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-catonin-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-PCB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.
Variant expression verification and functional research of ICAT protein before and after monocytic differentiation of HL60 cells
AIM: To verify the different expressing protein Beta-catenin-interacting protein 1 (ICAT) when monocytic differentiation was induced by NSC67657 on HL60 cells, and to research the role of ICAT protein in the differentiation. METHODS: The expression pattern of ICAT gene and protein were observed by RT-PCR and Western Blot when HL60 cells were treated by NSC67657. The different expression and location of ICAT protein were analyzed by immunofluorescence. Eukaryotic expressing vector pDsRed-ICAT was constructed and transfected into HL60 cells. The positive clones were screened. The morphology, proliferation , cellular cycle and ultrastructure of HL60 cells were investigated. RESULTS: The expression level of ICAT protein was promoted when HL60 cell line was induced by NSC67657 to differentiate into monocytic cells, and it mainly located in the nucleus and cytoplasm. The eukaryotic expressing vector was successfully constructed. G418 screening displayed more than 90% positive clones following electro-transfection. The proliferation of transfected HL60 cells was inhibited; heterochromatin was intensive under the electron microscope; karyoplasmic ratio was reduced. The expression of cellular surface antigen CD14 was not different between transfected cells and control cells. However,the expression of CD14 was significantly increased up to 71.3% when transfected HL60 cells were treated by NSC67657 for 24 h, significantly higher than that in control cells. The differentiated cells were easily found through Wright's staining. CONCLUSION: The expression of ICAT protein is promoted when HL60 cell line is treated by NSC67657. But only overexpression of ICAT protein could not induce the differentiation of HL60 cells into monocyte, but could promote the sensitivity of HL60 cells to NSC67657 treatment.
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