梅毒螺旋体膜蛋白Tpp17调节血管内皮屏障通透性的机制研究

In vitro treatment with Treponema paUidum membrane protein Tpp17 increased the permeability of vascular endothelial barrier

目的研究梅毒螺旋体膜重组蛋白Tpp17（rTpp17）对血管内皮屏障的调节作用，探讨膜蛋白Tppl7在梅毒免疫病理学发病机制中的作用。方法利用人脐静脉内皮细胞（HUVEC）构建体外细胞单层模型，将采用基因工程技术重组合成的rTppl7刺激HUVEC单层模型，ELISA检测Transwell小室的下室培养基中辣根过氧化物酶（HRP）流量；Cell-ELISA检测细胞表面VE．钙黏蛋白（VE-cadhefin）表达水平；rTppl7预处理HUVEC单层模型，加入Calcein—AM标记的THP-1细胞共培养，荧光倒置显微镜下计数THP-1细胞穿过HUVEC单层的迁移率；将HUVEC接种激光共聚焦专用培养皿，rTpp17处理后，加入荧光染料罗丹明-鬼笔环肽工作液，共聚焦显微镜下观察细胞骨架蛋白F-actin排列变化。结果rTpp17可提高HUVEC单层模型通透性及下调细胞膜VE-cadhefin的表达水平；促进THP-1细胞穿越HUVEC单层的迁移率，与阴性对照组比较，差异有统计学意义（P〈0．05）；rTppl7可促进细胞骨架蛋白F-actin重排。结论梅毒螺旋体膜蛋白Tpp17可提高血管内皮屏障通透性，下调血管内皮细胞VE-cadhefin的膜表达水平，促进细胞骨架蛋白F-actin重排及单核-巨噬细胞穿过血管内皮细胞单层，可能在梅毒的免疫病理学发病机制中起重要作用。

Objective To investigate the in vitro effects of Treponema pallidum membrane protein Tpp17 on the permeability of endothelial barrier for further investigation on the immunopathogenesis of syphi- lis. Methods A cellular model of in vitro mouolayer was established by using human umbilical vein endo- thelial cells （HUVECs）. Cell-ELISA and a TMB kit were respectively used to measure the expression of VE- cadherin and the flux of horseradish peroxidase （HRP） by monolayer HUVECs after stimulation with the re- combinant Tppl7 （rTpp17） protein. THP-1 cells stained with Calcein AM were added to the top of HUVEC monolayer in Transwell culture. Then, the numbers of THP-1 cells in the upper wells and beneath the HUVEC monolayer were counted by using a fluorescence microscope. The rTppl7 protein-treated HUVECs were fixed in 4% buffered paraformaldehyde and stained with rhodamine-phalloidin for observing the distri- bution of F-actin under a confocal laser scanning microscope. Results Compared with the control group, the expression of VE-cadherin in HUVECs was decreased, while the permeability of HUVEC monolayer was increased upon the stimulation with rTppl7 protein （P〈0.05）. Moreover, rTpp17 protein-induced F-actin redistribution and increased transendothelial migration of THP-1 cells were observed in rTpp17 protein-trea- ted HUVECs as compared with those of the control group （ P〈0.05 ）. Conclusion Treponema pallidum membrane protein Tpp17 could suppress the expression of VE-cadherin and enhance the redistribution of F-actin, resulting in an enhanced transendothelial migration of THP-1 ceUs and an increased permeability of HUVEC monolayer. The Tpp17 protein might play an important role in the immunopathogenesis of syphilis.