【摘要】：目的觀察構(gòu)建的DNA載體介導(dǎo)的小干擾RNA Tet-on基因表達(dá)系統(tǒng)的調(diào)控效應(yīng)。方法構(gòu)建DNA載體介導(dǎo)的小干擾RNA(siRNA)Tet-on表達(dá)載體,pDIRS4.1 siRNA。以增強(qiáng)綠色熒光蛋白(EGFP)基因和白細(xì)胞介素6(IL-6)基因?yàn)槟康幕?應(yīng)用脂質(zhì)體介導(dǎo)的瞬時(shí)轉(zhuǎn)染方法,分別將pDIRS4.1/EGFP siRNA載體和p EGFP-N3質(zhì)粒、pDIRS4.1IL-6siRNA載體和p IRESIL-6質(zhì)粒共轉(zhuǎn)染人成骨肉瘤細(xì)胞,加入系列濃度的四環(huán)素(Dox)后,觀察其誘導(dǎo)效應(yīng)。結(jié)果 pDIRS4.1 siRNA在人成骨肉瘤細(xì)胞中能夠高效轉(zhuǎn)染;隨著Dox濃度升高,pDIRS4.1 siRNA載體在人成骨肉瘤細(xì)胞中對目的基因的沉默作用有較好的調(diào)控性,且目的基因的表達(dá)與Dox具有嚴(yán)格的劑量依賴關(guān)系,高濃度Dox時(shí),IL-6表達(dá)水平降低約12倍。結(jié)論該研究構(gòu)建的以DNA載體介導(dǎo)的能夠高效調(diào)控siRNA沉默基因效應(yīng)的Tet-On基因表達(dá)系統(tǒng),能夠促進(jìn)其在更多模型系統(tǒng)中進(jìn)行基因功能研究的應(yīng)用。
[Abstract]:Objective to investigate the regulatory effects of small interfering RNA Tet-on gene expression system mediated by DNA vector. Methods the expression vector of small interfering RNA (siRNA) Tet-on mediated by DNA vector and pDIRS4.1 siRNA. were constructed. To enhance green fluorescent protein (EGFP) gene and interleukin 6 (IL-6) gene as target genes, pDIRS4.1/EGFP siRNA vector and p EGFP-N3 plasmid were transfected by liposome-mediated transient transfection, respectively. Human osteosarcoma cells were co-transfected with pDIRS4.1IL-6siRNA vector and p IRESIL-6 plasmid. The induction effect was observed by adding a series of tetracycline (Dox) to human osteosarcoma cells. Results pDIRS4.1 siRNA was highly transfected into human osteosarcoma cells. With the increase of Dox concentration, the silencing effect of pDIRS4.1 siRNA vector on target gene in human osteosarcoma cells was better, and the expression of target gene was in strict dose-dependent manner with Dox. The expression level of IL-6 was reduced by about 12 times. Conclusion the Tet-On gene expression system mediated by DNA vector can effectively regulate the gene effect of siRNA silencing, which can promote the application of Tet-On gene function research in more model systems.
【作者單位】： 鄭州大學(xué)第一附屬醫(yī)院藥學(xué)部;
【基金】：河南省優(yōu)秀創(chuàng)新型科技團(tuán)隊(duì)醫(yī)學(xué)科技攻關(guān)計(jì)劃項(xiàng)目(No:201303013)
【分類號】：Q78

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