摘要
本研究旨在探讨Notch1信号通路的配体delta-like ligand 4(DLL4)基因过表达对K562细胞增殖的影响及其可能的作用机制。采用脂质体介导将含配体DLL4基因的质粒pBudCE4.1-DLL4转染K562细胞,通过RTPCR和Western blot检测转染48 h后DLL4基因的mRNA及蛋白表达,同样检测转染后Notch1受体胞内区(NICD)、下游靶基因Hes1的mRNA及蛋白表达;通过Western blot检测与Notch信号通路相关的细胞转录因子YY1、原癌基因C-MYC及抑癌基因Rb蛋白的表达水平;用CCK-8法检测转染后细胞的增殖情况;用流式细胞术检测转染质粒48 h后各组细胞的凋亡情况。结果表明,DLL4基因转染后的K562细胞中DLL4、NICD及下游靶基因Hes1的mRNA及蛋白表达量比对照组明显增多(P<0.05),说明DLL4的过表达激活了Notch1信号通路;Western blot方法检测显示,DLL4的转染增加了细胞转录因子YY1、原癌基因C-MYC及抑癌基因Rb蛋白的表达,从而抑制了K562细胞的生长,诱导细胞周期停滞于G1期及细胞凋亡的增加。结论:外源性DLL4基因过表达可有效活化K562细胞内Notch1信号通路,可能通过YY1、C-MYC及Rb等Notch信号通路相关基因的高表达诱导K562细胞的生长减慢及细胞凋亡。
This study was aimed to explore the effect of DLL4/Notch1 ligand on cell growth in leukemia cell line K562 and its relevant mechanism.The pBudCE4.1-DLL4 plasmid was transfected into K562 cells by lipofectamine 2 000,RT-PCR and Western blot were applied to monitor the mRNA and the protein expression of exogenous DLL4 gene,as well as the expression of Notchl-ICD and target gene Hesl.Expression levels of Rb,YY1 and C-MYC protein in K562 cells were also detected by Western blot.Cell counting Kit-8 was used to detect the proliferation of K562 cells,and flow cytometry with Annexin V staining was used to detect the cell apoptosis.The results showed that the mRNA and protein expression levels of DLL4,Notch1-ICD and Hesl in cells of experimental group were significantly higher than those of control groups (P 〈 0.05),indicating the successful activation of the Notch1 signaling pathway.The protein expression levels of Rb,YY1 and C-MYC in cells of experimental group significantly increased when compared with that of control group cells (P 〈 0.05).After transfection,the proliferation of K562 cells was obviously inhibited,and apoptosis rate in DLL4-transfected cells was significantly enhanced.DLL4 transfection significantly increased the number of cells in G1 phase and decreased that in S phase.It is concluded that the over-expression of DLL4 ligand gene in K562 cells results in successful activation of the Notch1 signaling pathway,increases expression of Rb,YY1 and CMYC genes,which induces apoptosis and reduces proliferation.
出处
《中国实验血液学杂志》
CAS
CSCD
北大核心
2014年第5期1256-1260,共5页
Journal of Experimental Hematology
基金
福建省高等学校新世纪优秀人才支持计划(NCE7FJ0707)