钩端螺旋体对血管内皮细胞单层的侵袭性研究

Study on invasiveness of leptospires to endothelial monolayers

目的比较不同毒力的钩端螺旋体(钩体)对血管内皮细胞单层的侵袭能力和通透性改变的影响。方法利用Transwell小室和EAhy926细胞构建完整血管内皮细胞单层模型,分别加入钩体有毒株56606v株、减毒株56606 a株、腐生型PatocⅠ株、伤寒沙门菌14028菌株和大肠埃希菌DH5α菌株,作用2、4、8 h后在暗视野显微镜下计数到达Transwell下室细菌的数量,并检测内皮细胞单层对荧光颗粒的通透性改变。56606v株和56606 a株分别与EAhy926细胞共同孵育24 h,流式细胞术Annexin V和PI染色检测内皮细胞凋亡率。透射电镜下观察钩体穿透内皮细胞单层过程中的形态特征。结果感染后2 h,56606v和14028菌株开始在下室出现,而56606 a株和PatocⅠ株到4 h后才出现。随时间的延长,下室的细菌数目持续增加;感染8 h后,56606v株到达下室的数量最多,且明显多于56606 a株和PatocⅠ株(P<0.05)。不同毒力的钩体均未引起内皮细胞单层通透性明显改变;流式细胞术检测表明,钩体56606v株和56606 a株均未引起内皮细胞凋亡;电镜观察显示,钩体穿透内皮细胞单层主要是通过穿透内皮细胞胞体而非细胞间紧密连接处。结论有毒株钩体对内皮细胞单层的穿透能力明显强于减毒株和腐生型钩体;不同毒力的钩体对内皮细胞单层通透性及内皮细胞凋亡无明显影响。

Objective To investigate the invasive ability of leptospires with different virulence to endothelial monolayers and the changes in permeability of endothelial monolayers. Methods Intact endothelial monolayer models were constructed with EAhy926 cells and Transwell chamber,and were infected by Leptospira virulent 56606v strains,attenuated 56606a strains,saprophytic Patoc I strains,Salmonella 14028 strains and E.coli DH5α strains,respectively.The numbers of bacteria translocated into the lower Transwell chamber were counted by dark field microscopy after infection for 2 h,4 h and 8 h,and the permeability of endothelial monolayers to fluorescent particles were detected.56606v strains and 56606a strains were used to infect EAhy926 cells for 24 h,and apoptosis rates were detected by flow cytometry with Annexin V and PI staining.The morphology of the leptospires penetrating the endothelial monolayers were observed by transmission electron microscopy. Results Two hours after infection,56606v strains and 14028 strains began to appear in the lower chamber,and 56606a strains and Patoc I strains were not observed until 4 h after infection.The number of strains in the lower chamber increased with time,and the number of 56606v strains in the lower chamber was much larger than those of 56606a strains and Patoc I strains 8 h after infection(P<0.05).Leptospires with different virulence did not cause significant changes in permeability of endothelial monolayers.No apoptosis was detected in endothelial cells infected by 56606v strains or 56606a strains.Transmission electron microscopy revealed that the leptospires mainly penetrated the cytoplasm of endothelial monolayers instead of the intercellular tight junctions. Conclusion The invasive ability of virulent leptospires is much stronger than both attenuated and saprophytic leptospires.There are no changes in permeability and apoptosis of endothelial monolayers infected by leptospires with different virulence.