Wang Wei-jia, Zhang Xiu-ming, Wang Qian, Wen Dong-mei, Qiu Zong-yin
Department of Laboratory Medicine, Zhongshan People's Hospital, The Affiliated Hospital of Zhongshan University, Zhongshan 528402, China.
Zhonghua Zhong Liu Za Zhi. 2010 Apr;32(4):262-6.
To figure out the function of C/EBPalpha in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.
The differentiation of HL60 cells was induced by NSC67657, and the cell surface antigen CD14 expression was detected by flow cytometry. The gene and protein expressions of CCAAT enhancer binding protein alpha (C/EBPalpha) before and after the induction of cell differentiation were determined by RT-PCR and Western blot. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells, and its expression was verified. The effect of C/EBPalpha overexpression in HL60 cells was assessed by MTT assay, Wright's staining and flow cytometry before and after NSC67657 transfection.
HL60 cells could be induced into monocytes by 10 micromol/L ATRA within 5 days, and the coverage of CD14 positive cells reached 93.9% after 5 days of drug treatment. The eukaryotic expressing vector was successfully constructed, and over 90% positive clones were obtained after screening by G418 and electrotransfection. The results of proliferative analysis, chemical staining, ultrastructural observation, and CD11b detection confirmed that HL60 cells could be induced into granulocytic differentiation by overexpression of C/EBPalpha protein. Moreover, in the drug treatment group, transfected cells could not be induced into monocytic differentiation, and their granulocytic differentiation was also inhibited.
The monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPalpha protein-mediated signal transduction. However, the overexpression of CEBPalpha may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.
明确新型甾体药物NSC67657诱导HL60细胞向单核细胞分化过程中C/EBPα的作用。
用NSC67657诱导HL60细胞分化，采用流式细胞术检测细胞表面抗原CD14的表达。通过RT-PCR和蛋白质免疫印迹法检测细胞分化诱导前后CCAAT增强子结合蛋白α（C/EBPα）的基因和蛋白表达。构建真核表达载体pDsRed-ICAT并转染至HL60细胞，验证其表达。在转染NSC67657前后，通过MTT法、瑞氏染色和流式细胞术评估HL60细胞中C/EBPα过表达的影响。
10 μmol/L全反式维甲酸（ATRA）可在5天内将HL60细胞诱导为单核细胞，药物处理5天后CD14阳性细胞覆盖率达93.9%。成功构建真核表达载体，经G418筛选和电转染后获得90%以上的阳性克隆。增殖分析、化学染色、超微结构观察及CD11b检测结果证实，C/EBPα蛋白过表达可诱导HL60细胞向粒细胞分化。此外，在药物处理组中，转染细胞无法被诱导为单核细胞分化，其粒细胞分化也受到抑制。
NSC67657诱导HL60细胞向单核细胞分化可能并非通过C/EBPα蛋白介导的信号转导调控。然而，C/EBPα的过表达可能抑制NSC67657诱导HL60细胞向单核细胞分化的过程。
