利用小干扰RNA表达框鉴定小鼠骨髓树突状细胞有效沉默RelB基因的实验研究

Identification of effective siRNA sequence for RelB silencing in murine dendritic cells with siRNA cassette

目的构建靶向小鼠RelB基因的小干扰RNA(siRNA)表达框,鉴定针对小鼠骨髓树突状细胞(DC)的RelB基因最有效的siRNA序列。方法利用PCR方法构建3个不同位点的表达框,R1/siRNA、R2/siRNA和R3/siRNA分别位于1027、302和1121位点。利用阳离子脂质体AdvantGene转染小鼠骨髓DC,转染24h后,脂多糖(LPS)刺激DC,用RT-PCR和免疫荧光方法检测DC RelB基因表达的变化。结果经LPS刺激后DC成熟,RelB基因表达显著高于未成熟DC;R1/siRNA和R3/siRNA转染DC后,LPS刺激仍然可以增强RelB基因表达,而R2/siRNA转染DC后,LPS刺激不能增加RelB基因表达。结论R2/siRNA可有效抑制RelB基因表达,有望用于构建新的耐受性DC应用于临床免疫耐受的诱导。

Objective To construct small interfering RNA （siRNA） expression cassette targeting murine RelB gene and identify the most effective siRNA sequence against RelB gene in murine bone marrow-derived dendritic cells （DCs）. Methods Three expression cassettes namely R1/siRNA, R2/siRNA and R3/siRNA targeting the sites 1027, 302 and 1121 ofRelB gene, respectively, were constructed by PCR approach and transfected into cultured murine myeloid DCs by catione liposome Advant- Gene. After incubation for 24 hours in a incubator containing 5% CO2 at 37℃, the DCs were stimulated by lipopolysaccharide （LPS）, and RelB gene expression in DCs were then detected by RT-PCR and immunofluorescence. Results RT-PCR and immunofluorescence assay showed that the expression ofRelB gene in DCs transfected with R2/siRNA could not be upregulated by LPS stimulation, but transfection with R1/siRNA or R3/siRNA failed to produce such effect. Conclusion R2/siRNA is an effective sequence for RelB silencing, and can be a useful means to construct new tolerogenic DC, RNAi RelB DC, for clinical immunotolerance induction.