敲减干扰素调节因子3对脂多糖刺激Raw264.7细胞核内Irak1bp1表达的影响  

Effects of IRF3 knockdown on the nuclear expression of Irak1bp1 protein in LPS-stimulated Raw 264.7 cells

作　　者：谈志丽 王迎迎[1] 钟欢 何玉 施青青[1] 杨雪 徐国荣[1] 刘亮明[1] TAN Zhi-li;WANG Ying-ying;ZHONG Huan;HE Yu;SHI Qing-qing;YANG Xue;XU Guo-rong;LIU Liang-ming(Department of Infection Diseases,Songjiang Hospital Affiliated to First People's Hospital,Shanghai Jiao Tong University School of Medicine,Shanghai 201600,China)

机构地区：[1]上海交通大学附属第一人民医院松江分院感染科,上海201600

出　　处：《上海交通大学学报（医学版）》2018年第6期589-593,共5页Journal of Shanghai Jiao tong University:Medical Science

基　　金：国家自然科学基金(81770612;81070357;30660066);上海市松江区科技攻关项目(16SJGG57)~~

摘　　要：目的·扩增干扰素调节因子3(interferon regulator factor 3,IRF3)短发夹RNA(short hairpin RNA,sh RNA)腺病毒,并研究该病毒对脂多糖(lipopolysaccharide,LPS)刺激诱导Raw264.7细胞核内白介素受体相关激酶1结合蛋白1(interleukin-1 receptor associated kinase 1 binding protein 1,Irak1bp1)表达的影响。方法·IRF3 sh RNA腺病毒的扩增在人胚肾293 T(HEK293T)细胞中进行,并采用TCID 50法测定病毒滴度。Raw 264.7细胞随机分为4组,1组为腺病毒(-)LPS(-),2组为腺病毒(-)LPS(+),3组为腺病毒(+)LPS(-),4组为腺病毒(+)LPS(+)。细胞IRF3基因表达采用real-time PCR方法检测;核内IRF3及Irak1bp1的表达采用Western blotting方法检测。结果·经计算扩增腺病毒滴度为2.2×10^(11) PFU/m L,最佳MOI为300。LPS刺激后Raw 264.7细胞内IRF3 m RNA较对照组明显增加,核内IRF3蛋白及Irak1bp1表达也明显增加;IRF3 sh RNA腺病毒应用后,细胞对IRF3 m RNA的组成性表达及LPS刺激诱导的IRF3 m RNA和核内蛋白质表达均明显受抑,但未刺激状态下IRF3蛋白核内组成性表达无明显影响;IRF3 sh RNA腺病毒应用对细胞静息及LPS刺激诱导的核内Irak1bp1表达并无影响。结论·IRF3 sh RNA腺病毒能够有效抑制LPS刺激诱导的核内IRF3的表达,但并不影响核内Irak1bp1的表达。Objective·To amplify the interferon regulator factor 3(IRF3)short hairpin RNA(shRNA)virus and investigate the effect of the virus on the nuclear expression of Irak1bp1 protein in lipopolysaccharide(LPS)-stimulated Raw 264.7 cells.Methods·Adenovirus was amplified in HEK293T cells and the virus titer was detected by TCID 50 assay.The Raw 264.7 cells were randomly divided into four groups including adenovirus(-)LPS(-)group,adenovirus(-)LPS(+)group,adenovirus(+)LPS(-)group and adenovirus(+)LPS(+)group.The expression of intracellular IRF3 mRNA was detected by real-time PCR,and the nuclear expression of IRF3 and Irak1bp1 protein were detected by Western blotting.Results·The titer of adenovirus was 2.2×1011 PFU/mL and the best MOI was 300.The expression of IRF3 mRNA and nuclear IRF3 protein in LPS-stimulated Raw 264.7 cells were significantly higher than those of the control group.The cellular constitutive expression of IRF3 at mRNA level and the LPS-induced expression of IRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA.However,the nuclear constitutive expression of IRF3 protein was not affected by IRF3 shRNA in the unstimulated state.The expression of nuclear Irak1bp1 protein was significantly higher than that of the control group.The nuclear constitutive expression and the LPS-induced expression of Irak1bp1 protein were not affected by IRF3 shRNA.Conclusion·Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the expression of IRF3,but not affect the nuclear expression of Irak1bp1 protein.

关 键 词：干扰素调节因子3 短发夹RNA腺病毒 白介素受体相关激酶1结合蛋白1(Irak1bp1) 脂多糖 

分 类 号：R575.3[医药卫生—消化系统]