ABSTRACT
Objective To evaluate the effect of Treponema pallidum membrane protein Tpp47 on vascular endothelial cells.Methods Human umbilical vein endothelial cells (HUVECs) were classified into multiple groups to be cultured with various concentrations (50,100,200,400 and 800 μg/L) of the recombinant protein Tpp47 or lipopolysaccharide (LPS) for different durations (3,6,12,24 and 48 hours).Then,enzyme-linked immunosorbent assay (ELISA) was performed to determine the levels of intercellular cell adhesion molecule-1 (ICAM-1) and E-selectin in the culture supernatant of,fluorescence-based real-time quantitative PCR to quantify the mRNA expressions of ICAM-1 and E-selectin in,HUVECs.The 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT) assay was used to evaluate the proliferation activity of HUVECs treated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours.To estimate the effect on adhesion ability,some HUVECs were pretreated with Tpp47 (400 μg/L) and LPS (200 μg/L) respectively for 24 hours followed by coculture with THP-1 human monocytic leukaemia cells for 6 hours,then,the adhesion of HUVECs to THP-1 cells was visualized by fluorescence microscopy.The cells receiving no treatment served as the blank control.Results A significant increase was observed in the supernatant level (expressed as the absorbance value at 450 nm) of ICAM-1 for HUVECs treated with Tpp47 of 400 μg/L for 24 hours (1.28 ± 0.03 vs.0.90 ± 0.01,t =18.28,P ＜ 0.05) and that of E-selectin for HUVECs treated with Tpp47 of 400 μg/L for 12 hours (0.51 ± 0.01 vs.0.13 ± 0.03,t =18.19,P＜ 0.05) compared with untreated HUVECs.The adhesion rate to THP-1 cells was significantly higher in HUVECs pretreated with Tpp47 for 24 hours than in untreated HUVECs (56.1％ ± 1.9％ vs.16.3％ ± 2.1％,x2 =12.65,P ＜ 0.05).The cell proliferation rate was 19.5％ ± 1.7％ in HUVECs treated with Tpp47,significantly higher than that in untreated HUVECs (10.0％ ± 3.1％,x2 =3.92,P＜ 0.05),but lower than that in those treated with LPS (41.2％ ± 3.7％,x2 =10.42,P ＜ 0.05).Conclusions The recombinant membrane protein Tpp47 could enhance HUVECs to proliferate and adhere to monocytic THP-1 cells,suggesting a certain role of Tpp47 in the pathogenesis 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.