Gao P, Shivers R R
Department of Biology, University of Western Ontario, London, Ontario, Canada.
J Submicrosc Cytol Pathol. 2004 Jan;36(1):7-15.
Tight junctions are regarded as the primary anatomical structure responsible for the blood-brain barrier (BBB). The molecular components that have been defined include ZO-1, a peripheral membrane protein associated with the cytoplasmic surface of the tight junction in epithelial and endothelial cells. It has been localized to the points of membrane contact with the fibrils seen by freeze-fracture. Examination of passaged endothelial cells with freeze-fracture failed to locate the intramembrane specializations associated with tight junctions. For this reason, immunocytochemistry and freeze-fracture were used to study the correlation of ZO-1 expression with the presence of tight junctions in bovine brain and aorta endothelial cells. Indirect immunofluorescence analysis showed ZO-1 to be localized at sites of cell-cell contact. Images of freeze-fractured sites of endothelial cell-cell contacts in identical passage numbers did not display characteristic tight junctions. When bovine aorta endothelial cells were cultured in astrocyte-conditioned medium on a complete extracellular matrix, platinum replicas displayed profiles of tight junctions. The elements of tight junctions were arranged as parallel ridges which displayed free ends. The immunofluorescence staining of ZO-1 was identical to that obtained on the endothelial cells that displayed no tight junction profiles. These results suggest that ZO-1 may be present at putative junction-containing sites before the junctional structures appear in the surface membrane. Therefore, ZO-1 expression does not a priori reflect assembly of the tight junctions identified by freeze-fracture.
紧密连接被视为构成血脑屏障（BBB）的主要解剖结构。已确定的分子成分包括ZO-1，一种与上皮细胞和内皮细胞紧密连接的细胞质表面相关的外周膜蛋白。它已被定位到通过冷冻蚀刻观察到的与原纤维的膜接触点。用冷冻蚀刻法检查传代培养的内皮细胞未能定位与紧密连接相关的膜内特化结构。因此，采用免疫细胞化学和冷冻蚀刻技术来研究ZO-1表达与牛脑和主动脉内皮细胞中紧密连接存在之间的相关性。间接免疫荧光分析显示ZO-1定位于细胞间接触部位。相同传代数的内皮细胞间接触的冷冻蚀刻部位图像未显示出典型的紧密连接。当牛主动脉内皮细胞在完全细胞外基质上的星形胶质细胞条件培养基中培养时，铂复制品显示出紧密连接的形态。紧密连接的成分排列成平行的嵴，呈现出自由端。ZO-1的免疫荧光染色与在未显示紧密连接形态的内皮细胞上获得的染色相同。这些结果表明，在紧密连接结构出现在表面膜之前，ZO-1可能存在于假定的含连接部位。因此，ZO-1的表达并不必然反映通过冷冻蚀刻鉴定的紧密连接的组装情况。
