MODULATION OF BLOOD-BRAIN BARRIER

为了调节血脑屏障，即紧密连接，我们分析了occludin的功能。Occludin是唯一已知的紧密连接(TJ)完整膜蛋白，目前被认为直接参与TJ的屏障和屏障功能。Occludin缺陷的胚胎干细胞是通过靶向破坏occludin基因的两个等位基因而产生的。在悬浮培养下，这些细胞聚集成简单的囊状胚体(EBS)，其形成过程与野生型ES细胞形成EB的过程相同。免疫荧光显微镜和超薄切片电子显微镜显示极化的上皮细胞(内脏内胚层样细胞)被分化为EBS，不仅来自野生型，也来自闭合素缺乏的ES细胞。冷冻断裂分析表明，野生型和闭合素缺陷型上皮细胞的TJ链在数量和形态上没有显著差异。此外，ZO-1，一种与TJ相关的外周膜蛋白，在闭合素缺乏的上皮细胞中仍仅集中在TJ。与这些形态观察相一致的是，封闭蛋白缺乏的上皮细胞中的TJ是低分子质量示踪剂通过细胞旁途径扩散的主要屏障。这些结果表明，目前尚未鉴定的TJ完整膜蛋白(S)可以形成链结构，募集ZO-1，并发挥无封闭屏障的功能。

To modulate the blood-brain barrier, i.e.tight junctions, we analyzed the functions of occludin. Occludin is the only known integral membrane protein of tight junctions (TJ), and is now believed to be directly involved in the barrier and fence functions of TJ.Occludin-deficient embryonic stem (ES) cells were generated by targeted disruption of both alleles of the occludin gene. When these cells were subjected to suspension culture, they aggregated to form simple, and then cystic embryoid bodies (EBs) with the same time course as EB formation from wild-type ES cells. Immunofluorescence microscopy and ultrathin section electron microscopy revealed that polarized epithelial (visceral endoderm-like) cells were differentiated to delineate EBs not only from wild-type but also from occludin-deficient ES cells. Freeze fracture analyzes indicated no significant differences in number or morphology of TJ strands between wild-type and occludin-deficient epithelial cells. Furthermore, ZO-1, a TJ-associated peripheral membrane protein, was still exclusively concentrated at TJ in occludin-deficient epithelial cells. In good agreement with these morphological observations, TJ in occludin-deficient epithelial cells functioned as a primary barrier to the diffusion of a low molecular mass tracer through the paracellular pathway. These findings indicate that there are as yet unidentified TJ integral membrane protein(s) which can form strand structures, recruit ZO-1, and function as a barrier without occludin.