产气荚膜梭菌肠毒素肽段与Claudin4特异性结合对哺乳动物表皮屏障通透性的调控

Modulation of mammalian epidermal barrier by specific binding of Clostridium perfringens enterotoxin fragment to Claudin4

目的 :探讨产气荚膜梭菌肠毒素肽段(Clostridium perfringens enterotoxin,c CPE194-319)对哺乳动物表皮屏障通透性的影响。方法:首先利用SWISS-MODEL软件模拟分析了c CPE194-319与Claudin4(Cldn4)结合的结构模型,然后通过细胞免疫荧光染色法和跨内皮细胞电阻法(trans-epithelial electrical resistance,TEER)检测c CPE194-319对人源永生化表皮细胞Ha Ca T膜蛋白Cldn4表达定位的影响和细胞层紧密性的影响,最后在小鼠中利用葡聚糖-罗丹明荧光素扩散法观察经c CPE194-319处理后的小鼠表皮的荧光素渗透变化情况。结果:c CPE194-319可与Ha Ca T细胞中Cldn4蛋白特异性结合,随着作用时间的增加,c CPE194-319结合Cldn4蛋白后形成复合体从Ha Ca T细胞膜上向细胞质中转移,TEER值降低;当洗脱c CPE194-319后,新合成的Cldn4蛋白可重新定位到细胞膜上,TEER值升高。小鼠体内实验表明,c CPE194-319处理后的小鼠表皮渗透性增强。结论:c CPE194-319能够通过与Cldn4蛋白的特异性结合,从而调控哺乳动物表皮屏障的通透性。

Objective： To investigate the regulation effect of Clostridium perfringens enterotoxin fragment（c CPE194-319） on the function of mammalian epidermal barrier. Methods： The three dimensional（3D） binding model of c CPE194-319 to Claudin 4（Cldn4） was simulated using WISS-MODEL software first. The bind effect of c CPE194-319 to Claudin 4 and the monolayer cell permeability were analyzed by immunofluorescence staining and trans-epithelial electrical resistance（TEER） measurement in Ha Ca T cell line. To investigate the regulation effects of c CPE194-319 on mammalian skin, dye diffusion assay with Rhodamine B Dextran was performed to detect the diffusion of fluorescent dye on mice epidermis after c CPE194-319 application. Results： c CPE194-319 could specifically bind to mammalian Cldn4 and remove Cldn4 from cellular membrane,which resulted in TEER reduction. In vivo analysis displayed a strong diffusion of fluorescent dye in mice epidermis after treatment of c CPE194-319. Conclusion： c CPE194-319 can modulate the function of mammalian epidermal barrier by specific binding to Cldn4.