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目的人巨细胞病毒(human cytomegalovirus,HCMV)感染可抑制神经干细胞(neural stem cell,NSC)的增殖和分化,Notch信号对NSC的增殖和分化具有重要的调控功能。文中研究HCMV感染对人NSC向星形胶质细胞分化过程中Notch信号相关分子Notch1~3、Jagged(JAG)1、delta-like(DLL)1及早老素(presenilin,PS)1表达的影响。方法体外分离培养海马NSC,并诱导其向星形胶质细胞分化。同时以感染复数(multiplicity of infection,MOI)为5的HCMV AD169株感染NSC,分别在感染后0、1、3、5、7 d收获细胞,用实时RT-PCR法检测细胞内Notch1-3及其受体JAG1、DLL1的转录水平,Westernblot法检测Notch1细胞内段(Notch 1 intracellular domain,NICD)含量的变化。结果未分化状态的NSC含有大量Notch1NICD,Notch1-3、JAG1、DLL1mRNA呈高表达。诱导分化1d后,Notch各受体、配体mRNA表达均显著下降,3 d后表达回升,至第7天显著升高且可检出大量激活的NICD。HCMV感染组细胞在诱导分化1 d后Notch相关受体、配体mRNA水平亦明显下降,与对照组相比Notch1、Notch2和DLL1的表达更少。在以后各个时间点Notch相关受体、配体mRNA水平均低于对照组。PS1的表达仅在1 d时低于对照组。病毒感染7 d后,细胞内NICD含量明显低于对照组。结论HCMV感染可抑制Notch相关受体、配体的表达与活化,Notch信号异常表达参与HCMV感染所致NSC分化和增殖异常。
The target human cytomegalovirus (HCMV) infection can inhibit the proliferation and differentiation of neural stem cells (NSCs), and Notch signaling plays an important regulatory role in the proliferation and differentiation of NSCs. The aim of this study was to investigate the effects of HCMV infection on the expression of Notch1, Jagged1, delta-like (DLL) 1 and presenilin (PS) 1 in human astrocyte during NSC. Methods The hippocampal NSCs were isolated and cultured in vitro and their differentiation into astrocytes was induced. At the same time, NSC was infected with HCMV strain AD169 with a multiplicity of infection (MOI) of 5, and the cells were harvested at 0, 1, 3, 5 and 7 days after infection. Real-time RT- The transcription level of JAG1 and DLL1 was detected by Western blot. The content of Notch1 intracellular domain (NICD) was detected by Western blot. Results The undifferentiated NSCs contained a large number of Notch1NICDs, and Notch1-3, JAG1 and DLL1 mRNA were highly expressed. After induced for 1 day, the mRNA expression of Notch receptors and ligands decreased significantly. After 3 days, the mRNA expression increased and increased significantly on day 7, and a large number of activated NICDs could be detected. Notch1, Notch2 and DLL1 were less expressed in HCMV infected cells than those in control group after 1 day of differentiation. At various time points after Notch related receptors, ligand mRNA levels were lower than the control group. The expression of PS1 was lower than the control group only at 1 d. Seven days after virus infection, the intracellular NICD content was significantly lower than the control group. Conclusion HCMV infection can inhibit the expression and activation of Notch related receptors and ligands, and abnormal expression of Notch is involved in the differentiation and proliferation of NSC caused by HCMV infection.