【摘要】 目的观察可诱导共刺激因子(inducible co-stimulator factor,ICOS)短发夹状RNA(short hairpin RNA,shRNA)对大鼠活化T细胞中ICOS基因的沉默效应,筛选有效干扰靶点。方法设计合成ICOS RNA寡核苷酸片段,经退火、连接等步骤克隆至线性化的pRNAT-U6.1/Neo真核表达载体上,测序鉴定。应用Lipofectamine2000将该质粒转染活化的大鼠T细胞,荧光定量RT-PCR检测ICOS mRNA表达情况,Western blot检测ICOS蛋白表达情况。结果成功构建了3个靶点的重组质粒pRNAT-U6.1/Neo-ICOS-siRNA1、2、3,荧光定量RT-PCR和Western blot显示转染这3种重组质粒的T细胞ICOS mRNA和蛋白表达均较对照组有不同程度的下降,其中1号靶点的干扰效应最为明显。在转染后的T细胞中,其抑制率分别达到了51.3%、78.3%.结论重组质粒pRNAT-U6.1/Neo-ICOS-siRNA能有效抑制大鼠活化T细胞中ICOS基因的表达,并筛选出了有效干扰靶位。RNA干扰技术有望成为研究ICOS在实验性自身免疫性葡萄膜视网膜炎中作用的有效手段。更多还原

【Abstract】 Objective To investigate inhibitory effect of inducible co-stimulator factor (ICOS) short hairpin RNA on the expression of ICOS in activated rat T cell,and to screen suitable interference targets in ICOS gene.Methods Oligonucleotides targeting ICOS were cloned into linearized pRNAT-U6.1/Neo eukaryotic expression vector after annealing.The recombinant plasmids were identified by sequencing.Activated rat T cells were transfected with the recombinant plasmids.The transcription of ICOS gene and the expression of ICOS protein were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.Results DNA sequencing showed that three recombinant plasmids (pRNAT-U6.1/Neo-ICOS-siRNA 1,2,3) were successfully constructed.The transcription of ICOS gene and the expression of ICOS protein were down-regulated by RNA interference.The pRNAT-U6.1/Neo-ICOS-siRNA 1 showed highest interfering efficiency than the others.The inhibition rates of pRNAT-U6.1/Neo-ICOS-siRNA 1 were 51.3% and 78.3% in T cells at mRNA level and at protein level,respectively.Conclusion The recombinant plasmid pRNAT-U6.1/Neo-ICOS-siRNA can effectively suppress the expression of ICOS in activated rat T cells,and the efficient interfering targets in ICOS gene are screened out.RNAi will become an effective tool for searching the function of ICOS relating to experimental autoimmune uveoretinitis.更多还原