人Notch4基因转录调控的研究
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摘要
目的研究药物和细胞因子对人Notch4信号通路基因表达及其转录调控的影响,为干预Notch4基因在细胞增殖分化中的作用开拓新的分子药理学途径。
方法采用实时定量PCR方法分析Notch4基因在不同细胞系的表达谱,筛选影响HUVEC和HL-60细胞Notch4基因表达的几种药物和细胞因子。采用MTT法、流式细胞仪、实时定量PCR和Western bolt等分析细胞学和分子生物学技术,观察全反式视黄酸(alltrans retinoic acid, ATRA)和三氧化二砷(Arsenic Trioxide, As)对人早幼粒细胞白血病HL-60细胞增殖、分化和Notch4、Notch1信号通路基因表达的影响。利用PCR技术扩增人脐带血CD34+造血干/祖细胞Notch4启动子,采用基因重组技术构建Notch4启动子绿色荧光蛋白报告基因系统,用流式细胞仪和倒置荧光显微镜观测重组质粒在细胞系的表达规律。
结果①Notch4、Jagged1和Delta4在HUVEC、MCF-10、HK-2、MT-/-、MT-/-CdR、MT-/-CdRev细胞中表达较高,而在HacaT、HacaT-As、HL-60、RWPE-1、RWPE-As和RWPE-Cd细胞中则表达较低。②1μM氢化可的松(Hydrocortisone, Hyd)明显增加HUVEC细胞Notch4 mRNA相对定量的百分率(P < 0.05),500pg/ml肿瘤坏死因子-α(tumor necrosis factor- alpha,TNF-α)减少Notch4 mRNA的相
AIM To investigate the effects of drugs and cytokines on Notch4 signaling and regulation. It will open a new way of molecular pharmacology for regulating the Notch4 gene on cell proliferation and differentiation.
METHODS The Notch4 gene expression was studied by real time RT-PCR, and the effects of drugs and cytokines were screened on Nocth4 gene expression in HUVEC and HL-60 cells. Using analytical cytology and molecular biology, the MTT assay,flow cytometry, real time-RT PCR and western blot, The effects of alltans retinoic acid (ATRA) and arsenic trioxide (As) were investigated on cell proliferation, differentiation and the expression of Notch4 and Notch1 signaling pathways in HL-60 cells. The Notch4 promoter of human cord blood CD34 + hematopoietic stme/progenitor cell was amplified by polymerase chain reaction, and GFP report vectors of Notch4 promoter were reconstructed by genetic recombination technology. Expression methods of recombinant vectors were observed by flow cytometry and fluorescent microscope in fluorescence and bright field in cells transfected with GFP-Notch 4 plasmid.
方法采用实时定量PCR方法分析Notch4基因在不同细胞系的表达谱,筛选影响HUVEC和HL-60细胞Notch4基因表达的几种药物和细胞因子。采用MTT法、流式细胞仪、实时定量PCR和Western bolt等分析细胞学和分子生物学技术,观察全反式视黄酸(alltrans retinoic acid, ATRA)和三氧化二砷(Arsenic Trioxide, As)对人早幼粒细胞白血病HL-60细胞增殖、分化和Notch4、Notch1信号通路基因表达的影响。利用PCR技术扩增人脐带血CD34+造血干/祖细胞Notch4启动子,采用基因重组技术构建Notch4启动子绿色荧光蛋白报告基因系统,用流式细胞仪和倒置荧光显微镜观测重组质粒在细胞系的表达规律。
结果①Notch4、Jagged1和Delta4在HUVEC、MCF-10、HK-2、MT-/-、MT-/-CdR、MT-/-CdRev细胞中表达较高,而在HacaT、HacaT-As、HL-60、RWPE-1、RWPE-As和RWPE-Cd细胞中则表达较低。②1μM氢化可的松(Hydrocortisone, Hyd)明显增加HUVEC细胞Notch4 mRNA相对定量的百分率(P < 0.05),500pg/ml肿瘤坏死因子-α(tumor necrosis factor- alpha,TNF-α)减少Notch4 mRNA的相
AIM To investigate the effects of drugs and cytokines on Notch4 signaling and regulation. It will open a new way of molecular pharmacology for regulating the Notch4 gene on cell proliferation and differentiation.
METHODS The Notch4 gene expression was studied by real time RT-PCR, and the effects of drugs and cytokines were screened on Nocth4 gene expression in HUVEC and HL-60 cells. Using analytical cytology and molecular biology, the MTT assay,flow cytometry, real time-RT PCR and western blot, The effects of alltans retinoic acid (ATRA) and arsenic trioxide (As) were investigated on cell proliferation, differentiation and the expression of Notch4 and Notch1 signaling pathways in HL-60 cells. The Notch4 promoter of human cord blood CD34 + hematopoietic stme/progenitor cell was amplified by polymerase chain reaction, and GFP report vectors of Notch4 promoter were reconstructed by genetic recombination technology. Expression methods of recombinant vectors were observed by flow cytometry and fluorescent microscope in fluorescence and bright field in cells transfected with GFP-Notch 4 plasmid.
引文
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