缺氧缺血对体外血脑屏障通透性的影响及其机制的研究

目的：探讨血脑屏障紧密连接（BBB-tight junction，BBB—TJ）蛋白和基质金属蛋白酶-9（MMP-9）在缺氧缺血诱导的体外血脑屏障（BBB）模型通透性增加中的作用及相互关系。方法：ECV304与原代培养的星形胶质细胞共培养建立体外BBB模型。实验分为正常对照组（C组）、缺氧缺血组（H／I组）、BB-1101预处理组（P组）3组。透射电镜分别观察各组BBB-TJ变化；通过吖计数仪测定核素标记牛血清白蛋白（^125I-BSA）通透量检测BBB屏障功能。直接免疫荧光观察细胞骨架蛋白Actin分布的改变，Westemblot检测Actin、Occludin、ZO-1和MMP-9表达量的改变。结果：电镜观察见C组内皮细胞间形成光滑、连续、较高密度的紧密连接，免疫荧光检测见细胞骨架蛋白Actin主要分布在细胞膜及细胞核周边。形成肌动蛋白丝带，轮廓内包含大量柬状排列有序的微丝结构，细胞间连接紧密。缺氧缺血后．H／I组细胞间连接开放，形成裂隙，而胞膜及胞核周边肌动蛋白丝带模糊、部分断裂，并有应力纤维形成，出现细胞间裂隙，BBB对岱I—BSA通透性明显增高，与C组和P组比较，差异有显著性（P〈0．01），同时ZO-1的表达量显著减少，MMP-9的表达显著增多，而Occludin和Actin的表达量无改变。BB-1101预处理后，P组细胞BBB-TJ破坏减少．周边肌动蛋白丝带重新出现，细胞间裂隙较少，岱I-BSA通透量较H／I组明显下降（P〈0．01）．MMP-9的表达较H／I组也明显减少（P〈0.01）。结论：（1）缺氧缺血能促使BBB—TJ的开放进而导致BBB通透性增加；（2）BBB-TJ的开放可能与BBBActin的重组及ZO-1的表达量降低有关；（3）缺氧缺血能增加MMP-9的表达：（4）MMP-9与缺氧缺血时BBBActin的重组及ZO—1的表达减少相关。

The influence of hypoxia-ischemia on the permeability of blood-brain barrier in vitro and its mechanism

Objective To investigate the effect of blood-brain barrier tight junction (BBB-TJ) proteins and matrix metalloproteinase-9 (MMP-9) on the permeability of blood-brain barrier(BBB) under hypoxia-ischemia in vitro. Methods ECV304 and primary cultured astrocytes were co-cultured to establish BBB model in vitro, then were divided into 3 groups: normal BBB model group(group C), hypoxic-ischemic group (group H/I), and BB-1101 pretreated group(group P). The change of BBB-TJ was detected by transmission electron microscope, the barrier function of BBB by radionuclide gamma counting 125I - BSA detection, the distribution of actin by direct immunofluorescence, and the expressed levels of Actin, Occludin, ZO-1 and MMP-9 by Western blot. Results The tight junctions, which were smooth, continuous and high density, between endothelial cells were formed. Actin distributed maily in cell membrane and nuclear ambitus and formed the actin band, which included considerable fasciculated and orderly arranged microfilament microfilament. Cell junctions opened, actin band got fuzzy and fractured partly, stress fibers were formed, and inter-cellular fissure appeared after hypoxia-ischemia. The permeability of BBB to 125I -BSA increased obviously, the expressions of ZO-1 decreased but MMP-9 increased markedly in group H/I as compared to group C and P(P < 0.01). The destruction of BBB-TJ was diminished, actin band reappeared, inter-cellular fissure declined, the permeability to 125I -BSA and expression of MMP-9 decreased in group P in contrast with group H/I group(P < 0.01). Conclusions Hypoxia-ischemia can promote the opening of BBB-TJ and increase the permeability of BBB. The opening of BBB-TJ maybe related to the reorganization of actin and decreased levle of ZO-1. Hypoxia-ischemia can increase the expression of MMP-9. MMP-9 is involved in the reorganization of actin and decreased level of ZO-1 in BBB under hypoxia-ischemia.