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目的:运用干扰腺病毒沉默THP1细胞中SCARF1基因研究其在体外抗烟曲霉中的作用。方法:用灭活的烟曲霉分生孢子(1×105 CFU/m L)于不同时间点处理THP-1细胞,RT-PCR分别检测SCARF1和TNF-αm RNA的表达;将Ad-si RNA-SCARF1转导细胞24 h后给予烟曲霉孢子刺激24 h,通过RT-PCR法检测细胞中TNF-αm RNA表达,Western blot法检测细胞中SCARF1表达以及NF-κB通路相关信号分子的活性。结果:RT-PCR证实烟曲霉孢子刺激能时间依赖性增强THP1细胞中SCARF1和TNF-α表达;Western法证实与Ad-GFP组比较Ad-si RNA-SCARF1组SCARF1的表达量显著降低(P<0.05),沉默效率为71%;与Ad-GFP组比较,Ad-GFP+Af组NF-κB亚单位p65的磷酸化水平明显升高(P<0.05),在Ad-si RNA-SCARF1+Af组,磷酸化p65的产生明显减少,SCARF1沉默后细胞因子TNF-α的分泌明显减少。结论:烟曲霉孢子刺激能诱导巨噬细胞SCARF1的表达增加,诱导信号分子NF-κB的活化,导致相应的细胞因子分泌增加,从而在巨噬细胞抗烟曲霉中发挥作用。
AIM: To investigate the role of SCARF1 gene in the inhibition of Aspergillus fumigatus in vitro by interfering with the expression of SCARF1 gene in THP1 cells. METHODS: THP-1 cells were treated with inactivated Aspergillus fumigatus conidia (1 × 105 CFU / mL) at different time points. The expression of SCARF1 and TNF-αmRNA were detected by RT-PCR. The Ad-si RNA- SCARF1 transduced cells were stimulated with Aspergillus fumigatus spores for 24 h after 24 h. The expression of TNF-αmRNA was detected by RT-PCR. The expression of SCARF1 and NF-κB pathway-related signal molecules were detected by Western blot. Results: RT-PCR confirmed that Aspergillus fumigatus spores stimulated the expression of SCARF1 and TNF-α in THP1 cells in a time-dependent manner. Western-blot showed that the expression of SCARF1 in Ad-si RNA-SCARF1 group was significantly lower than that in Ad-GFP group 0.05), and the silencing efficiency was 71%. Compared with Ad-GFP group, phosphorylation of NF-κB subunit p65 in Ad-GFP + Af group was significantly increased (P <0.05) Group, phosphorylation of p65 production was significantly reduced, SCARF1 silencing cytokine TNF-α secretion was significantly reduced. CONCLUSION: Aspergillus fumigatus spores can induce the expression of SCARF1 in macrophages and induce the activation of NF-κB, resulting in the increase of corresponding cytokine secretion and thus play a role in the inhibition of A. fumigatus by macrophages.