Notch4受体激活对K562细胞增殖的影响及机制研究  

Effects of Notch4 receptor activation on proliferation of K562 cells and the mechanism study

作　　者：杨春秀[1] 胡建娥 伞景辉[1] 陈建斌[3] 

机构地区：[1]遵义医学院附属医院血液内科,贵州遵义563099 [2]重庆市三峡中心医院检验科,重庆404100 [3]重庆医科大学附属第一医院血液内科,重庆400016

出　　处：《遵义医学院学报》2017年第2期172-176,共5页Journal of Zunyi Medical University

基　　金：重庆市自然科学基金资助项目(NO:CSTC2009BB5401)

摘　　要：目的研究Notch4受体激活后对慢性髓系白血病细胞株K562增殖的影响及可能的作用机制。方法用脂质体分别将携带Notch4胞内段(ICN4)的质粒pc DNA 3.1-ICN4及空载体质粒pc DNA 3.1转染入K562细胞,用G418筛选出稳定表达ICN4的细胞株。观察K562细胞ICN4转染后的形态变化,MTT法分析细胞增殖水平,RT-PCR法和Western blot法检测Notch4受体、Hes1、Hey1下游靶基因mRNA及蛋白的表达,流式细胞仪检测细胞周期分布。结果筛选出稳定表达ICN4的细胞株K562/ICN4细胞,与对照组及空载体组比较,ICN4转染组K562细胞数量减少,胞膜透亮度减小,核染色质固缩,核浆比例减低;ICN4转染后K562细胞生长受抑(P<0.05),且随时间延长而明显;Notch4受体及Hes1下游靶基因mRNA及蛋白表达水平相似,均较对照组及空载体组表达增强,但Hey1下游靶基因mRNA及蛋白表达水平未见明显变化;细胞周期检测结果示,转染48 h后细胞阻滞于G1期(P<0.05),S期细胞减少(P<0.05)。结论 Notch4受体激活可抑制K562细胞增殖,其机制可能是通过激活下游靶基因Hes1表达,而调控细胞于G1期而实现的。Objective To investigate the effects of activation of Notch4 receptor on cell proliferation and possible mechanisms in human chronic myeloid leukemia cell line K562.Methods The ICN4 expression plasmid pcDNA3.1-ICN4 and the empty vector pcDNA3.1 were individually transfected in K562 cells with LipofectamineTM 2000 and the ICN4 stable expression cell line was screened with G418.The morphology change of K562 cells was observed after trasnsfected with ICN4.The cell proliferation of K562 cells was detected by MTT assays.RT-PCR and Western-blot were used to detect the mRNA and protein expression of Notch4 receptor,Hes1 and Hey1 target genes.The cell cycle distribution was detected by flow cytometry.Results The stable expression of cell line K562/ICN4 cells was screened.Compared with the control group and empty vector transfected group,the cell number of ICN4 transfected K562 cells decreased with nuclear chromatin aggregated.The proliferation of transfected group K562 cells were inhibited （P〈0.05）.With time extending,the cells were significantly inhibited.Both mRNA and protein expression of Notch4 receptor and Hes1 target genes were up-regulated in ICN4 transfected group,while the Hey1 mRNA and protein expression level showed no significant change.After transfected with ICN4 for 48 h,the cell cycle was blocked in G1 phase （P〈0.05）,while S phase cells were reduced （P〈0.05）.Conclusion The Notch4 receptor activation could inhibit K562 cells proliferation by up-regulating the target gene Hes1 expression and arresting cells at G1 phase.

关 键 词：NOTCH4 K562细胞 基因转染 靶基因 

分 类 号：R733.72[医药卫生—肿瘤]