小干扰RNA对大鼠OX40基因表达的沉默作用

Interference of OX40 gene expression induced by small interfering RNA

目的:研究靶向大鼠OX40基因的二聚体小干扰RNA(siRNA-OX40)对靶基因表达的沉默作用。方法:建立并筛选稳定表达OX40基因的293T转染细胞株,用脂质体转染法将体外合成的siRNA-OX40转染上述细胞株,采用实时荧光定量PCR检测靶基因OX40mRNA表达情况。结果:用不同浓度的siRNA-OX40转染细胞48小时,10nmol/L浓度对293T细胞靶基因表达的抑制作用最强[抑制率(68.3±8.7)%],1nmol/L浓度仍有一定的抑制作用[(37.2±4.8)%];25、10nmol/LsiRNA转染293T细胞后,均在24小时内起效[(21.4±3.2)%,(26.8±3.8)%],抑制作用至少能维持72小时[(61.7±8.4)%,(39.6±5.6)%]。结论:siRNA-OX40对293T细胞外源性靶基因表达有较强的特异性沉默作用,siRNA在24小时内起效,48～72小时达高峰,抑制作用至少能维持72小时。本研究结果为下一步在动物或临床进行siRNA干扰试验提供了实验依据。

Objective：To investigate the specific interference of OX40 gene expression induced by RNAi technique in 293T cell lines transfected with rat OX40 gene. Methods： 293T cells were transfected with recombined plasmid pEGFP-N1-GFP/OX40, and the positive cell clones were selected by fluorescence prntein observation and RT-PCR. One specific dicer siRNA targeted to OX40 mRNA was designed and synthesized, which shared no homology with exons of known human gene. Quantitative real-time PCR was performed to measure the inhibitory rate of target gene expression by comparing OX40 mRNA concentrations before and after siRNA transfection. Results： 10 nmol/L siRNA-OX40 elicited the highest level of gene silence in 293T cells which was transfected with siRNA after 48 h （68.3 - 8.7） % ） ; The time of maximal inhibitory effect was at 48-72 h [ （ 61.7 ±8.4 ） %, （ 39.6 ±5.6） % ]. Conclusion： The exogenous OX40 expression can be significantly inhibited by treatment with specific siRNA in a dose and time -dependent manner in 293T cells,which may provide a useful profile for further investigation of inhibition of OX40 protein, and a promising control approach for preventing immune reaction.