慢病毒介导的siRNA靶向CD47基因对人喉癌细胞Hep-2增殖、凋亡影响的实验研究
发布时间：2018-08-21 07:04
【摘要】：目的探究慢病毒载体介导siRNA抑制CD47基因表达后对人喉癌Hep-2细胞体外增殖凋亡的影响，为喉癌的基因治疗提供有效靶点。 方法构建慢病毒载体，将靶向CD47基因的siRNA慢病毒转染至喉癌细胞株Hep-2；用荧光显微镜行Hep-2细胞形态学变化观察；RT-PCR检测喉癌细胞CD47mRNA表达的变化情况；Western blotting检测CD47蛋白表达的变化情况；MTT检测CD47-siRNA基因沉默喉癌Hep-2细胞的生存率；体外吞噬试验检测巨噬细胞对CD47沉默后Hep-2细胞的影响。 结果CD47-siRNA慢病毒转染人喉癌细胞株Hep-2后，荧光显微镜从形态学显示CD47-siRNA能有效抑制喉癌Hep-2细胞增殖，细胞变形明显，体积变小，可见细胞坏死及凋亡；RT-PCR和Western blotting检测：喉癌Hep-2细胞CD47的mRNA和蛋白表达水平显著降低(P0.05)，使CD47mRNA表达减少76%~82%，，CD47蛋白表达减少77%；慢病毒转染细胞在48h后MTT检测细胞生存率明显降低(P0.01)；CD47-siRNA基因沉默Hep-2细胞更易被巨噬细胞所吞噬。 结论慢病毒介导的siRNA干扰CD47基因表达后，明显抑制了喉癌Hep-2细胞CD47的表达，增强了巨噬细胞对CD47-siRNA基因沉默Hep-2细胞的吞噬；CD47-siRNA慢病毒载体介导的基因治疗有希望成为喉癌靶向性CD47基因治疗的新策略，为今后应用RNAi基因治疗提供一定的实验参考。
[Abstract]:Objective To investigate the effect of lentiviral vector-mediated siRNA on proliferation and apoptosis of human laryngeal carcinoma Hep-2 cells in vitro, and to provide an effective target for gene therapy of laryngeal carcinoma.
Methods Lentiviral vector was constructed and transfected into laryngeal cancer cell line Hep-2 with siRNA lentiviruses targeting CD47 gene. The morphological changes of Hep-2 cells were observed by fluorescence microscope; the expression of CD47 mRNA in laryngeal cancer cells was detected by RT-PCR; the expression of CD47 protein was detected by Western blotting; the expression of CD47-siRNA gene was detected by MTT. The survival rate of cancer Hep-2 cells and the effect of macrophages on CD47 silenced Hep-2 cells were detected by phagocytic assay in vitro.
Results CD47-siRNA lentivirus transfected human laryngeal carcinoma cell line Hep-2, fluorescence microscopy showed that CD47-siRNA could effectively inhibit the proliferation of Hep-2 cells, the cell deformation was obvious, the volume became smaller, cell necrosis and apoptosis were observed; RT-PCR and Western blotting detection: the expression of CD47 mRNA and protein in Hep-2 cells were significantly decreased (P The expression of CD47 mRNA and CD47 protein were decreased by 76%~82% and 77% respectively, and the survival rate of the cells transfected with lentiviruses was significantly decreased by MTT assay after 48 hours (P 0.01). Hep-2 cells with CD47-siRNA gene silencing were more susceptible to phagocytosis by macrophages.
Conclusion Lentiviral-mediated siRNA interferes with the expression of CD47 gene and significantly inhibits the expression of CD47 in Hep-2 cells and enhances the phagocytosis of CD47-silenced Hep-2 cells by macrophages. CD47-siRNA lentiviral vector-mediated gene therapy may become a new strategy for targeting CD47 gene therapy in laryngeal cancer, and may be a new strategy for the future application of RNAi-based gene therapy. It provides some experimental reference for treatment.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.65
[Abstract]:Objective To investigate the effect of lentiviral vector-mediated siRNA on proliferation and apoptosis of human laryngeal carcinoma Hep-2 cells in vitro, and to provide an effective target for gene therapy of laryngeal carcinoma.
Methods Lentiviral vector was constructed and transfected into laryngeal cancer cell line Hep-2 with siRNA lentiviruses targeting CD47 gene. The morphological changes of Hep-2 cells were observed by fluorescence microscope; the expression of CD47 mRNA in laryngeal cancer cells was detected by RT-PCR; the expression of CD47 protein was detected by Western blotting; the expression of CD47-siRNA gene was detected by MTT. The survival rate of cancer Hep-2 cells and the effect of macrophages on CD47 silenced Hep-2 cells were detected by phagocytic assay in vitro.
Results CD47-siRNA lentivirus transfected human laryngeal carcinoma cell line Hep-2, fluorescence microscopy showed that CD47-siRNA could effectively inhibit the proliferation of Hep-2 cells, the cell deformation was obvious, the volume became smaller, cell necrosis and apoptosis were observed; RT-PCR and Western blotting detection: the expression of CD47 mRNA and protein in Hep-2 cells were significantly decreased (P The expression of CD47 mRNA and CD47 protein were decreased by 76%~82% and 77% respectively, and the survival rate of the cells transfected with lentiviruses was significantly decreased by MTT assay after 48 hours (P 0.01). Hep-2 cells with CD47-siRNA gene silencing were more susceptible to phagocytosis by macrophages.
Conclusion Lentiviral-mediated siRNA interferes with the expression of CD47 gene and significantly inhibits the expression of CD47 in Hep-2 cells and enhances the phagocytosis of CD47-silenced Hep-2 cells by macrophages. CD47-siRNA lentiviral vector-mediated gene therapy may become a new strategy for targeting CD47 gene therapy in laryngeal cancer, and may be a new strategy for the future application of RNAi-based gene therapy. It provides some experimental reference for treatment.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.65
【参考文献】
相关期刊论文 前9条
1 高树峰;张少容;徐莲;李黎;万俊;杨春平;刘建国;汪美群;刘月辉;;慢病毒介导的siRNA靶向CD47基因对人喉癌细胞Hep-2增殖、凋亡的影响[J];解放军医学杂志;2013年08期
2 熊辉强;王鹤龄;罗t
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