CAJ | 학술논문

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.
목적 탐토nm23-H1침묵대K562세포향거핵세포분화적영향.방법 채용Lipofectamine2000장파향nm23-H1기인적RNA간우질립pSileneerTM 4.1-CMV-sinm23급공질립전염K562세포,경G418사선건립해기인은정하조적K562세포(K562-sinm23세포)급공질립전염K562세포(K562-siNC세포),실시정량PCR、면역조직화학、단백인적반응등방법증실료nm23기인침묵세포구건성공.NBT환원비색시험검측세포분화능력.류식세포술검측재유도제불파지작용하K562-sinm23세포표면거핵세포분화항원GP Ⅱ b-Ⅲa(CD41)적표체.단백인적법검측세포재불파지유도후ERK1/2린산화활성.결과 여K562세포화K562-siNC세포비교,pSilencerTM 4.1-CMV-sinm23능구침묵내원성nm23-H1 mRNA적표체,기인수평화단백수평적침묵효솔분별체도75%화70%.경불파지유도,여K562-siNC세포비교K562-sinm세포적분화능력명현증강(NBT환원능력A치분별위0.23±0.05화0.31±0.07).nm23-H1기강조공K562세포향거핵세포분화여ERK1/2린산화활성증강유관.결론 성공구건료nm23-H1기인은정하조표체적K562세포주,병차증명nm23-H1삼여료K562세포향거핵세포계적분화.
Objective 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.