M1表型的小胶质细胞外泌体对血脑屏障功能的影响

Effects of M1 microglia-derived exosomes on the function of the blood-brain barrier

摘要:

目的 研究M1表型的小胶质细胞外泌体（M1 microglia-derived exosome，M1-exo）对体外血脑屏障（blood-brain barrier，BBB）功能及血管内皮细胞间紧密连接蛋白表达的影响。方法 用脂多糖（lipopolysaccharide，LPS）刺激小鼠小胶质细胞来源的细胞系BV2细胞，流式技术检测其向M1型极化情况，分离提取外泌体。用小鼠脑微血管内皮细胞系b.End3细胞与原代培养的小鼠星形胶质细胞构建体外BBB模型，随机分为3组：b.End3细胞正常培养组（b.End3组）、b.End3细胞+25 μg/mL正常BV2细胞来源外泌体（BV2-derived exosome，BV2-exo）组（b.End3+BV2-exo组）、b.End3细胞+25 μg/mL M1表型的小胶质细胞来源外泌体组（b.End3+M1-exo组）。按实验分组将不同来源外泌体与BBB模型共培养，检测各组的跨膜电阻（trans-endothelial electrical resistance，TEER）、荧光黄通过率，免疫印迹法（Western blot，WB）检测紧密连接复合物蛋白Claudin-1、Occludin、ZO-1及JAM蛋白的表达。结果 ①小胶质细胞向M1型极化成功，其细胞标志物CD16/32阳性率较对照组明显升高（P=0.023）；②与M1-exo共培养后，体外BBB模型的TEER明显下降（P=0.000），对荧光黄的透过率明显增加（P=0.000）；③与b.End3组和b.End3 + BV2-exo组相比，b.End3+M1-exo组的Claudin-1、Occludin及ZO-1蛋白的表达水平明显下降。结论 M1-exo可以破坏BBB的完整性，影响其功能。

Abstract:

Objective To investigate the effects of M1 microglia-derived exosomes（M1-exo） on the function of an in-vitro blood-brain barrier（BBB） model and the expression of tight junction proteins between vascular endothelial cells.Methods Mouse microglia-derived BV2 cells were stimulated with lipopolysaccharide to be polarized towards the M1 phenotype，and exosomes were isolated and extracted after the phenotype was confirmed by flow cytometry. An in-vitro BBB model was constructed using the mouse brain microvascular endothelial cell line b.End3 and primary cultured mouse astrocytes. The BBB model was co-cultured with exosomes of different sources in three groups：normal culture for b.End3 cells（b.End3 group）；b.End3 cells + 25 μg/mL exosomes derived from normal BV-2 cells（b.End3+BV2-exo group）；and b.End3 cells+25 μg/mL M1-exo（b.End3+M1-exo group）. Trans-endothelial electrical resistance（TEER） and Lucifer yellow permeability were examined. The expression of tight junction complex proteins（claudin-1，occludin，zonula occludens-1［ZO-1］，and junctional adhesion molecules） was measured by Western blot.Results Microglia were successfully polarized to the M1 type，and the positivity rate of the M1 marker CD16/32 was significantly increased compared with the control group（P=0.023）. After co-culture with M1-exo，the TEER of the in-vitro BBB model was significantly decreased（P<0.001），and Lucifer yellow permeability was significantly increased（P<0.001）. Compared with the b.End3 group and b.End3+BV2-exo group，the b.End3+M1-exo group showed significantly decreased expression levels of claudin-1，occludin，and ZO-1.Conclusion M1-exo can disrupt the integrity of BBB and interfere with its normal function.