蛋白酶激活受体在人单核细胞和T细胞上的表达及功能
发布时间：2018-11-25 08:18
【摘要】：凝血酶、胰蛋白酶、类胰蛋白酶和弹性蛋白酶等丝氨酸蛋白酶能调节凝血和纤维蛋白溶解平衡、降解神经肽以参与神经性水种，调控补体系统从而在炎症反应时发挥免疫调节作用。近年的研究显示，它们还能通过蛋白酶激活受体(proteinase activated receptor，PARs)调节细胞的功能而在炎症和免疫反应中起重要作用。PARs是新的G蛋白偶联受体，目前已发现有四种PARs，分别为PAR-1、PAR-2、PAR-3和PAR-4，凝血酶可以激活PAR-1、PAR-3和PAR-4，胰蛋白酶可以激活PAR-1、PAR-2和PAR-4，类胰蛋白酶和弹性蛋白酶可以激活PAR-2。单核细胞和T淋巴细胞是炎征和免疫反应重要的效应细胞，但是PARs在人外周血单核细胞和T细胞上表达及它们分泌的细胞因子上的影响则知之甚少。 采用磁激活的细胞分选法(magnetic activated cell sorting，MACS)纯化人外周血单核细胞和T细胞，分别用RT-PCR和流式细胞术检测外单核细胞和T细胞上的PARs在基因和蛋白质水平的表达；用凝血酶、胰蛋白酶、类胰蛋白酶和弹性蛋白酶及PARs的激动肽分别激发MACS纯化后单核细胞和淋巴细胞16小时后，用ELISA检测单核细胞培养上清液中的IL-6、IL-1β和IL-12，，T细胞培养上清液中的IL-6和IL-13。 实验显示，人外周血单核细胞表达PAR-1、PAR-3和PAR-4但不表达PAR-2蛋白质，单核细胞表达PAR-1和PAR-3，但是不表达PAR-2和PAR-4 mRNAs。凝血酶、胰蛋白酶、类胰蛋白酶和弹性蛋白酶呈浓度依赖性地诱导单核细胞产生IL-6，当这些蛋白酶酶活性被它们的抑制剂抑制后，它们诱导单核细胞产生IL-6的能力被抑制。PAR-1激动肽SFLLR-NH_2和PAR-4激动肽GYPGQV-NH_2可浓度依赖性地诱导单核细胞产生IL-6，PAR-1反激动肽RLLFS-NH_2和PAR-4反激动肽VQGPYG-NH_2不能诱导单细胞产生IL-6，而PAR-3激动肽TFRGAP-NH_2和反激动肽PAGRFT-NH_2对于单核细胞产生IL-6无明显作用。蛋白酶及PAR激动肽在各自工作浓度对于单核细胞产生IL-1β和IL-12无明显作用。 实验还显示，人外周血T细胞表达PAR-1、PAR-和PAR-3但不表达PAR-4蛋白质，与流式细胞结果一致，T细胞表达PAR-1、PAR-2和PAR-3，但是不表达PAR-4 mRNAs。凝血酶、胰蛋白酶、类胰蛋白酶呈浓度依赖性地诱导T细胞产生IL-6，当这些蛋白酶酶活
[Abstract]:Serine proteases such as thrombin, trypsin, trypsin and elastase regulate coagulation and fibrinolysis balance, and degrade neuropeptides to participate in neurotic water. Regulate the complement system and thus play an immunomodulatory role in the inflammatory response. Recent studies have shown that they also play an important role in inflammation and immune response through protease activated receptor (proteinase activated receptor,PARs) regulation of cell function. PARs is a new G-protein-coupled receptor, and four kinds of PARs, have been found. PAR-1,PAR-2,PAR-3 and PAR-4, thrombin can activate PAR-1,PAR-3 and PAR-4, trypsin can activate PAR-1,PAR-2 and PAR-4, trypsin and elastase can activate PAR-2.. Monocytes and T lymphocytes are important effectors of inflammation and immune response, but the effect of PARs on the expression of PARs on human peripheral blood monocytes and T cells and the cytokines secreted by them is little known. Human peripheral blood monocytes and T cells were purified by magnetic activated cell sorting (magnetic activated cell sorting,MACS), and the expression of PARs on outer monocytes and T cells was detected by RT-PCR and flow cytometry, respectively. Monocytes and lymphocytes were stimulated by thrombin, trypsin, elastase and PARs activation peptide after MACS purification for 16 hours respectively. ELISA was used to detect IL-6,IL-1 尾 and IL-12, in the supernatant of monocyte culture. IL-6 and IL-13. in supernatant of T cell culture Human peripheral blood monocytes expressed PAR-1,PAR-3 and PAR-4 but did not express PAR-2 protein. Monocytes expressed PAR-1 and PAR-3, but did not express PAR-2 and PAR-4 mRNAs.. Thrombin, trypsin, trypsin and elastase induce monocytes to produce IL-6, in a concentration-dependent manner when the activity of these proteases is inhibited by their inhibitors. Their ability to induce IL-6 production in monocytes was inhibited. PAR-1 activator SFLLR-NH_2 and PAR-4 GYPGQV-NH_2 could induce IL-6, production in a concentration-dependent manner. PAR-1 RLLFS-NH_2 and PAR-4 VQGPYG-NH_2 could not induce the production of IL-6, in monocytes, but PAR-3 TFRGAP-NH_2 and PAGRFT-NH_2 had no effect on IL-6 production in monocytes. Protease and PAR activator peptide had no significant effect on the production of IL-1 尾 and IL-12 in monocytes at their respective working concentrations. The results also showed that human peripheral blood T cells expressed PAR-1,PAR- and PAR-3 but did not express PAR-4 protein. In accordance with the flow cytometry results, T cells expressed PAR-1,PAR-2 and PAR-3, but did not express PAR-4 mRNAs.. Thrombin, trypsin, trypsin induce T cells to produce IL-6, in a concentration-dependent manner when these proteases are active.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
本文编号：2355425
[Abstract]:Serine proteases such as thrombin, trypsin, trypsin and elastase regulate coagulation and fibrinolysis balance, and degrade neuropeptides to participate in neurotic water. Regulate the complement system and thus play an immunomodulatory role in the inflammatory response. Recent studies have shown that they also play an important role in inflammation and immune response through protease activated receptor (proteinase activated receptor,PARs) regulation of cell function. PARs is a new G-protein-coupled receptor, and four kinds of PARs, have been found. PAR-1,PAR-2,PAR-3 and PAR-4, thrombin can activate PAR-1,PAR-3 and PAR-4, trypsin can activate PAR-1,PAR-2 and PAR-4, trypsin and elastase can activate PAR-2.. Monocytes and T lymphocytes are important effectors of inflammation and immune response, but the effect of PARs on the expression of PARs on human peripheral blood monocytes and T cells and the cytokines secreted by them is little known. Human peripheral blood monocytes and T cells were purified by magnetic activated cell sorting (magnetic activated cell sorting,MACS), and the expression of PARs on outer monocytes and T cells was detected by RT-PCR and flow cytometry, respectively. Monocytes and lymphocytes were stimulated by thrombin, trypsin, elastase and PARs activation peptide after MACS purification for 16 hours respectively. ELISA was used to detect IL-6,IL-1 尾 and IL-12, in the supernatant of monocyte culture. IL-6 and IL-13. in supernatant of T cell culture Human peripheral blood monocytes expressed PAR-1,PAR-3 and PAR-4 but did not express PAR-2 protein. Monocytes expressed PAR-1 and PAR-3, but did not express PAR-2 and PAR-4 mRNAs.. Thrombin, trypsin, trypsin and elastase induce monocytes to produce IL-6, in a concentration-dependent manner when the activity of these proteases is inhibited by their inhibitors. Their ability to induce IL-6 production in monocytes was inhibited. PAR-1 activator SFLLR-NH_2 and PAR-4 GYPGQV-NH_2 could induce IL-6, production in a concentration-dependent manner. PAR-1 RLLFS-NH_2 and PAR-4 VQGPYG-NH_2 could not induce the production of IL-6, in monocytes, but PAR-3 TFRGAP-NH_2 and PAGRFT-NH_2 had no effect on IL-6 production in monocytes. Protease and PAR activator peptide had no significant effect on the production of IL-1 尾 and IL-12 in monocytes at their respective working concentrations. The results also showed that human peripheral blood T cells expressed PAR-1,PAR- and PAR-3 but did not express PAR-4 protein. In accordance with the flow cytometry results, T cells expressed PAR-1,PAR-2 and PAR-3, but did not express PAR-4 mRNAs.. Thrombin, trypsin, trypsin induce T cells to produce IL-6, in a concentration-dependent manner when these proteases are active.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R392
【引证文献】
相关硕士学位论文 前1条
1 杨雪;伤寒沙门氏菌致病仔猪胃肠道组织中蛋白酶激活受体2的定位研究[D];华中农业大学;2007年
本文编号：2355425
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