梅毒螺旋体膜蛋白Tpp47对血管内皮细胞黏附功能影响的实验研究

Treponema pallidum membrane protein Tpp47 promotes the adhesion ability of vascular endothelial cells invitro： an experimental study

目的探讨梅毒螺旋体膜蛋白Tpp47对血管内皮细胞的作用。方法利用基因工程技术重组合成的梅毒螺旋体膜蛋白rrpp47及脂多糖分别刺激人脐静脉内皮细胞（HUVEC），ELISA检测上清中细胞间黏附分子1（ICAM-1）及E选择素的水平；荧光定量PCR检测HUVEC中ICAM-1及E选择素mRNA的转录水平；M1Tr法检测HUVEC增殖水平；将重组蛋白Tpp47及脂多糖预处理的HUVEC与钙黄绿素AM标记的THP-1细胞共培养，荧光倒置显微镜下观察HUVEC与THP-1细胞的黏附情况。结果梅毒螺旋体膜重组蛋白Tpp47刺激HUVEC后，其分泌的黏附分子ICAM-1（1．28±0．03）及E选择素（0．51±0．01）水平显著提高，与空白对照组（0．90±0．01与0．13±0．03）比较，差异均有统计学意义（t值分别为18．28和18．19，均P〈0．05）；将重组蛋白Tpp47刺激24h后的HUVEC与THP-1细胞共培养，HUVEC与THP-1细胞的黏附率为56．1％±1．9％，较空白对照组（16．3％±2．1％）明显提高，差异有统计学意义（r=12．65，P〈0．05）。M1Tr试验结果显示，重组蛋白Tpp47刺激HUVEC后，其增殖率（19．5％±1．7％）高于空白对照组（10．0％±3．1％），差异有统计学意义（X^2=3．92，P〈0．05）；较脂多糖低（41．2％±3．7％），差异有统计学意义（X^2=10．42，P〈0．05）。结论梅毒螺旋体膜重组蛋白Tpp47可体外上调HUVEC与THP-1的黏附能力及促进HUVEC增殖，可能在梅毒的发病中起一定作用。

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 Ug/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-dimethyhhiazol-2-yl）-2,5 diphenyl tetrazolium bromide （MTY） assay was used to evaluate the proliferation activity of HUVECs treated with Tpp47 （400 Ug/L） and LPS （200 Ug/L） respectively for 24 hours. To estimate the effect on adhesion ability, some HUVECs were pretreated with Tpp47 （400 ug/L） and LPS （200 ug/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 ug/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 bg/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%, ）（2 = 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.