基础医学与临床 ›› 2022, Vol. 42 ›› Issue (1): 120-125.

• 研究论文 • 上一篇    下一篇

重组梅毒螺旋体蛋白Tp47通过诱导uPA增加血管内皮细胞通透性

于宝丹1,陈聪2,柯吴坚2,吕萍2   

  1. 1. 广州医科大学附属第一医院检验科
    2. 南方医科大学皮肤病医院
  • 收稿日期:2021-03-23 修回日期:2021-06-01 出版日期:2022-01-05 发布日期:2022-01-05
  • 通讯作者: 吕萍 E-mail:lvping75@163.com
  • 基金资助:
    广东省公益研究与能力建设资金

Recombinant Treponema pallidum protein Tp47 increases permeability of vascular endothelial cells through inducing uPA

  • Received:2021-03-23 Revised:2021-06-01 Online:2022-01-05 Published:2022-01-05

摘要: 目的 探讨重组梅毒螺旋体蛋白Tp47(rTpp47)对THP-1巨噬细胞合成uPA的调控及其对人血管内皮细胞通透性的影响。方法 用rTpp47刺激THP-1巨噬细胞24小时后,分别收集细胞培养上清和THP-1细胞,用ELISA和Western blot方法检测THP-1细胞表达的upa含量;用THP-1细胞培养上清刺激人单层血管内皮细胞后,使用FITC-葡聚糖评价单层内皮细胞通透性的变化,用Western blot方法观察uPA对HUVEC细胞紧密连接蛋白Claudin-5表达的影响以及PKC信号通路是否参与rTpp47诱导的THP-1细胞表达uPA。结果 重组蛋白rTpp47刺激THP-1细胞合成和分泌的uPA显著高于对照组(p<0.05 和p<0.001);用rTpp47与THP-1巨噬细胞共培养24小时后收集的细胞培养上清刺激单层血管内皮细胞12小时和24小时,实验组与对照组的血管内皮细胞相对通透性差异有显著性(p<0.05和p<0.0001);uPA活性抑制剂阿米洛利(Amiloride)显著抑制了rTpp47刺激THP-1细胞分泌uPA(p<0.0001),改善了rTpp47对HUVEC的Claudin-5蛋白表达的抑制作用(p<0.05),显著抑制了rTpp47诱导的THP-1细胞表达PKC的磷酸化(p<0.05)。结论 重组蛋白rTpp47可能通过激活PKC信号通路刺激THP-1细胞合成和分泌的uPA,uPA作用于血管内皮细胞,可能导致血管内皮细胞通透性升高。

关键词: 梅毒螺旋体, 重组蛋白Tp47, 尿激酶型纤溶酶原激活物, 巨噬细胞, 人脐静脉内皮细胞

Abstract: Objective To investigate the effect of recombinant Treponema pallidum protein Tp47 (rTpp47) on the expression of uPA in THP-1 macrophages and on the permeability of vascular endothelial cells. Methods THP-1 cells were stimulated with rTpp47 for 24 hours and then the culture supernatant and THP-1 cells were collected respectively. The expression of Urokinase-type plasminogen activator (uPA) in THP-1 cells was detected by ELISA and Western blot. After THP-1 Cell culture supernatant was used to stimulate human umbilical vein endothelial cells (HUVEC) monolayers, FITC dextran was used to evaluate the permeability of HUVEC . Western blot method was used to observe the effect of uPA on the expression of tight junction protein claudin-5 in HUVEC cells and on the PKC signaling pathway in THP-1 cells stimulated by rTpp47.Results The synthesis and secretion of uPA in THP-1 cells stimulated by recombinant protein rTpp47 were significantly higher than those in the control group (P < 0.05). When HUVECS were stimulated by the THP-1 cell supernatant for 12 hours and 24 hours , the relative permeability of vascular endothelial cells in the experimental group was significantly higher than the control group (P < 0.05 for 12 hours and P < 0.0001 for 24 hours). Amiloride , the synthetic inhibitor of uPA, significantly inhibited the uPA expression and secretion of THP-1 cells stimulated by rTpp47 (P < 0.0001) and significantly suppressed PKC phosphorylation (P < 0.05) in THP-1 cells induced by rTpp47. Conclusion Our data indicate that the recombinant protein rTpp47 can stimulate the synthesis and secretion of uPA in THP-1 cells by activating PKC signaling pathway and uPA may increase the permeability of vascular endothelial cells.

Key words: Treponema pallidum, recombinant protein TP47, Urokinase-type plasminogen activator, macrophages, human umbilical vein endothelial cells

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