大蹼铃蟾皮肤分泌物βγ-晶状体蛋白与三叶因子复合物和天然免疫关系探讨
文献类型：学位论文
| 作者 | 向阳 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-06 |
| 授予单位 | 中国科学院研究生院 |
| 授予地点 | 北京 |
| 导师 | 张云 |
| 关键词 | 非晶状体βγ-晶状体蛋白 βγ-CAT 炎症小体 促炎性程序性细胞 促炎性程序性细胞 死亡 自身危险信号 区域性自身危险信号 caspase-1 |
| 其他题名 | Non-lens betagamma–crystallin and Trefoil factor Complex: a possible role in innate immunity |
| 学位专业 | 动物学 |
| 中文摘要 | bg-CAT是一个从中国西南部特有的两栖动物大蹼铃蟾(Bombina maxima)皮肤分泌物中分离得到的，由非晶状体bg-晶状体蛋白(a-亚基, CAT-a)和三叶因子蛋白(b-亚基, CAT-b)结合在一起形成的天然复合物。其a-亚基与b-亚基通过非共价的方式交联形成ab2, 分子量为72-kDa。CAT-a的N-末端部分(CAT-aN, 1-170 氨基酸残基)是两个bg-晶状体结构域，C-末端部分(CAT-aC)与产气荚膜梭菌ε毒素的膜插入结构域有序列同源性。 本实验室前期工作发现bg-CAT处理后的人脐静脉内皮细胞多种应激蛋白以及免疫相关分子表达上调，同时，bg-CAT也可以强烈地激活caspase-1。基于以上研究基础，我们推测bg-CAT可能参与免疫调节，而其在大蹼铃蟾自身的生理功能也很可能就是调节皮肤免疫。 炎症小体是指可以激活炎症性caspase以及细胞因子并促进白细胞介素1-beta（IL1b）、白细胞介素18以及白细胞介素33成熟与分泌的大分子复合物。近年来的研究发现炎症小体在天然免疫系统中发挥着重要作用，它可以多种被病原相关分子模式（PAMPs）激活，也可以被很多种危险信号相关分子模式激活（DAMPs）。bg-CAT可以激活THP-1细胞NLRP3炎症小体，并促进白细胞介素1-beta以及白细胞介素18的成熟与分泌。bg-CAT刺激后的THP-1细胞表现出钾离子外流、胞外钙离子内流以及活性氧释放等特征。胞外高浓度钾离子以及胞内钙离子螯合剂都可以明显抑制NLRP3炎症小体的激活以及白细胞介素1-beta的成熟与分泌，说明钾离子的外流和胞质中钙离子浓度的增加都是bg-CAT诱导的NLRP3炎症小体激活所必需的。 依赖caspase-1的促炎性程序性细胞死亡即pyroptosis，是近年来发现的一种新的程序性细胞死亡方式。Pyroptosis发生过程中，依赖于caspase-1活性的孔道在细胞膜上形成并释放细胞内容物，触发免疫反应。bg-CAT可以诱导人真皮成纤维细胞死亡，而这种死亡可以被caspase-1抑制剂所抑制，因此认为bg-CAT诱导成纤维细胞死亡的方式为pyroptosis。bg-CAT诱导的成纤维细胞的死亡也可以被胞外钙离子螯合剂EGTA以及胞内钙离子螯合剂BAPTA-AM所抑制，说明钙离子在bg-CAT诱导的成纤维细胞pyroptosis过程中起到重要作用。bg-CAT可以引起明显的细胞溶酶体破裂，而且胞吞抑制剂M-beta-CD和细胞松弛素D都可以明显抑制bg-CAT诱导的pyroptosis，说明内吞过程在bg-CAT形式功能过程中能够起到重要作用。 以上的结果有力地支持了我们认为bg-CAT参与天然免疫的推测，前期的实验证明bg-CAT对表皮层的角质形成细胞没有任何生物学效应，因此我们推测bg-CAT是区域性危险信号，在正常的生理状态下，bg-CAT存在于皮肤表面不发挥作用，而当皮肤组织受损时，bg-CAT进入皮肤更深层次，诱导真皮层成纤维细胞发生炎症性死亡，诱导免疫细胞分泌促炎症性细胞因子，进一步诱发免疫反应。为了进一步验证我们的假设，我们培养了大蹼铃蟾成纤维细胞，以在以后的研究中更深入研究bg-CAT的免疫调节功能。 在本研究中，我们首次提出了区域性危险信号（Regional innate danger signal）的概念，bg-CAT结构相似的分子并不只是存在于大蹼铃蟾，在其他两栖动物、爬行类、鸟类以及哺乳类都有类似结构的分子，因此我们推测这种区域性危险信号可能广泛存在于脊椎动物中而参与免疫调节。另外，本研究也为揭示很多细节尚不清楚的炎症小体以及pyroptosis的相关信号通路提供一些基础数据。最后，我们对bg-CAT的研究也为最终揭示非晶状体bg-晶状体蛋白的生理功能提供新的线索。 |
| 英文摘要 | bg-CAT is a naturally existing 72-kDa complex of non-lens bg-crystallin (a-subunit, CAT-a) and trefoil factor (b-subunit, CAT-b), with a non-covalently linked form of ab2, identified from frog Bombina maxima skin secretions. The N-terminal part (CAT-aN, 1-170 residues) of CAT-a are two bg-crystallin domains, while the rest C-terminal part (CAT-aC) shows sequence homology to membrane insertion domain of Clostridium perfringens epsilon toxin. It was previously found that bg-CAT is able to upregulate many cell-stress related and proinflammatory proteins in Human umbilical vein endothelial cells. And bg-CAT can also strongly activate caspase-1 in Human umbilical vein endothelial cells and human colon cancer cell line HCT116 cells. Based on these findings, we hypothesized that bg-CAT may be involved in innate immune system. And we can further suppose that immune regulation may be the physiological function of bg-CAT in Bombina maxima skin. The term inflammasome was coined to describe a high molecular molecular weight complex that activates inflammatory caspases ultimately caspase-1 and induces the maturation and secretion of proinflammatory cytokines such as IL1b and IL18. Both pathogen associated molecular patterns (PAMPs) and Danger associated molecular patterns (DAMPs) activate inflammasomes. In the present study, bg-CAT was found to activate NLRP3 inflammasomes in THP-1 cells, which subsequently induced the maturation and secretion of proinflammatory cytokines IL1b and IL18. After exposure to 1nM concentration of bg-CAT, THP-1 cells showed the phenomena of potassium efflux, calcium influx and ROS generation. Extracellular high concentration of potassium and calcium chelator BAPTA-AM strongly inhibited the activation of NLRP3 inflammasome and IL1b secretion in THP-1 cells. These results suggested that potassium efflux and increase of the calcium concentration inside the cell were essential for the activation of NLRP3 inflammaosomes by bg-CAT. Pyropotosis is a more recently recognized form of regulated cell death dependent of the activation of caspase-1. Caspase-1 dependent pores are formed on the plasma membrane of pyroptotic cells. Subsequently, cell contents are released out of the cells through these pores. The released cell contents would activate inflammatory responses. bg-CAT was able to induce cell death of human dermal fibroblasts and cell death effect could be inhibited with caspase-1 inhibitor. Thus, we deduced that the bg-CAT induced cell death type in fibroblasts was pyroptosis. The cell death effect could also be strongly inhibited with both extracellular calcium chelator EGTA and intracellular calcium chelator BAPTA-AM. These findings strengthened the importance of calcium in the process of pyroptosis induced by bg-CAT. Lysosomes inside the fibroblast cells were disrupted and the endocytosis inhibitor M-beta-CD and cytochalasin D could inhibit the pyroptosis, which suggested that endocytosis of bg-CAT may be essential for it to exert its function. The findings that bg-CAT could activate NLRP3 inflammasome and induce pyroptosis strongly supported the hypothesis that bg-CAT may act as immune modulator. Thus, we proposed a new concept, Regional innate danger signal (RIDS). RIDS locates in limited organs or tissues without biological function in its resident location. But when the body is on pathological condition, the RIDS may reach other tissues or organs, and trigger immune responses. bg-CAT might be a typical example. To further prove our hypothesis, fibroblast cells originated from Bombina maxima skin were cultured. In the present study, we proposed the concept Regional innate danger signal (RIDS) for the first time. And also, our present study may be beneficial to understand the basic mechanisms of inflammasome activation and pyroptosis. The methods of amphibian cell culture generated may be useful for amphibian physiological study. |
| 语种 | 中文 |
| 公开日期 | 2013-04-24 |
| 源URL | [http://159.226.149.42:8088/handle/152453/7388]  |
| 专题 | 昆明动物研究所_动物活性蛋白多肽组学 |
| 推荐引用方式 GB/T 7714 | 向阳. 大蹼铃蟾皮肤分泌物βγ-晶状体蛋白与三叶因子复合物和天然免疫关系探讨[D]. 北京. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：昆明动物研究所
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