ABSTRACT
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 monolayer 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 Tpp17 (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 rTpp17 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 rTpp17 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 cells and an increased permeability of HUVEC monolayer.The Tpp17 protein might play an important role in the immunopathogenesis of syphilis.
ABSTRACT
Objective To study the effects of Treponema pallidum membrane protein Tpp17 on ac-tivation of human umbilical vein endothelial cells (HUVECs) in vitro and to understand its role in the immu-nopathogenesis of syphilis .Methods HUVECs were co-cultured with recombinant protein Tpp 17.Then the expressions of TNF-α, MCP-1, ICAM-1 and E-selectin at mRNA and protein levels in supernatants were re-spectively detected by enzyme-linked immunosobent assay ( ELISA ) and fluorescent real-time quantitative PCR.The adhesive ability of THP-1 cells was observed by fluorescence microscopy after co-cultured pretrea-ted HUVECs with recombinant protein Tpp 17 with Calcein-AM labeled THP-1.Pretreated HUVECs were cultured in the lower chamber of Transwell with recombinant protein Tpp 17 and monocytic THP-1 cells were cultured in the upper chamber of Transwell .After that, the migration of monocytic THP-1 cells was evalua-ted by using fluorescence microscopy .Results Compared with the blank control group , the expression of TNF-α, ICAM-1, E-selectin, especially the MCP-1, were enhanced by recombinant protein Tpp 17 of Trepo-nema pallidum.Moreover, Tpp17 improved the adhesive ability and migration of monocytic THP-1 cells, es-pecially the latter.Conclusion Treponema pallidum membrane protein Tpp17 might play a certain role in the immunopathogenesis of syphilis by enhancing the expression of TNF-α, MCP-1, ICAM-1 and E-selectin, and by promoting the adherence ability and migration of monocytic THP-1 cells.