAC4 played an important role in the differentiation of C3H10T1/2 cells into cardiomyocyte-like cells.更多还原