Abstract: The purpose of this study was to explore the effect of meningitis-causing Enterococcus faecalis on the function of blood-brain barrier in vitro,and find a stable method to construct the model of blood-brain barrier injury in vitro.One week old ICR mice were used to isolate primary brain microvascular endothelial cells (BMEC),and the BMEC specific coagulation factor Ⅷ(Factor Ⅷ) was used for fluorescent labeling to identify the isolated cells and determine the isolation rate.Primary astrocytes were isolated from ICR mice aged 1-3 days and labeled with glial fibrillary acidic protein (GFAP) to identify the isolated cells and determine the isolation rate.The primary cells were passaged to the third generation,and the blood-brain barrier models of BMEC monolayer and BMEC co-cultured with astrocytes were constructed by Transwell two-dimensional pore.Meningitis-causing Enterococcus faecalis,non meningitis-causing Enterococcus faecalis and E.coli DH5α competent cells were co-cultured with the blood-brain barrier model in vitro to evaluate the penetration of meningitis-causing Enterococcus faecalis to the blood-brain barrier model in vitro.The effect of meningitis-causing Enterococcus faecalis on blood-brain barrier model in vitro was evaluated by 4 h penetration test,sodium fluorescein penetration test and detection of matrix metalloproteinase 2 (MMP-2) expression.The results showed that BMEC and astrocytes were successfully isolated,and the purity were above 90% and 95%,respectively.The penetration test showed that only meningitis-causing Enterococcus faecalis could cross the blood-brain barrier model in vitro.4 h penetration test and fluorescein sodium penetration test showed that the co-culture model was superior to the monolayer culture model of BMEC.Compared with other groups,the penetration of fluorescein sodium was higher and the expression of MMP-2 was significantly increased in the co-culture group.In conclusion,the co-culture model of BMEC and astrocytes was better than BMEC monolayer culture model,meningitis-causing Enterococcus faecalis could cross the blood-brain barrier model in vitro and mediate the blood-brain barrier damage,which provided a theoretical basis for revealing the mechanism of the blood-brain barrier damage induced by meningitis-causing Enterococcus faecalis.

Key words: Enterococcus faecalis; cell isolation and identification; blood-brain barrier in vitro; damage model