目的:对HL60细胞在NSC67657作用下向单核系分化前后,双向电泳分离的表达差异蛋白β-catenin相关蛋白1(Beta-catenin-interacting protein 1,ICAT)进行验证,并对ICAT在细胞分化中的功能进行研究.方法:通过RT-PCR和Western blot方法验证药物作用细胞前后ICAT基因和蛋白的表达差异;通过免疫荧光协同分析目的蛋白的表达水平,并对其进行初步定位.构建pDsRed-ICAT真核表达载体,转染HL60细胞,筛选阳性克隆.对ICAT基因重组质粒转染细胞作细胞形态学、细胞增殖改变的观察和细胞周期检测以及超微结构观察.结果:NSC67657诱导HL60细胞向单核系分化,ICAT蛋白表达上调,其主要定位于细胞核和胞质.真核表达载体构建成功,电转后G418筛选可得90%以上阳性克隆.转染重组质粒的HL60细胞增殖受抑,电镜下胞核异染色质密集,核质比减小,表面抗原CD14表达和对照组无差异,但在药物处理后24h即可表达71.3%,明显高于对照组,瑞氏染色可见明显分化细胞.结论:ICAT蛋白在NSC67657诱导HL60细胞分化中表达上调,但仅是过表达的ICAT基因并不能诱导HL60细胞向单核系分化,却能提高HL60细胞对NSC67657诱导作用的敏感性. Objective: To verify the differential expression of beta-catenin-interacting protein 1 (ICAT) in HL60 cells by two-dimensional electrophoresis before and after differentiation of mononuclear cells under the action of NSC67657. .Methods: The expression of ICAT gene and protein was tested by RT-PCR and Western blot before and after the drug-induced cells were treated with pDsRed-ICAT, and the expression of the target protein was analyzed by immunofluorescence. The eukaryotic expression vector was transfected into HL60 cells and the positive clones were screened.The changes of cell morphology and cell proliferation of ICAT gene recombinant plasmids were observed and cell cycle detection and ultrastructure were observed.Results: NSC67657 induced HL60 cells to single Nuclear differentiation, ICAT protein expression upregulation, which is mainly located in the nucleus and cytoplasm.Eukaryotic expression vector was constructed successfully, more than 90% positive clones were screened by G418 after electroporation.The proliferation of HL60 cells transfected with the recombinant plasmid was inhibited under electron microscope The nucleus heterochromatin was dense and the ratio of nuclear to cytoplasm was decreased. There was no difference between the expression of surface antigen CD14 and the control group, but it was 71.3% after treatment for 24 h In control group, Wright’s staining showed obvious differentiated cells.Conclusion: ICAT protein was up-regulated in NSC67657-induced HL60 cell differentiation, but only the over-expressed ICAT gene did not induce differentiation of HL60 cells into mononuclear cells, but increased HL60 cells Sensitivity to induction of NSC67657.