摘要/Abstract

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，shRNA）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 shRNA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2.2×1011 PFU/mL，最佳MOI为300。LPS刺激后Raw 264.7细胞内IRF3 mRNA较对照组明显增加，核内IRF3 蛋白及Irak1bp1表达也明显增加；IRF3 shRNA腺病毒应用后，细胞对IRF3 mRNA的组成性表达及LPS刺激诱导的IRF3 mRNA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 shRNA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 shRNA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。
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
Abstract:
Objective · To amplify the interferon regulator factor 3 (IRF3) short hairpin RNA (shRNA) virus and investigate the effect of the virus on the nuclear of Irak1bp1 protein in lipopolysaccharide (LPS)-stimulated Raw 264.7 cells. Methods · Adenovirus was amplified in HEK293Tcells and the virus titer was detectedTCID 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 of intracellular IRF3 mRNA was detectedreal-time PCR, and the nuclear of IRF3 and Irak1bp1 protein were detectedWestern blotting. Results · The titer of adenovirus was 2.2×1011 PFU/mL and the best MOI was 300. The 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 of IRF3 at mRNA level and the LPS-induced ofIRF3 were significantly inhibited after transfection of Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA. However, the nuclear constitutive of IRF3 protein was not affectedIRF3 shRNA in the unstimulated state. The of nuclear Irak1bp1 protein was significantly higher than that of the control group. The nuclear constitutive and the LPS-induced of Irak1bp1 protein were not affectedIRF3 shRNA. Conclusion · Transfection of LPS-stimulated Raw 264.7 cells with adenovirus strains carrying IRF3 shRNA could effectively inhibit the ofIRF3, but not affect the nuclear of Irak1bp1 protein.
Key words: interferon regulator factor 3, short hairpin RNA , interleukin-1 receptor associated kinase 1 binding protein 1 (Irak1bp1), lipopolysaccharide (LPS)


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