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

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DCReIB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。

Identification of effective siRNA-mediated RelB silencing in murine dendritic cells by siRNA cassette

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 of RelB 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% CO(2) at 37 degrees C, 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 of RelB 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.