K562细胞nm23-H1基因沉默对其向巨核细胞分化的影响  

Influence of nm23-H1 gene silence in K562 cell on its differentiation toward megakaryocyte

作　　者：金琳[1] 刘格[1] 张传海[1] 熊盛[1] 张美英[1] 刘秋英[1] 钱垂文[1] 王一飞[1] 

机构地区：[1]暨南大学生物医药研究开发基地药学院,广州510632

出　　处：《中华血液学杂志》2008年第6期384-387,共4页Chinese Journal of Hematology

基　　金：基金项目：国家自然科学基金（30400071）;博士后科学基金（20060390737）;广东省科技计划（2005850301017）;2005年度人事部留学人员科技活动项目（国人厅发[2005]129号）

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Lipo-fectamine2000将靶向nm23-H1基因的RNA干扰质粒pSilencer^TM 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GPⅡb-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，pSilencer^TM 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基因调控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。Objective To construct a stable nm23-Hl-knock-down cell model with K562 cell line and study its differentiation toward megakaryocyte. Methods Eukaryotic expression vector pSilencer^TM 4.1- CMV-sinm23 expressing siRNA targeting nm23-H1 was transfected into K562 cells with lipofectamine2000. Cells with stably nm23-H1 silence were screened out by G418. Real-time quantitative PCR, immunocyto-chemistry,western blot were used to confirm the nm23-Hl-knock-down K562 model. Cell differentiation capacity was detected by NBT reduction assay. Surface antigen Gp Ⅱ b-Ⅲa （CD41） of knock-down cells treated with phorbol 12-myristate 13-acetate was analyzed by flow cytometry. Western blot was used to detect the ERK1/2 signal pathway after the stimulation of phorbol 12-myristate 13-acetate. Results Endogenous nm23- H1 was silenced by pSilencer^TM 4. 1-CMV-sinm23 and the silence efficiency was up to 75% and 70% in mRNA and protein levels 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 （0.31±0.07 ） and （0.23±0.05 ） respectively. Further results revealed that nm23-H1 gene regulating the megakaryocytic differentiation was due in part to the increased ERK1/2 phosphorylation. Conclusions A stable nm23-Hl-knock-down K562 cell model is success- fully constructed, nm23-H1 involves in regulating the megakaryocytic differentiation of K562 cell line.

关 键 词：基因 NM23-H1 K562细胞 细胞分化 RNA干扰 

分 类 号：R686[医药卫生—骨科学]