蛋白酶在自身免疫病基底膜损伤中的作用

发布时间：2018-05-14 05:35

本文选题：大疱性类天疱疮 + 趋化作用　；参考：《大连理工大学》2012年博士论文

【摘要】：大疱性类天疱疮(Bullous Pemphigoid, BP)是一种皮肤水疱性自身免疫性疾病,其主要症状表现为张力性水疱的形成、表皮和真皮的分离、严重的炎症细胞浸润及半桥粒和细胞外基质的破坏,该病的发生和抗半桥粒蛋白BP180和BP230的自身性抗体有关。通过IgG被动转移动物模型,我们了解到一些BP的发病机制。在BP的IgG被动转移动物模型中,抗BP180的IgG诱导水疱的形成,而水疱的形成依赖于补体的激活、肥大细胞的脱颗粒和嗜中性粒细胞的浸润。此外,在BP病人和实验动物的水疱液中还发现了许多蛋白水解酶,如血纤维蛋白酶、胶原蛋白酶、嗜中性粒细胞弹性蛋白酶(Neutrophil Elastase, NE)和基质金属蛋白酶(Matrix Metalloproteinase, MMP-9)的存在。由于这些蛋白水解酶能够降解细胞外基质组分,因此他们对于BP水疱的形成有重要的作用。如果能够找到这些蛋白酶在BP中产生及参与病症的途径,并抑制这些途径的发生,则对BP的治疗具有很大的帮助。本论文通过基因敲除和炎症细胞重建的方法分析了NE和肥大细胞蛋白酶-4(Mouse mast cell protease-4, mMCP-4)在BP动物模型中参与疾病发生的机制,更重要的是这一发现为BP的治疗提供了潜在的靶点。我们的实验结果显示NE能够降解鼠BP180重组蛋白,其酶切位点在BP180细胞外结构域的第506和561号氨基酸上,产生的小肽分别命名为p506和p561。在体内和体外的实验结果都表明,p561小肽对嗜中性粒细胞有趋化活性。向B6小鼠体内局部注射NE能够将嗜中性粒细胞招募至皮肤,而如果向小鼠体内同时注射NE的抑制剂α1-PI(al-proteinase inhibitor)则无嗜中性粒细胞的浸润。更重要的是,NE能够直接降解人和鼠的BP180以及天然状态存在的人BP180三聚体分子。这些结果表明：1)NE能够直接造成细胞外基质的损伤；2)NE降解BP180后产生具有嗜中性粒细胞趋化活性的小肽,这个小肽能够加重发病初期的病症强度。除了NE外,mMCP-4也和许多自身免疫性疾病和炎症反应有关,然而其参与疾病的机制尚不清楚。在实验中,我们发现mMCP-4对于自身免疫性疾病BP动物模型中水疱的形成至关重要。mMCP-4缺陷型小鼠能够抑制BP的发生,但mMCP-4-/-小鼠中补体的激活、肥大细胞的脱颗粒和嗜中性粒细胞的早期浸润都和野生型类似。而向mMCP-4-/-小鼠体内注射致病性IgG后,由于mMCP-4的缺失,激活的MMP-9的量明显低于野生型。野生型和mMCP-4-/-小鼠的肥大细胞重建不能完全恢复激活的MMP.9的数量。在mMCP-4-/-(?)勺小鼠体内重建mMCP-4+/+小鼠的肥大细胞,能够使机体产生水疱和嗜中性粒细胞的浸润,而重建mMCP-4-/-小鼠的肥大细胞则不能。mMCP-4也能够在体内和体外降解半桥粒蛋白BP180,并造成表皮和真皮的分离。这些实验结果表明,mMCP-4在BP的发病机制中发挥两个作用：它既能够激活MMP-9,又能直接降解BP180,从而造成半桥粒和基底膜的细胞外基质损伤。
[Abstract]:Bullous pemphigoid (BPP) is a skin blistering autoimmune disease characterized by the formation of tensional blisters, the separation of epidermis and dermis, severe inflammatory cell infiltration and the destruction of semidomere and extracellular matrix. The development of the disease is related to the autoantibodies against hemidopontin BP180 and BP230. Through the IgG passive transfer animal model, we know some BP pathogenesis. In BP IgG passive transfer animal model, the formation of vesicles was induced by anti-BP180 IgG, and the formation of vesicles depended on the activation of complement, degranulation of mast cells and infiltration of neutrophils. In addition, many proteolytic enzymes such as blood fibrinase, collagenase, neutrophil elastase (NEUtrophil) and matrix metalloproteinase (MMP-9) were found in the blister fluid of BP patients and laboratory animals. Because these proteolytic enzymes can degrade extracellular matrix components, they play an important role in the formation of BP blister. If we can find the pathway that these proteases produce and participate in the disease in BP, and inhibit the occurrence of these pathways, it will be of great help to the treatment of BP. In this paper, the mechanism of NE and mast cell protease-4mouse mast cell protease-4 (mMCP-4) involved in the development of disease in BP animal model was analyzed by gene knockout and inflammatory cell reconstruction. More importantly, this discovery provided a potential target for the treatment of BP. Our results showed that NE could degrade mouse BP180 recombinant protein, and its restriction site was on amino acids 506 and 561 of the extracellular domain of BP180, and the resulting peptides were named p506 and p561respectively. The results in vivo and in vitro showed that p561 small peptide was chemoattractant to neutrophils. Local injection of NE into B6 mice could recruit neutrophils into the skin, but no infiltration of neutrophils was observed if the inhibitor 伪 1-PI(al-proteinase inhibitor of NE was injected simultaneously into the skin of B6 mice. More importantly, it can directly degrade BP180 of human and mouse and human BP180 trimer in natural state. These results suggest that the small peptide with neutrophil chemotactic activity can be produced after the degradation of BP1 80 by 1: 1 ne, which can aggravate the intensity of the disease in the early stage of the disease. In addition to NE, mMCP-4 is also associated with many autoimmune diseases and inflammatory reactions, but the mechanism of its involvement in the disease is unclear. In our experiment, we found that mMCP-4 is very important to the formation of blisters in the animal model of autoimmune disease BP. MMCP-4 deficient mice can inhibit the occurrence of BP, but the complement activation in mMCP-4-r-mice. Degranulation of mast cells and early infiltration of neutrophils are similar to wild type. However, after injection of pathogenic IgG into mMCP-4-r-mice, the amount of activated MMP-9 was significantly lower than that of wild type due to the absence of mMCP-4. In wild type and mMCP-4-r-mice, mast cell remodeling did not fully restore the number of activated MMP.9. At mMCP-4-r-p-) Reconstructing mast cells in mMCP-4 / mice in spoons can cause blisters and neutrophils to infiltrate the body. But the mMCP-4-r-mouse mast cells could not. MMCP-4 could also degrade BP180 in vivo and in vitro, resulting in the separation of epidermis and dermis. These results suggest that mMCP-4 plays two roles in the pathogenesis of BP: it not only activates MMP-9, but also directly degrades BP180, thus causing damage to the extracellular matrix of the semidopontine and basement membrane.
【学位授予单位】：大连理工大学
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R758.6

【共引文献】