Development of a new drug delivery method by the use of occludin molecules

封闭蛋白是唯一已知的紧密连接（TJ）的膜整合蛋白，并且现在被认为直接参与TJ的屏障和栅栏功能。通过靶向破坏封闭蛋白基因的两个等位基因来产生封闭蛋白缺陷型胚胎干（ES）细胞。当这些细胞进行悬浮培养时，它们聚集形成简单的，然后是囊性胚状体（EB），其时间过程与野生型ES细胞形成EB的时间过程相同。免疫荧光显微镜和透射电镜显示，极化上皮（内脏内胚层样）细胞分化描绘EB不仅从野生型，但也从occludin缺陷的ES细胞。冷冻断裂分析表明，野生型和闭合蛋白缺陷的上皮细胞之间的TJ链的数量或形态没有显着差异。此外，ZO-1，TJ相关的外周膜蛋白，仍然只集中在TJ在闭塞素缺陷的上皮细胞。在良好的协议与这些形态学观察，TJ在闭塞素缺陷的上皮细胞作为一个主要的障碍，低分子量示踪剂通过细胞旁途径的扩散。这些发现表明，存在尚未鉴定的TJ整合膜蛋白，其可以形成链结构，募集ZO-1，并且在没有occludin的情况下起屏障作用。

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 analyses 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 barrier without occludin.