甾醇类新药NSC67657诱导THP-1细胞分化及ICAT蛋白在细胞分化中的作用研究

目的 研究新的甲磺酸甾醇类药物NSC67657对白血病细胞的诱导分化作用及可能机制.方法 采用MTT法分析在不同浓度NSC67657作用下THP-1细胞的增殖水平,通过细胞表面分化抗原的检测,观察不同药物浓度、不同作用时间处理的THP-1细胞分化程度,并对完全分化细胞做形态学分析.通过RT-PCR和Western blot方法观察药物作用细胞前后β-catenin相关蛋白1(ICAT)基因和蛋白的表达情况;构建pDsRed-ICAT真核表达载体,测序后转染THP-1细胞,筛选阳性克隆,并做表达验证.采用流式细胞术,瑞特染色和超微结构观察,分析重组质粒转染细胞在药物处理前和处理24 h后THP-1细胞的分化情况.结果 通过比较可见药物处理后的THP-1细胞增殖明显受抑;细胞表面分化抗原CD14的表达水平随药物处理时间的延长和药物浓度的升高而增加,结合增殖分析,以10 μmol/L药物、连续诱导5 d为宜,细胞分化可达到90%以上.形态学观察验证了THP-1细胞在NSC67657的作用下向单核系分化.真核表达载体构建成功,电转后THP-1细胞ICAT基因和蛋白表达升高.药物作用前后,重组质粒转染THP-1细胞CD14的表达与对照组比较无明显差异;通过瑞特染色和超微结构观察,发现药物作用重组质粒转染THP-1细胞24 h后,细胞仍处初级分化阶段.结论 NSC67657可诱导THP-1细胞向单核系分化,并激活ICAT基因的表达,但仅是该基因的高表达并不 足以诱导THP-1细胞分化,也不会增加THP-1细胞对NSC67657 药物作用的敏感性.

Study on the mechanism of THP-1 cell differentiation induced by a new steroidal drug NSC67657

Objective To study the potential mechanism of the new steroidal drug NSC67657 induced leukemic cels differentiation.Methods Cell proliferation was assayed by MTT assay.Surface antigen CD14 on THP-1 cells treated by NSC67657 at different time different concentration,was detected by flow cytometry(FCM).The expression of beta-catenin-interacting protein 1(ICAT)gene and protein were detected by RT-PCR and Western blot.Eukaryotic expressing vector pDsRed-ICAT was constructed and transfeeted into HL60 cell line.FCM,Wright's staining and electronmicroscope were employed to analyse the difierentiation of transfected THP-1 cells after they were treated with NSC67657 for 24 hours.Results The proliferation of THP-1 cells was significantly inhibited by NSC67657 treatment.The level of CD14 expression was elerated in line with the increasing drug concentration and treatment time.10 μmol/L NSC67657 treatment for five days was the optimal condition for the induction of THP-1 cells ditierentiation,when the CD14+ THP-1cells were more than 90%.Morphological study indentified the THP-1 cells of monocvtic differentiation.The eukaryotic expressing vector pDSRed-ICAT was successfully constructed,and almost 90%positive clone could be obtained after G418 screening.Electro-transfection was employed for transfecting the vector into THP-1cells.After the transfection the expression of ICAT gene and protein was increased.On the NSC67657 treatment,there was not significant differenee in CD14 expression on transfected THP-1 cells compared to that on the control groups.After 24h treatment,the transfected THP-1 cells remained in early differentiated stage.Conclusion NSC67657 can induce THP-1 cell to monocytic differentiation and activate the expression of ICAT gene,but overexpression of ICAT itself is not sufficient to induce such differentiation.