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MODULATION OF BLOOD-BRAIN BARRIER

血脑屏障的调节

基本信息

批准号：
06557014
负责人：
TSUKITA Shoichiro
金额：
$ 8万
依托单位：
KYOTO UNIVERSITY (1995)Okazaki National Research Institutes (1994)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1995
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-06557014/
关键词：
blood-brain barrier tight junction occludin claudin knock-out cell adhesion phosphrylation endothelial cell アンチセンスDNA 機能阻害抗体上皮細胞 Z0-1 脳血管バリアー

项目摘要

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.

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）的完整膜蛋白，现在被认为直接参与TJ的屏障和栅栏功能。通过靶向破坏occludin基因的两个等位基因，产生了occludin缺陷胚胎干细胞（ES）。当这些细胞进行悬浮培养时，它们聚集形成简单胚状体，然后形成囊性胚状体（EBs），其形成过程与野生型ES细胞形成EB的时间相同。免疫荧光显微镜和超薄切片电镜显示极化上皮（内脏内胚层样）细胞分化，不仅从野生型分化出EBs，而且从occludin缺乏的ES细胞分化出EBs。冻裂分析表明，野生型和occludin缺陷型上皮细胞在TJ链的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occludin缺乏的上皮细胞中仍然只集中在TJ。与这些形态学观察结果很好地吻合，occludin缺陷上皮细胞中的TJ作为低分子质量示踪剂通过细胞旁途径扩散的主要屏障。这些发现表明，存在尚未确定的TJ整体膜蛋白，它可以形成链结构，招募ZO-1，并在不闭塞的情况下起屏障作用。