丝氨酸蛋白酶3及蛋白酶活化受体-2激动剂对韦格纳肉芽肿病患者树突状细胞样单核细胞的影响

目的 探索丝氨酸蛋白酶3(PR3)对人外周血树突状细胞(DC)样单核细胞-CD14+CD16high单核细胞的蛋白酶活化受体(PAR)-2表达、成熟及细胞因子产生的作用.方法 密度梯度离心法分离健康人和韦格纳肉芽肿病(WG)患者外周血单个核细胞(PBMC);分别以脂多糖、PR3、胰蛋白酶、PAR-2激动肽(PAR-2-AP)、脂多糖+PR3、脂多糖+胰蛋白酶在体外刺激PBMC 24h;流式细胞仪检测刺激后的CD14+CD16high单核细胞表面PAR-2及CD80、CD83、人类白细胞抗原(HLA)-DR的表达;酶联免疫吸附试验(ELISA)检测培养上清中细胞因子白细胞介素(IL)-6的分泌.统计学分析采用Mann Whitney 非参数检测方法.结果 PR3、胰蛋白酶和PAR-2-AP对外周血DC样单核细胞表面PAR-2和CD80、CD83、HLA-DR表达均无影响;脂多糖能够显著升高健康人细胞表面PAR-2[(5.8±1.5)%升至(24.5±4.5)%,P=0.002]及WG患者和健康人CD80、CD83、HLA-DR的表达;PR3、胰蛋白酶、PAR-2-AP及脂多糖均能诱导IL-6的分泌.结论 PR3和PAR-2通路激动剂不能诱导DC样单核细胞的PAR-2的表达及成熟,但能诱导IL-6分泌.

Abstract：

Objective To assess the effects of protease 3(PR3)and protease-activated receptor (PAR)-2-activator on the maturation and functions of peripheral blood dendritic cell(DC)-like monocytes.Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMC)from Wegener's granulomatosis(WG)patients and healthy controls(HC).PBMC were stimulated by LPS,human PR3,trypsin,PAR-2-agonist peptide (PAR-2-AP),LPS+PR3 or LPS+trypsin for 24 h.Flow cytometry was used to analyze the expression of PAR-2,CD80,CD83,HLA-DR on stimulated DC-like monocytes-CD14+CD16high monocytes.ELISA kit was used to test the concentration of IL-6 in the culture supernants.Mann-Whitney non-parameteric test was used fur statistical analysis.Resuits No effect of PR3,trypsin and PAR-2-AP on the expression of PAR-2,CD80,CD83,HLA-DR of DC-like monoeytes was found.LPS could significantly induce PAR-2 expression in HC[from(5.8±1.5)%to(24.5±4.5)%,P=0.002]and the expression of CD80,CD83,HLA-DR in HC and WG;PR3,trypsin,PAR-2-AP and LPS could all stimulate the secretion of IL-6.Conclusion PR3 and PAR-2 pathway-activators can not promote PAR-2expression and maturation of DC-tike monocytes,but they can induce the secretion of IL-6.

Effects of PR3 and protease-activated receptor-2-activators on the maturation and functions of dendritic cell-like monocytes

Objective To assess the effects of protease 3(PR3)and protease-activated receptor (PAR)-2-activator on the maturation and functions of peripheral blood dendritic cell(DC)-like monocytes.Methods Density gradient centrifugation was used to isolate peripheral blood mononuclear cells(PBMC)from Wegener's granulomatosis(WG)patients and healthy controls(HC).PBMC were stimulated by LPS,human PR3,trypsin,PAR-2-agonist peptide (PAR-2-AP),LPS+PR3 or LPS+trypsin for 24 h.Flow cytometry was used to analyze the expression of PAR-2,CD80,CD83,HLA-DR on stimulated DC-like monocytes-CD14+CD16high monocytes.ELISA kit was used to test the concentration of IL-6 in the culture supernants.Mann-Whitney non-parameteric test was used fur statistical analysis.Resuits No effect of PR3,trypsin and PAR-2-AP on the expression of PAR-2,CD80,CD83,HLA-DR of DC-like monoeytes was found.LPS could significantly induce PAR-2 expression in HC[from(5.8±1.5)%to(24.5±4.5)%,P=0.002]and the expression of CD80,CD83,HLA-DR in HC and WG;PR3,trypsin,PAR-2-AP and LPS could all stimulate the secretion of IL-6.Conclusion PR3 and PAR-2 pathway-activators can not promote PAR-2expression and maturation of DC-tike monocytes,but they can induce the secretion of IL-6.