探讨脂多糖诱导的中枢神经系统炎症反应对体外血脑屏障功能的影响

Effect of lipopolysaccharide-induced central nervous system inflammation response on blood-brain barrier function in vitro

目的利用bEND.3小鼠脑微血管内皮细胞及星形胶质细胞建立体外血脑屏障模型,并观察脂多糖(lipopolysaccharide,LPS)诱导的中枢炎症对血脑屏障功能的影响。方法培养小鼠原代脑微血管星形胶质细胞,利用流式细胞术对原代星形胶质细胞标记物胶质纤维酸性蛋白进行细胞纯度鉴定后,将其与bEND.3小鼠脑微血管内皮细胞进行共培养构建体外血脑屏障模型。将小鼠原代星形胶质细胞、bEND.3小鼠脑微血管内皮细胞、共培养的血脑屏障模型均分为对照组和LPS处理组。LPS处理浓度为20μg/ml,处理时间为24 h。通过跨内皮细胞电阻(trans-epithelium electrical resistant,TEER)检测血脑屏障功能,Western blot检测血脑屏障连接蛋白Occludin和ZO-1蛋白含量。结果原代培养的星形胶质细胞呈典型的多突起形态,流式细胞学鉴定细胞纯度为96%。三种细胞分别进行LPS处理后,TEER值均较各自对照组明显下降(P<0.05),Occludin和ZO-1蛋白相对表达量均较各自对照组明显下降(P<0.05)。结论bEND.3小鼠脑微血管内皮细胞和星形胶质细胞共培养可成功建立一种体外血脑屏障模型,LPS诱导的中枢神经系统炎症反应会使血脑屏障完整性受损,破坏其功能。

Objective To establish a blood-brain barrier(BBB)model in vitro using bEND.3 mouse brain microvascular endothelial cells and astrocytes,and observe the effects of central nervous system inflammation induced by lipopolysaccharide(LPS)on blood-brain barrier function.Methods The primary brain microvascular astrocytes of mice were cultured,and their cell purity was identified by flow cytometry using glial fibrillary acidic protein(GFAP)as marker.The primary astrocyte was co-cultured with bEND.3 mouse brain microvascular endothelial cells to establish an in vitro blood-brain barrier model.Then the primary astrocytes,bEND.3 brain microvascular endothelial cells,and co-cultured blood-brain barrier models were divided into control group and LPS treatment group.The LPS treatment group was stimulated with LPS(20μg/ml)for 24 hours.The blood-brain barrier function was measured by trans-epithelium electrical resistant(TEER),and western blot was used to measure the relative expression of Occludin and ZO-1 protein.Results The primary cultured astrocytes had a typical polymorphic morphology,and the purity of the cells identified by flow cytometry was 96%.After treated with LPS,the TEER values of three types of cells were significantly lower than those of the control group(P<0.05),and the relative expression levels of Occludin and ZO-1 proteins were also significantly lower than those of the control group(P<0.05).Conclusion Co-culture of bEND.3 mouse brain microvascular endothelial cells and astrocytes can successfully establish an in vitro blood-brain barrier model.LPS-induced central nervous system inflammation can impair the integrity of the blood-brain barrier and destroy its function.