体外血脑屏障模型的构建及致脑膜炎粪肠球菌对其的影响

doi:10.16431/j.cnki.1671-7236.2021.07.031

摘要/Abstract

摘要： 试验旨在探索致脑膜炎粪肠球菌对体外血脑屏障功能的影响，寻求稳定的构建体外血脑屏障损伤模型的方法。选用1周龄ICR小鼠进行原代脑微血管内皮细胞（BMEC）分离并通过BMEC特有的凝血因子Ⅷ（coagulation factor Ⅷ，Factor Ⅷ）进行荧光标记，鉴定分离的细胞并判定分离率。选用1~3日龄ICR小鼠分离星形胶质细胞并通过其特有的胶质纤维酸性蛋白（glial fibrillary acidic protein，GFAP）对进行荧光标记，鉴定分离的细胞并判定分离率。将原代细胞传至3代，用Transwell二维小孔分别构建单层BMEC和BMEC与星形胶质细胞共培养的血脑屏障模型。选用致脑膜炎粪肠球菌、非致脑膜炎粪肠球菌和大肠杆菌DH5α感受态细胞分别与构建的体外血脑屏障模型共培养，评估致脑膜炎粪肠球菌对体外血脑屏障模型的穿透性。通过4 h渗透试验、荧光素钠通透性试验和血脑屏障损伤相关标志基因基质金属蛋白酶2（MMP-2）表达量的检测，评估致脑膜炎粪肠球菌对体外血脑屏障模型的影响。结果显示，试验成功分离到BMEC和星形胶质细胞，纯度分别达90%和95%以上。细菌穿透试验结果显示，所选的3株细菌只有致脑膜炎粪肠球菌可穿越体外血脑屏障模型。4 h渗透试验和荧光素钠通透性试验结果显示，共培养模型优于单层BMEC模型。与其他组相比，构建的体外血脑屏障与致脑膜炎粪肠球菌共培养组荧光素钠的穿透力更高，MMP-2基因的表达量明显上升。综上，BMEC与星形胶质细胞共培养的模型优于单层BMEC模型，致脑膜炎粪肠球菌可穿越体外血脑屏障模型，并介导体外血脑屏障损伤，本试验结果为揭示致脑膜炎粪肠球菌对体外血脑屏障损伤机制提供了理论依据。

关键词: 粪肠球菌; 细胞分离鉴定; 体外血脑屏障; 损伤模型

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

中图分类号:

S855.1

李益涛, 曹梦园, 陈明杰, 齐亚银. 体外血脑屏障模型的构建及致脑膜炎粪肠球菌对其的影响[J]. 中国畜牧兽医, 2021, 48(7): 2549-2558.

LI Yitao, CAO Mengyuan, CHEN Mingjie, QI Yayin. Establish of a Blood-brain Barrier Model in vitro and the Influence of Meningitis-causing Enterococcus faecalis on the Model[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(7): 2549-2558.

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