Wu Li-Wen, Yin Fei, Peng Jing, Wang Wei-Dong, Gan Na
Department of Pediatrics, Xiangya Hospital, Central South University, Changsha 410008, China.
Zhongguo Dang Dai Er Ke Za Zhi. 2008 Aug;10(4):513-6.
To 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.
BBB 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 gamma events-per-unit-time meter. Expression of actin, ZO-1 and occludin was detected by Western blot.
After 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 immunofluorescence showed that the peripheral filament bands became blurred, the cell-cell junction loosened 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.
The 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.
研究缺氧缺血（HI）后血脑屏障紧密连接（BBB-TJ）蛋白ZO-1、闭合蛋白和肌动蛋白的变化，以探讨HI诱导血脑屏障（BBB）通透性增加的可能机制。
体外将ECV304细胞与星形胶质细胞（AS）共培养建立BBB模型，然后随机分为对照组和HI组。用透射电子显微镜观察BBB-TJ的变化。用直接免疫荧光显微镜检测肌动蛋白的分布。用γ单位时间计数仪检测125I-BSA对BBB模型的通透性。用蛋白质免疫印迹法检测肌动蛋白、ZO-1和闭合蛋白的表达。
培养10天后，BBB模型中的内皮细胞紧密连接，有大量光滑、连续且高密度的TJ。HI 5小时后，TJ开放，细胞间间隙形成。直接免疫荧光显示，HI组外周丝状带模糊，细胞间连接松散，出现裂隙。HI组125I-BSA的通透性较对照组显著增加（P<0.01）。HI组ZO-1表达明显降低，而肌动蛋白和闭合蛋白的表达与对照组相比无差异。
闭合蛋白分布的变化和ZO-1表达的降低导致BBB-肌动蛋白蛋白的重组，这可能是HI后BBB通透性增加的机制之一。
