摘要目的 探讨nm23-H1沉默对K562细胞向巨核细胞分化的影响.方法 采用Lipofectamine2000将靶向nm23-H1基因的RNA干扰质粒pSileneerTM 4.1-CMV-sinm23及空质粒转染K562细胞,经G418筛选建立该基因稳定下调的K562细胞(K562-sinm23细胞)及空质粒转染K562细胞(K562-siNC细胞),实时定量PCR、免疫组织化学、蛋白印迹反应等方法证实了nm23基因沉默细胞构建成功.NBT还原比色试验检测细胞分化能力.流式细胞术检测在诱导剂佛波酯作用下K562-sinm23细胞表面巨核细胞分化抗原GP Ⅱ b-Ⅲa(CD41)的表达.蛋白印迹法检测细胞在佛波酯诱导后ERK1/2磷酸化活性.结果 与K562细胞和K562-siNC细胞比较,pSilencerTM 4.1-CMV-sinm23能够沉默内源性nm23-H1 mRNA的表达,基因水平和蛋白水平的沉默效率分别达到75%和70%.经佛波酯诱导,与K562-siNC细胞比较K562-sinm细胞的分化能力明显增强(NBT还原能力A值分别为0.23±0.05和0.31±0.07).nm23-H1基冈调控K562细胞向巨核细胞分化与ERK1/2磷酸化活性增强有关.结论 成功构建了nm23-H1基因稳定下调表达的K562细胞株,并且证明nm23-H1参与了K562细胞向巨核细胞系的分化.

abstractsObjective To construct a stable nm23-H1-knock-down cell model with K562 cell 1ine and study its differentiation toward megakaryocyte.Methods Eukaryotic expression vector pSilencerTM 4.1-CMV-sinm23 expressing siRNA targeting nm23-Hl was transfected into K562 cells with lipofectamine2000.Cells with stably nm23-H1 silence were screened out by G4l8.Real-time quantitative PCR.immunocyto-.chemistry.western blot were used to confirm the nm23-H1-knock-down K562 model.Cell differentiation ca-pacity was detected by NBT reduction assay.Surface antigen Gp Ⅱb-Ⅲa (CD41)of knock-down cells trea-ted with phorbol 12-myristate 13-acetate was analyzed by flow cytometry.Western blot was used to detect the ERKl/2 signal pathway after the stimulation of phorbol 12-myristate 13-acetate.Results Endogenous nm23-H1 was silenced by pSilencerTM 4.1-CMV-sinm23 and the silence efficiency was up to 75％and 70％in mRNA and protein Ievels respectively compared with the mock cells.Under phorbol 12-myristate 13-acetate treatment,the knock-down cells displayed a significantly increased differentiation ability toward megakaryocyte compared with control.The NBT reduction values were(0.3 1±0.07)and(0.23±0.05)respectively.Fur-ther results revealed that nm23-H1 gene regulating the megakaryocytic differentiation was due in part to the in-creased ERKl/2 phosphorylation.Conclusions A stable nm23-H1-knock-down K562 cell model iS SUCCESS-fully constructed.nm23-H1 involves in regulating the megakaryocytic differentiation of K562 cell line.