CAJ | 학술논문

目的探讨CCAAT增强子结合蛋白α(C/EBPα)在甾醇类新药NSC67657诱导HL60细胞向单核系分化中的作用.方法采用NSC67657诱导HL60细胞分化,流式细胞仪检测细胞表面分化抗原CD14的表达.应用逆转录聚合酶链反应(RT-PCR)和Western blot方法连续检测C/EBPα基因和蛋白在细胞分化前后的表达情况.构建pDsRed-C/EBPα真核表达载体,基因测序后采用电转染技术,将真核表达载体转入HL60细胞中,并进行表达验证.采用四甲基偶氮唑蓝(MTT)法、瑞氏染色和流式细胞技术,观察转染细胞在NSC67657作用前后的增殖和分化情况.结果 10 μmol/L NSC67657可以诱导HL60细胞向单核系分化,连续诱导5 d后,有93.9%的细胞有CD14阳性表达.分化后的HL60细胞中,C/EBPα基因和蛋白表达均先升高后下降,分别在第2天和第3天达到峰值.真核表达载体构建成功,通过电转染和G418筛选,可获得90%以上阳性克隆.C/EBPα高表达可使HL60细胞增殖明显减缓,表面抗原CD11b的表达可达到(50.44±4.92)%.在NSC67657处理的重组质粒转染HL60细胞中,细胞单核系分化受到抑制,保留部分粒系分化.结论 NSC67657诱导HL60细胞向单核系分化不一定通过C/EBPα蛋白的协调作用,但C/EBPα蛋白高表达可以阻止NSC67657诱导HL60细胞向单核系分化的进程.
목적 탐토CCAAT증강자결합단백α(C/EBPα)재치순류신약NSC67657유도HL60세포향단핵계분화중적작용.방법 채용NSC67657유도HL60세포분화,류식세포의검측세포표면분화항원CD14적표체.응용역전록취합매련반응(RT-PCR)화Western blot방법련속검측C/EBPα기인화단백재세포분화전후적표체정황.구건pDsRed-C/EBPα진핵표체재체,기인측서후채용전전염기술,장진핵표체재체전입HL60세포중,병진행표체험증.채용사갑기우담서람(MTT)법、서씨염색화류식세포기술,관찰전염세포재NSC67657작용전후적증식화분화정황.결과 10 μmol/L NSC67657가이유도HL60세포향단핵계분화,련속유도5 d후,유93.9%적세포유CD14양성표체.분화후적HL60세포중,C/EBPα기인화단백표체균선승고후하강,분별재제2천화제3천체도봉치.진핵표체재체구건성공,통과전전염화G418사선,가획득90%이상양성극륭.C/EBPα고표체가사HL60세포증식명현감완,표면항원CD11b적표체가체도(50.44±4.92)%.재NSC67657처리적중조질립전염HL60세포중,세포단핵계분화수도억제,보류부분립계분화.결론 NSC67657유도HL60세포향단핵계분화불일정통과C/EBPα단백적협조작용,단C/EBPα단백고표체가이조지NSC67657유도HL60세포향단핵계분화적진정.
Objective To figure out the function of C/EBPα in the monocytic differentiation of HL60 cells induced by a new steroidal drug NSC67657.Methods 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 α(C/EBPα)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/EBPα overexpression in HL60 cells was assessed by MTT assay,Wright's staining and flow cytometry before and after NSC67657 transfection.Results HL60 cells could be induced into monocytes by 10 μmol/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/EBPα protein.Moreover,in the drug treatment group,transfected cells could not be induced into monocytic differentiation,and their granulocytic differentiation was also inhibited.Conclusion The monocytic differentiation of HL60 cells induced by NSC67657 may not be via the regulation by C/EBPα protein-mediated signal transduction.However,the overexpression of CEBPα may inhibit the process of NSC67657-induced monocytic differentiation in HL60 cells.