【摘要】：目的探究慢病毒載體介導(dǎo)siRNA抑制CD47基因表達(dá)后對(duì)人喉癌Hep-2細(xì)胞體外增殖凋亡的影響，為喉癌的基因治療提供有效靶點(diǎn)。 方法構(gòu)建慢病毒載體，將靶向CD47基因的siRNA慢病毒轉(zhuǎn)染至喉癌細(xì)胞株Hep-2；用熒光顯微鏡行Hep-2細(xì)胞形態(tài)學(xué)變化觀察；RT-PCR檢測(cè)喉癌細(xì)胞CD47mRNA表達(dá)的變化情況；Western blotting檢測(cè)CD47蛋白表達(dá)的變化情況；MTT檢測(cè)CD47-siRNA基因沉默喉癌Hep-2細(xì)胞的生存率；體外吞噬試驗(yàn)檢測(cè)巨噬細(xì)胞對(duì)CD47沉默后Hep-2細(xì)胞的影響。 結(jié)果CD47-siRNA慢病毒轉(zhuǎn)染人喉癌細(xì)胞株Hep-2后，熒光顯微鏡從形態(tài)學(xué)顯示CD47-siRNA能有效抑制喉癌Hep-2細(xì)胞增殖，細(xì)胞變形明顯，體積變小，可見細(xì)胞壞死及凋亡；RT-PCR和Western blotting檢測(cè)：喉癌Hep-2細(xì)胞CD47的mRNA和蛋白表達(dá)水平顯著降低(P0.05)，使CD47mRNA表達(dá)減少76%~82%，，CD47蛋白表達(dá)減少77%；慢病毒轉(zhuǎn)染細(xì)胞在48h后MTT檢測(cè)細(xì)胞生存率明顯降低(P0.01)；CD47-siRNA基因沉默Hep-2細(xì)胞更易被巨噬細(xì)胞所吞噬。 結(jié)論慢病毒介導(dǎo)的siRNA干擾CD47基因表達(dá)后，明顯抑制了喉癌Hep-2細(xì)胞CD47的表達(dá)，增強(qiáng)了巨噬細(xì)胞對(duì)CD47-siRNA基因沉默Hep-2細(xì)胞的吞噬；CD47-siRNA慢病毒載體介導(dǎo)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(yīng)用RNAi基因治療提供一定的實(shí)驗(yàn)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(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R739.65

【參考文獻(xiàn)】

相關(guān)期刊論文 前9條

1 高樹峰;張少容;徐蓮;李黎;萬(wàn)俊;楊春平;劉建國(guó);汪美群;劉月輝;;慢病毒介導(dǎo)的siRNA靶向CD47基因?qū)θ撕戆┘?xì)胞Hep-2增殖、凋亡的影響[J];解放軍醫(yī)學(xué)雜志;2013年08期

2 熊輝強(qiáng);王鶴齡;羅t

本文編號(hào)：2194917