摘要
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 Francisella tularensis LVS infection of mouse macrophages. Methods F. tularensis LVS expressing green fluorescent protein was used to infect murine macrophage J774A. 1 cells. Transferrin receptor 1 （Tfrl） was detected with mono-antibody and visualized with a goat-anti mouse IgG conjugated to Alexa 594. The expression profile of 5 iron metabolism related genes of J774A. 1 murine maerophages uninfected or infected with F. tularensis LVS was determined with real-time PCR. Immunoblot analysis was used to compare the Tfrl expression of live Franciselta infected macrophage with dead bacteria. Tfrl knock-off in J774A. 1 cells was performed with siRNA. The transfected cells were infected with FranciseUa for immunoblotting and microscopy and infection assay. Results It was revealed that the live vaccine strain of F. tularensis induced the expression of Tfrl in host macrophages. Gene expression analysis indicated that F. tularensis INS drove an active iron acquisition program with induction of Tfrl and iron regulatory proteins （Irpl and Irp2）. It was shown by Westenwblotting that the siRNA Tfrc-1 could knock off about 75% of Tfrl in J774A. 1 cells. It was determined by infection assay that, Tfrl was knocked off, the bacteria number at lh infection with Francisella was not different from that of control （F=1. 06, P=0. 326 5）, while it was decreased significantly after 24h of infection （F=24. 12, P=0. 000 6）. Conclusions It is demonstrated that upregulation of the Tfrl may be mediated by post-transcriptional regulation during early infection, but sustained later through increased expression of Irp 1 and Irp 2. Increased expression of Tfrl expands the intracellnlar iron pool through transferrirrmediated delivery and may thus be readily available for uptaking by Francisella. Knocldng off the expression of Tfrl does not affect bacterial invasion. Francisdla, however, may fail to proliferate in macrophages in which the expression of transfe
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2009年第8期932-935,共4页
Medical Journal of Chinese People's Liberation Army
基金
Supported by the Foundation for Supporting Returned Overseas Chinese Scholars,State Education Ministry(2008-890-2)
the National Natural Science Foundation of China(30170891)
