【摘要】：目的探究慢病毒載體介導siRNA抑制CD47基因表達后對人喉癌Hep-2細胞體外增殖凋亡的影響，為喉癌的基因治療提供有效靶點。 方法構建慢病毒載體，將靶向CD47基因的siRNA慢病毒轉染至喉癌細胞株Hep-2；用熒光顯微鏡行Hep-2細胞形態(tài)學變化觀察；RT-PCR檢測喉癌細胞CD47mRNA表達的變化情況；Western blotting檢測CD47蛋白表達的變化情況；MTT檢測CD47-siRNA基因沉默喉癌Hep-2細胞的生存率；體外吞噬試驗檢測巨噬細胞對CD47沉默后Hep-2細胞的影響。 結果CD47-siRNA慢病毒轉染人喉癌細胞株Hep-2后，熒光顯微鏡從形態(tài)學顯示CD47-siRNA能有效抑制喉癌Hep-2細胞增殖，細胞變形明顯，體積變小，可見細胞壞死及凋亡；RT-PCR和Western blotting檢測：喉癌Hep-2細胞CD47的mRNA和蛋白表達水平顯著降低(P0.05)，使CD47mRNA表達減少76%~82%，，CD47蛋白表達減少77%；慢病毒轉染細胞在48h后MTT檢測細胞生存率明顯降低(P0.01)；CD47-siRNA基因沉默Hep-2細胞更易被巨噬細胞所吞噬。 結論慢病毒介導的siRNA干擾CD47基因表達后，明顯抑制了喉癌Hep-2細胞CD47的表達，增強了巨噬細胞對CD47-siRNA基因沉默Hep-2細胞的吞噬；CD47-siRNA慢病毒載體介導的基因治療有希望成為喉癌靶向性CD47基因治療的新策略，為今后應用RNAi基因治療提供一定的實驗參考。
[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.
【學位授予單位】：南昌大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R739.65

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相關期刊論文 前9條

1 高樹峰;張少容;徐蓮;李黎;萬俊;楊春平;劉建國;汪美群;劉月輝;;慢病毒介導的siRNA靶向CD47基因對人喉癌細胞Hep-2增殖、凋亡的影響[J];解放軍醫(yī)學雜志;2013年08期

2 熊輝強;王鶴齡;羅t

本文編號：2194917