肥大细胞蛋白酶激活受体表达及功能初探

发布时间：2018-03-27 06:37

本文选题：蛋白酶激活受体　切入点：肥大细胞　出处：《南方医科大学》2010年硕士论文

【摘要】： 蛋白酶激活受体(protease activated receptors,PARs)是丝氨酸蛋白酶受体,目前在人和小鼠体内共发现了四种PARs,分别为PAR-1、PAR-2、PAR-3和PAR-4。它们的基因序列于1991、1994、1997和1998年被先后克隆出来。其中PAR-1是凝血酶和胰蛋白酶的受体,PAR-2是胰蛋白酶、类胰蛋白酶和弹性蛋白酶的受体,PAR-3是凝血酶受体,PAR-4是凝血酶和胰蛋白酶受体。很久以来就认识到肥大细胞分泌介质参与过敏反应。作为初级效应细胞,肥大细胞既参与急性过敏反应又参与慢性过敏反应。肥大细胞一旦被激活,即释放其已预先合成的和新产生的介质执行其生物学功能。这些介质包括组胺、肝素、蛋白酶以及一系列的细胞因子如IL-4、IL-5和IL-6等,这些细胞因子可以在生理和病理情况下参与调节细胞的生长、分泌和迁移等。有关PARs与过敏反应性疾病的研究发现,PARs尤其是PAR-2与过敏反应密切相关。一些过敏原如尘螨、蟑螂过敏原可以激活PAR-2。但通过疾病动物模型得到的实验结果却不尽相同：有些结果显示PAR-2激活引起呼吸道的过敏性炎症反应,有些则发现PAR-2的激活在过敏性疾病中起到保护作用。所以有待于进一步深入探讨PARs在过敏反应性疾病中的作用。虽然对于参与过敏反应的一些细胞如内皮细胞、上皮细胞、中性粒细胞、单核细胞、淋巴细胞等的PARs功能已经有所了解,然而对过敏性疾病的核心效应细胞—肥大细胞的PARs功能还知之甚少。因此本研究将进一步的探讨肥大细胞PARs的功能,为过敏性疾病发病机制的研究提供实验数据。一、蛋白酶激活受体在肥大细胞的表达用逆转录聚合酶链式反应、流式细胞术及免疫荧光细胞染色分析方法,在mRNA水平和蛋白水平检测PAR-1、PAR-2、PAR-3和PAR-4在小鼠肥大细胞MC/9和P815的表达。结果小鼠肥大细胞MC/9和P815在mRNA水平和蛋白水平均表达PAR-1、PAR-2、PAR-3和PAR-4。结论肥大细胞MC/9和P815表达PAR-1、PAR-2、PAR-3和PAR-4。二、丝氨酸蛋白酶对肥大细胞IL-4分泌的影响肥大细胞P815培养后用胰蛋白酶、类胰蛋白酶、凝血酶单独或与相应抑制剂凝血酶抑制剂、大豆胰蛋白酶抑制剂、α抗胰蛋白酶抑制剂、亮肽素结合处理肥大细胞,收集上清并用ELISA方法检测IL-4的水平。结果：胰蛋白酶和类胰蛋白酶能促进肥大细胞P815分泌IL-4明显增多(P0.05)；大豆胰蛋白酶抑制剂和α抗胰蛋白酶能阻断胰蛋白酶引起的肥大细胞P815 IL-4分泌(P0.05)；亮肽素能阻断类胰蛋白酶引起的肥大细胞P815 IL-4分泌(P0.05),而凝血酶对肥大细胞P815分泌IL-4无明显影响(P0.05)。结论：胰蛋白酶和类胰蛋白酶刺激肥大细胞IL-4分泌,而凝血酶对肥大细胞IL-4的分泌功能无影响。
[Abstract]:Protease activated receptor (PARs) is a serine protease receptor. At present, four species of pars have been found in human and mouse. They are PAR-1, PAR-2, PAR-3 and PAR-4. their gene sequences were cloned in 199119941997 and 1998.The receptor of PAR-1 is thrombin and trypsin, PAR-2 is trypsin. Trypsin and elastase receptors PAR-3 are thrombin receptors PAR-4 are thrombin and trypsin receptors. It has long been recognized that mast cell secretory mediators are involved in allergic reactions. As primary effector cells, mast cells are involved in both acute and chronic allergic reactions. That is, release its presynthesized and newly produced media for its biological functions. These mediums include histamine, heparin, protease, and a series of cytokines such as IL-4, IL-5 and IL-6. These cytokines are involved in the regulation of cell growth, secretion and migration, both physiologically and pathologically. Studies on PARs and allergic reactivity diseases have found that PARs, especially PAR-2, are closely related to allergic reactions. Some allergens such as dust mites, Cockroach allergens activate PAR-2. But the results from animal models of disease vary: some of the results show that PAR-2 activates allergic inflammation in the respiratory tract. Some have found that the activation of PAR-2 plays a protective role in allergic diseases. Therefore, it is necessary to further explore the role of PARs in allergic diseases, although for some cells involved in allergic reactions, such as endothelial cells, epithelial cells, The PARs function of neutrophils, monocytes, lymphocytes, etc. However, little is known about the PARs function of mast cells, which is the core effector of allergic diseases. Therefore, this study will further explore the function of mast cell PARs in order to provide experimental data for the study of pathogenesis of allergic diseases. The first is the expression of protease activated receptor in mast cells. Reverse transcriptase polymerase chain reaction, flow cytometry and immunofluorescence staining were used. The expressions of PAR-1PAR-2PAR-3 and PAR-4 in mouse mast cell MC/9 and P815 were detected at the level of mRNA and protein. Results the expression of PAR-1PAR-2PAR-3 and PAR-4in mouse mast cell MC/9 and P815 at the mRNA level and protein level was detected. Conclusion the expression of PAR-1PAR-2PAR-2PAR-3 and PAR-4Mast cell in mast cell MC/9 and P815 were detected. Effect of serine protease on IL-4 secretion of mast cells. Mast cells P815 were treated with trypsin, trypsin, thrombin alone or with the corresponding inhibitor thrombin inhibitor, soybean trypsin inhibitor, 伪 -antitrypsin inhibitor, and leucopeptide binding. The levels of IL-4 were detected by ELISA. Results: trypsin and trypsin could increase the secretion of IL-4 in mast cells, and soybean trypsin inhibitor and 伪 -antitrypsin could block the secretion of IL-4 by trypsin. Leucopeptide could block the P815 IL-4 secretion of mast cells induced by trypsin, while thrombin had no significant effect on the IL-4 secretion of mast cells. Conclusion: trypsin and trypsin stimulate the secretion of IL-4 in mast cells. Thrombin had no effect on the secretory function of mast cell IL-4.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R392

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