紧密连接蛋白ZO-1、occludin和actin参与缺氧缺血诱导的血脑屏障通透性增加

The tight junction proteins ZO-1, occludin and actin participate in the permeability increasing of blood-brain barrier induced by hypoxia-ischemia

摘要:

目的：探讨血脑屏障紧密连接（blood-brain barrier-tight junction，BBB-TJ）蛋白ZO-1、occludin和actin在缺氧缺血诱导的血脑屏障（blood-brain barrier，BBB）通透性增加中的变化及其机制。方法：利用人脐静脉内皮细胞系ECV304与星形胶质细胞（astrocytes, AS）共培养建立体外BBB模型，模型随机分为正常对照组和缺氧缺血两组。透射电镜观察两组间BBB-TJ的变化，直接免疫荧光观察细胞骨架蛋白actin分布的改变。γ计数仪检测大分子物质 125I-牛血清白蛋白（125I-BSA）通透曲线观察BBB通透性的改变， Western blot检测细胞骨架蛋白actin，胞浆附着蛋白ZO-1，跨膜蛋白occludin的表达量的改变。结果：透射电镜观察培养第10天的体外BBB模型，可见内皮细胞连接紧密，细胞间形成光滑、连续、较高密度的紧密连接。缺氧缺血后5 h，内皮细胞间连接开放，形成裂隙。直接免疫荧光下检测可见周边Actin丝带模糊，部分断裂，形成细胞间裂隙。缺氧缺血组 125I-BSA的通透量增加，与对照组比较差异有统计学意义（P<0.01）。同时ZO-1的表达量显著减少，而occludin和actin的表达量无明显改变。结论： 缺氧缺血诱导occludin的位置分布改变和ZO-1的表达量减少进而促使actin蛋白发生重排，是导致缺氧缺血后BBB通透性增加的可能机制之一。

Abstract:

ObjectiveTo study the changes of blood-brain barrier-tight junction (BBB-TJ) proteins ZO-1, occludin and actin following hypoxia-ischemia (HI) in order to explore the possible mechanism of permeability increasing of blood-brain barrier (BBB) induced by HI.MethodsBBB models were established by co-culture of cell ECV304 and astrocytes (AS) in vitro, then randomly assigned to control and HI groups. Transmission electron microscope was used to observe the changes of BBB-TJ. The distribution of actin was determined by direct-immunofluorescence microscope. Definite permeability of BBB models by 125I-BSA was detected by γ events-per-unit-time meter. Expression of actin, ZO-1 and occludin was detected by Western blot.ResultsAfter 10-day culture, endothelial cells connected tightly, with plenty of TJ which was smooth, continuous and of high density, in the BBB models. After 5 hrs of HI, the TJ was opened with intercellular gaps formation. The direct immunofluotescence showed that the peripheral filament bands became blurred, the cell-cell junction loosed and fissure appeared in the HI group. The permeability of 125I-BSA in the HI group increased significantly compared with the control group (P<0.01). Expression of ZO-1 decreased markedly, while expression of actin and occludin was not different in the HI group compared with the control group.ConclusionsThe changes in occludin distribution and decreased expression of ZO-1 lead the reorganization of BBB-actin protein, which may be one of the mechanisms of permeability increasing of BBB following HI.