具核梭杆菌体外对小鼠巨噬细胞超微形态和机能的影响

Effects of Fusobacterium nucleatum in vitro on ultrastructural morphology and function of mouse macrophages

本研究对具核梭杆菌(F.nucleatum)感染鼠巨噬细胞进行共同培养之后,分别在10个时段采取上清液和巨噬细胞,用ELISA、qRT-PCR法和扫描电镜检测TNF-α、 IL-1α、 IL-6和IL-8细胞因子,并观察巨噬细胞的超微形态变化。结果显示,4种细胞因子均有高水平表达;IL-8在共培养6~72 h呈高水平表达;IL-6和TNF-α表达水平也较高;IL-1从3 h表达,12 h达最高值,48 h后无表达。qRT-PCR检测结果显示,TNF-α的RNA表达呈波浪式,分别于6,60 h各出现1个高峰值;IL-1α和IL-6呈现正态表达,从3 h开始明显表达至60 h达峰值,此后缓慢下降;IL-8从6 h逐渐上升,至72 h仍呈高水平表达。超微形态观察显示,在F.nucleatum感染的前24 h,巨噬细胞呈收缩状,其表面和周围有大量发育良好F.nucleatum,并呈团块状黏附,巨噬细胞逐渐伸出伪足。30 h后F.nucleatum逐渐发育不良,小而细,而巨噬细胞表面粗糙,有许多大小不一的结节,伪足变粗大。48 h巨噬细胞表面出现胞吐孔,少量细小的F.nucleatum仍然黏附于巨噬细胞伪足。此后,发现少量巨噬细胞凋亡。结果表明,F.nucleatum可诱导巨噬细胞产生大量炎性细胞因子,并能长时间与巨噬细胞共生;巨噬细胞能够吞噬F.nucleatum,其胞吐作用可传递F.nucleatum抗原信息。

To investigate the influence of Fusobacterum nucleatum(F.nucleatum) on the morphology and function of mouse macrophage, mouse macrophages was infected with F.nucleatum and they were cultured together, the supernatant and macrophages were taken in 10 periods respectively.TNF-α,IL-1α,IL-6 and IL-8 were detected by ELISA,RNA of macrophages were tested by qRT-PCR,and the ultrastructural changes of macrophage were observed by scanning electron microscopy.The results showed that the four cytokines were expressed at high levels.IL-8 was highly expressed from 6 to 72 h.IL-6 and TNF-α expression levels were also higher.The expression of IL-1α appeared at 3 h and reached the peak at 12 h and no expression was observed after 48 h.qRT-PCR showed that TNF-α RNA expression was wavy mode, with a peak value at 6 and 60 h, respectively.The expression of IL-1α and IL-6 was orthostate, and the expression of IL-6 was obvious from 3 to 60 h, and then decreased slowly.IL-8 expression began at 6 h, then increased gradually, and remained at a high level until 72 h.Ultrastructural morphology showed that 24 h after F.nucleatuminfection,macrophages showed contraction,a large number of well-developed F.nucleatum were on the surface and around them,and adhered in clumps,and macrophages gradually extended pseudopod.30 hlater,F.nucleatum was gradually developed poorly,small and thin.The surface of macrophages was rough,with many nodules of different sizes,and the pseudopods became thicker.48 hlater,exocytosis pores appeared on the surface of macrophages,and a small amount of tiny F.nucleatum still adhered to the pseudopod of macrophages.Thereafter,apoptosis was observed in a small amount of macrophage.In conclusion,F.nucleatum might induce macrophages to produce a large number of inflammatory cytokines and could be symbiosis with macrophages for a long time.Macrophages were able to devour F.nucleatum,and its exocytosis might transmit F.nucleatum antigen information.