【作者】 夏仁品； 武聚山； 卢实春； 李宁； 陈永兵； 朱俊萍； 丁惠国；

【Author】 Xia ren pin Wu ju shan Lu shi chun Li ning Cheng yong bing Zhu jun ping Ding hui guo, Department of bioclogy and Immunology , Department of Surgery , Beijing You-An Hospital, Capital university of medical sciences , Department of biology and Immunology , Capital university of medical sciences, Beijing 100069.

【机构】 首都医科大学附属北京佑安医院肝胆外科； 首都医科大学微生物及免疫学系；

【摘要】 目的:研究小分子干扰RNA(siRNA)抑制293T细胞中OX40基因表达的可行性。方法: 根据大鼠OX40cDNA已知序列,设计并化学合成一条含19个核甘酸siRNA。将此siRNA包装入质粒载体后,转化质粒到大肠杆菌中,经克隆,扩增,纯化。应用Lipofectamine 2000将OX40 基因表达载体与其相应siRNA(OX40-siRNA)质粒共转染293T细胞,观察siRNA载体对OX40基因的抑制效应。在转染后48h用实时定量PCR从基因转录水平检测OX40基因的表达率,并以 western blot在蛋白水平检查293T细胞中OX40蛋白的表达水平。结果:siRNA转染293T细胞 28h后,OX40靶mRNA及OX40蛋白的表达水平明显下降;且siRNA所介导的抑制效应与干扰载体剂最大小有关。结论:siRNA能抑制293T细胞中OX40基因表达,从而为siRNA作为一种可能的治疗手段提供了理论依据。更多还原

【Abstract】 Objective To illustrate the feasibility of gene silence of Ox40 triggered by interfering RNA in 293 T cells .Methods One siRNA specifically targeted OX40 gene were designed and synthesized by transcription in vitro. The siRNA was inserted into pLVTH vector and the plasmid was be screened into E.Coli .After a whole night, the positive clone , the plasmid was obtained , purified and sequenced to determine whether the ligation between The siRNA insert and the vector was correct. If the sequence was correct, the purified plasmid and OX40-pLVTH plasmid was co-transfected into 293 T cells.The expression rate of OX40 was measured by real-time relative quantitative PCR and western blot. Results The expression level of OX40mRNA treated by siRNA for 48 hours was decreased from 98.5% to 27.3%. Besides, the gene silence of siRNA is relative to dose of siRNA .Conclusion siRNA can silence OX40 gene in 293 T cells, may become a new effective medical technique.更多还原