南方医科大学学报

南方医科大学学报 ›› 2006, Vol. 26 ›› Issue (03): 301-304.

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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

郑磊； 包杰； 王前； 杨红玲； 裘宇容；   

  1. 南方医科大学南方医院检验科； 南方医科大学南方医院检验科 广东广州510515； 广东广州510515；
  • 出版日期:2006-03-20 发布日期:2006-03-20
  • 基金资助:
    广东省自然科学基金(04020404)~~

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

ZHENG Lei, BAO Jie, WANG Qian, YANG Hong-ling,QIU Yu-rong. Laboratory Medicine Center, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China   

  1. 南方医科大学南方医院检验科； 南方医科大学南方医院检验科 广东广州510515； 广东广州510515；
  • Online:2006-03-20 Published:2006-03-20

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摘要： 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。 更多还原

Abstract: 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 AdvantGene. 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 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.

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