RNA干扰抑制小鼠OX40基因表达的实验研究  

Interference of OX40 gene expression induced by small interfering RNA

作　　者：夏仁品[1] 卢实春[1] 武聚山[1] 李宁[1] 郑静 

机构地区：[1]首都医科大学附属北京佑安医院外科,北京100069 [2]上海吉凯基因化学技术有限公司,上海201020

出　　处：《免疫学杂志》2008年第6期674-677,共4页Immunological Journal

基　　金：2007年教育部留学回国人员科研启动基金资助;首都医科大学基础与临床合作基金(2006JL55)资助

摘　　要：目的研究靶向小鼠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OX40 mRNA表达情况。结果用不同浓度的siRNA-OX40转染细胞48h,10nmol/L浓度对293T细胞靶基因表达的抑制作用最强(抑制率76.4%),1nmol/L浓度仍有一定的抑制作用(39.4%);25、10nmol/L siRNA转染293T细胞后,均在24h内起效(24.9%,20.5%),抑制作用至少能维持72h(74.4%,38.8%)。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24h内起效,48～72h达高峰,抑制作用至少能维持72h。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 293 T cell lines transfected with mice OX40 gene. Methods 293 T cells were transfected with recombined plasmid pEGFP-N1-GFP/ OX40, and then the positive cell clones were selected by fluorescence protein observation and RT-PCR. One specific dicer siRNA targeted to OX40 mRNA was designed and synthesized, sharing 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 293 T cells 48 h after siRNA transfection （76.4%） ; the time of maximal inhibitory effect was at 48 - 72 h （74.3 %, 38.8 % ）. Conclusion The exogenous OX40 expression could be significantly inhibited by treatment with specific siRNA in a dose- and time-dependent manner in 293 T cells, which may provide a useful profile for further investigation on inhibition of OX40 protein, and a promising control approach for preventing immune reaction.

关 键 词：OX40 基因沉默 小干扰RNA 

分 类 号：R346[医药卫生—基础医学]