副猪嗜血杆菌对猪气管上皮细胞的黏附作用及其黏附模型的建立

为了探讨副猪嗜血杆菌(Haemophilus parasuis,Hps)与原代培养的猪气管黏膜上皮细胞的相互作用,应用气管支气管结扎灌注酶冷消化法分离气管黏膜上皮细胞,于胶原覆盖的盖玻片上培养,使上皮细胞分化成假复层黏膜纤毛上皮细胞,然后以对数生长期不同Hps含量的菌液感染上皮细胞和猪脐静脉血管内皮细胞,分别作用1、2、34、h后,通过革兰氏染色法观察Hps对细胞的黏附情况。结果表明,Hps可黏附在上皮细胞和内皮细胞表面;Hps对气管上皮细胞有毒性作用,致细胞变性坏死和凋亡;Hps对细胞的黏附力与其在菌液中含量和作用时间有关,以菌落形成单位计,当含量为1.0×108mL-1且感染4 h后,黏附效果最好。高倍显微镜下可见大量Hps黏附于猪气管上皮细胞,只有个别Hps黏附于猪血管内皮细胞表面。说明Hps对猪气管黏膜上皮细胞的黏附能力远强于猪血管内皮细胞,可以利用猪气管黏膜上皮细胞建立副猪嗜血杆菌的黏附细胞模型。

Adhesion of Haemophilus parasuis to Primary Swine Tracheal Epithelial Cells and Establishment of a Cellular Adhesion Model

To explore the interaction of Haemophilus parasuis (Hps) with primary swine tracheal epithelial cell (STEC) , the tracheal epithelium was digested with enzyme by the method of douche for overnight, the tracheal epithelial cells were isolated and cultured on glass collagen coated coverslips. Under these conditions, STECs were proliferated and differentiated into a pseudostratified mucociliary epithelium. STECs and swine umbilical vein endothelical cell (SUVEC) were infected with different concentration Hps respectively. The cell adhesion was examined by Gram staining in STECs after infection 1 h, 2 h, 3 h and 4 h. The results indicated that Hps can attach to STECs and SUVEC;Hps has a toxic effect and can cause cell death or apoptosis on STECs. The cell adhesion of Hps was associated with the bacterial concentration and infection time. The Hps could be screened on the infected cell surface after infection 4 h which the clonal formation unit is 1.0×10⁸ mL^-1. More Hps were examined on the surface of STECs and just few on SUVEC under high power microscope, it suggested that the adhesion ability of Hps was higher to STECs than SUVEC. The pathogenic mechanism of Hps can be studied using this cell adhesion model in future research.