IL-12对肥大细胞蛋白酶激活受体表达和介质分泌的影响
发布时间：2018-08-13 09:46
【摘要】： 蛋白酶激活受体(proteinase activated receptors或protease activated receptors，PARs)，白细胞介素(interleukin，IL)-4，IL-6参与过敏反应的发生，IL-12参与后天获得性免疫反应。然而IL-12对酶引起的肥大细胞分泌细胞因子的影响，细胞因子对肥大细胞PAR表达的影响，Th1类细胞因子IL-12对肥大细胞Th2类细胞因子分泌的影响及其信号转导通路还不甚清楚。因此本研究探讨IL-12对肥大细胞PARs表达和IL-4，IL-6分泌以及组胺释放的影响，并探讨可能的信号转导通路。 肥大细胞激发后，收集各时间点的细胞和上清。细胞处理后采用流式细胞术和实时定量PCR的方法在蛋白水平和mRNA水平检测PAR-1，PAR-2，PAR-3和PAR-4表达情况，用细胞激发信号ELISA(eellular activation of signaling ELISA，CASE)方法检测信号转导通路蛋白Akt，，ERK，p38和STAT3磷酸化情况；上清用ELISA方法检测IL-4，IL-6和组胺水平。 结果显示：肥大细胞P815在蛋白水平和mRNA水平表达PAR-1，PAR-2，PAR-3和PAR-4。IL-12下调PAR-2蛋白和mRNA在肥大细胞P815上的表达；IL-12上调PAR-4蛋白和mRNA在肥大细胞P815上的表达；IL-12上调PAR-1和PAR-3 mRNA在肥大细胞P815的表达；以低浓度IL-12(1 ng／ml)预处理肥大细胞60分钟后，胰蛋白酶和类胰蛋白酶引起的PAR-2蛋白和mRNA在肥大细胞P815上的表达增强；IL-12抗体的应用可以阻断IL-12对PAR表达的上调作用。IL-12以浓度依赖的方式促进肥大细胞P815分泌IL-4；低浓度IL-12(1ng／ml)预处理P815细胞60分钟后，胰蛋白酶和类胰蛋白酶引起的IL-4分泌被抑制；PAR-2拮抗肽FSLLRY-NH_2的应用能阻断胰蛋白酶和类胰蛋白酶引起的IL-4分泌，但对IL-12引起的IL-4分泌无影响；IL-12对肥大细胞IL-6分泌和组胺释放无明显影响。应用PD98059，U0126和LY294002可以阻断IL-12引起的肥大细胞IL-4分泌并抑制IL-12引起的ERK和Akt磷酸化。 总之，IL-12能够调节肥大细胞PARs表达并刺激肥大细胞分泌IL-4。IL-12引起肥大细胞P815分泌IL-4很可能是通过激活Akt和ERK信号转导通路实现的，而胰蛋白酶和类胰蛋白酶引起肥大细胞分泌IL-4似乎是有赖于激活PAR-2途径实现的。这些发现提示在过敏性炎症反应中，IL-12可作为一种调节因子，通过调节PAR-2在肥大细胞表达和调节肥大细胞分泌IL-4，发挥其维持Th1细胞因子和Th2细胞因子分泌平衡的功能。同时，胰蛋白酶和类胰蛋白酶刺激肥大细胞分泌IL-4的发现为肥大细胞激活的自我放大机制提供了一个新的理论依据。
[Abstract]:Protease activated receptors (proteinase activated receptors or protease activated receptor PARs) and interleukin-4- (IL-6) were involved in the development of allergic reaction. IL-12 was involved in acquired immune response. However, the effect of IL-12 on the secretion of cytokines from mast cells induced by enzymes and the effect of cytokines on the expression of PAR in mast cells are unclear. The effect of Th1 cytokine IL-12 on the secretion of Th2 cytokines of mast cells and its signal transduction pathway are not clear. The present study was designed to investigate the effects of IL-12 on the expression of PARs, IL-4 and IL-6 secretion and histamine release in mast cells, and to explore the possible signal transduction pathways. After mast cells were stimulated, cells and supernatants were collected at each time point. Flow cytometry and real-time quantitative PCR were used to detect the expression of PAR-1, PAR-2, PAR-3 and PAR-4 at the protein level and mRNA level, and the phosphorylation of the signal transduction pathway proteins AktnERKP38 and STAT3 were detected by ELISA (eellular activation of signaling elisa case. The levels of IL-4, IL-6 and histamine in supernatant were detected by ELISA. The results showed that P815 and PAR-2PAR-3 and PAR-4.IL-12 down-regulated the expression of PAR-2 and mRNA in P815 and P815, respectively. IL-12 up-regulated the expression of PAR-4 and mRNA on P815. IL-12 up-regulated the expression of PAR-1 and PAR-3 mRNA in mast cell P815, and the expression of PAR-2 protein and mRNA on mast cell P815 was enhanced by trypsin and trypsin induced by low concentration IL-12 (1 ng/ml) for 60 minutes. The application of IL-12 antibody could block the up-regulation of PAR expression by IL-12. IL-12 promoted the secretion of IL-4 by mast cells in a concentration-dependent manner, and the secretion of IL-4 induced by trypsin and trypsin was inhibited after pretreatment of P815 cells with low concentration of IL-12 (1ng/ml) for 60 minutes. PAR-2 antagonistic peptide FSLLRY-NH_2 could block IL-4 secretion induced by trypsin and trypsin, but had no effect on IL-4 secretion induced by IL-12. IL-12 had no effect on IL-6 secretion and histamine release of mast cells. PD98059 U0126 and LY294002 could block IL-4 secretion induced by IL-12 and inhibit ERK and Akt phosphorylation induced by IL-12. In conclusion, IL-12 can regulate the expression of PARs in mast cells and stimulate the secretion of IL-4.IL-12 by mast cells. It is possible that IL-12 can activate the signal transduction pathways of Akt and ERK by activating the IL-4 secretion of mast cells P815. The secretion of IL-4 by mast cells induced by trypsin and trypsin seems to depend on the activation of PAR-2 pathway. These findings suggest that IL-12 may act as a regulatory factor in allergic inflammatory response by regulating the expression of PAR-2 in mast cells and regulating the secretion of IL-4 by mast cells, thus exerting its function of maintaining the balance between Th1 cytokines and Th2 cytokines. At the same time, the discovery of trypsin and trypsin stimulated mast cells to secrete IL-4 provides a new theoretical basis for the self-amplifying mechanism of mast cell activation.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
本文编号：2180614
[Abstract]:Protease activated receptors (proteinase activated receptors or protease activated receptor PARs) and interleukin-4- (IL-6) were involved in the development of allergic reaction. IL-12 was involved in acquired immune response. However, the effect of IL-12 on the secretion of cytokines from mast cells induced by enzymes and the effect of cytokines on the expression of PAR in mast cells are unclear. The effect of Th1 cytokine IL-12 on the secretion of Th2 cytokines of mast cells and its signal transduction pathway are not clear. The present study was designed to investigate the effects of IL-12 on the expression of PARs, IL-4 and IL-6 secretion and histamine release in mast cells, and to explore the possible signal transduction pathways. After mast cells were stimulated, cells and supernatants were collected at each time point. Flow cytometry and real-time quantitative PCR were used to detect the expression of PAR-1, PAR-2, PAR-3 and PAR-4 at the protein level and mRNA level, and the phosphorylation of the signal transduction pathway proteins AktnERKP38 and STAT3 were detected by ELISA (eellular activation of signaling elisa case. The levels of IL-4, IL-6 and histamine in supernatant were detected by ELISA. The results showed that P815 and PAR-2PAR-3 and PAR-4.IL-12 down-regulated the expression of PAR-2 and mRNA in P815 and P815, respectively. IL-12 up-regulated the expression of PAR-4 and mRNA on P815. IL-12 up-regulated the expression of PAR-1 and PAR-3 mRNA in mast cell P815, and the expression of PAR-2 protein and mRNA on mast cell P815 was enhanced by trypsin and trypsin induced by low concentration IL-12 (1 ng/ml) for 60 minutes. The application of IL-12 antibody could block the up-regulation of PAR expression by IL-12. IL-12 promoted the secretion of IL-4 by mast cells in a concentration-dependent manner, and the secretion of IL-4 induced by trypsin and trypsin was inhibited after pretreatment of P815 cells with low concentration of IL-12 (1ng/ml) for 60 minutes. PAR-2 antagonistic peptide FSLLRY-NH_2 could block IL-4 secretion induced by trypsin and trypsin, but had no effect on IL-4 secretion induced by IL-12. IL-12 had no effect on IL-6 secretion and histamine release of mast cells. PD98059 U0126 and LY294002 could block IL-4 secretion induced by IL-12 and inhibit ERK and Akt phosphorylation induced by IL-12. In conclusion, IL-12 can regulate the expression of PARs in mast cells and stimulate the secretion of IL-4.IL-12 by mast cells. It is possible that IL-12 can activate the signal transduction pathways of Akt and ERK by activating the IL-4 secretion of mast cells P815. The secretion of IL-4 by mast cells induced by trypsin and trypsin seems to depend on the activation of PAR-2 pathway. These findings suggest that IL-12 may act as a regulatory factor in allergic inflammatory response by regulating the expression of PAR-2 in mast cells and regulating the secretion of IL-4 by mast cells, thus exerting its function of maintaining the balance between Th1 cytokines and Th2 cytokines. At the same time, the discovery of trypsin and trypsin stimulated mast cells to secrete IL-4 provides a new theoretical basis for the self-amplifying mechanism of mast cell activation.
【学位授予单位】：汕头大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392
【引证文献】
中国硕士学位论文全文数据库 前1条
1 张旭;针刺对EAE小鼠模型IL-12和MIP-1α的实验研究[D];北京中医药大学;2010年
本文编号：2180614
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