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目的评估土拉弗朗西斯菌LVS感染鼠巨噬细胞期间获取铁的影响因素。方法用表达绿色荧光蛋白(GFP)的土拉弗朗西斯菌LVS感染鼠巨噬细胞J774A.1。结合单抗的转铁蛋白受体-1用键合了Alexa594的羊抗鼠二抗显色。用实时定量PCR法检测5个铁代谢相关基因在土拉弗朗西斯菌LVS感染或未感染J774A.1鼠巨噬细胞中的表达。用活的弗朗西斯菌和灭活菌分别感染巨噬细胞,免疫印迹分析比较Tfr1表达水平。用小干扰RNA下调转铁蛋白受体-1的表达,进而用土拉弗朗西斯菌LVS分别感染转铁蛋白受体-1表达下调的细胞和转染无关siRNA的细胞,并进行细菌计数。结果土拉弗朗西斯菌疫苗株可以诱导转铁蛋白受体-1在宿主巨噬细胞中表达。基因表达分析显示土拉弗朗西斯菌LVS随着时间的增加通过诱导转铁蛋白受体1(Tfr1)和铁调节蛋白(Irp1和IRP2)主动获取铁。免疫印迹结果表明小干扰RNA对转铁蛋白受体-1的表达下调了大约75%。细菌入侵试验显示,在感染1h时,转铁蛋白受体-1表达下调的细胞内细菌数量等同于对照(F=1.06,P=0.3265);而在感染24h时,Tfr1下调样本中的细菌数量明显低于对照样本(F=24.12,P=0.0006)。结论在感染早期Tfr1的上调是由翻译后调节介导的,并随着Irp1和IRP2表达的增加而增加。Tfr1表达的增加通过转铁蛋白介导的铁运输扩充了细胞内动态铁池,使弗朗西斯菌易于获取铁。转铁蛋白受体-1的下调不影响细菌与其他膜蛋白的结合而入侵,但抑制细菌在细胞内的增殖。
Objective To evaluate the influential factors of iron acquisition during the murine macrophages infected by Francisella tularensis LVS. Methods Murine macrophage J774A.1 was infected with L. tularensis LVS expressing green fluorescent protein (GFP). Transferrin receptor-1 binding mAb was developed with goat anti-mouse secondary antibody conjugated to Alexa594. Real - time quantitative PCR was used to detect the expression of five genes related to iron metabolism in the J774A.1 murine macrophages infected with LVS infected or not infected with. Macrophages were infected with viable Francisella and inactivated bacteria, respectively, and the level of Tfr1 expression was compared by immunoblot analysis. The expression of transferrin receptor-1 was downregulated with small interfering RNA, and then the cells transfected with unrelated siRNA were transfected with the down-regulated expression of transferrin receptor-1 and the count of bacteria with Francisella tularensis LVS respectively. Results The F. tulara vaccine strain can induce transferrin receptor-1 expression in host macrophages. Gene expression analysis showed that the F. tularensis LVS actively acquired iron by inducing transferrin receptor 1 (Tfr1) and iron regulatory proteins (Irp1 and IRP2) over time. Immunoblotting results showed that small interfering RNA down-regulated the expression of transferrin receptor-1 by about 75%. Bacterial invasion tests showed that the number of intracellular bacteria that the expression of transferrin receptor-1 was down-regulated at 1 h was equivalent to that of the control (F = 1.06, P = 0.3265); while at 24 h, Tfr1 down-regulated the number of bacteria Significantly lower than the control sample (F = 24.12, P = 0.0006). Conclusion The up-regulation of Tfr1 in early stage of infection is mediated by posttranslational regulation and increases with the expression of Irp1 and IRP2. Increased Tfr1 expression augments the intracellular dynamic iron pool with transferrin-mediated iron transport, making it easier for Francis to acquire iron. The downregulation of transferrin receptor-1 does not affect the invasion of bacteria in combination with other membrane proteins, but inhibits bacterial proliferation in the cell.