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-耳鼻咽喉科学专业论文.docxVIP

摘I 摘I要I 万方数据 万方数据 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 CD47 mRNA 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 CD47 mRNA 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。 关键词： CD47；慢病毒载体；RNA 干扰；Hep-2 细胞；基因沉默 II Ab Abstract ABSTRACT Objective To assess the effect of the lentiviral vectors expressing small interfering RNA (siRNA) for CD47 gene knockdown in inhibiting Hep-2 cell growth and apoptosis, to provide an effective target for gene therapy about laryngeal carcinoma. Methods After build lentiviral vector,a siRNA directed against CD47 gene was transfected into Hep-2 cells; Cell morphological changes were observed with fluorescence microscope; The changes of CD47mRNA expression were detected by semi-quantitive RT-PCR; The protein of CD47 was evaluated by means of Western blotting; The effect of suppressing proliferation of Hep-2 cells was detected by MTT assay;phagocytosis test in vitro detectioned macrophages that the influence of Hep-2 cells after CD47 silence. Result After CD47-siRNA lentiviral plasmid transfected into laryngeal carcinoma Hep-2 cells, The morphology result showed the CD47 siRNA to be able effectively to suppress the Hep-2 cell multiplication, the cell deformation was discovered, The tumor cell volume was significantly smaller, the necrosis and apoptosis of tumor cells were found under fluorescence microscope. The results of semi-quantitive RT-P