基质素2网络结构稳定性在放射肺损伤中的作用的机制研究

Matrilin 2 is a widely distributed extracellular matrix protein that participates in the formation of both collagen-dependent and collagen-independent filamentous macromolecular network with adaptor functions which are involved in the development and homeostasis of network of extracellular matrix. We first reported that two alternative splicing isoforms with unique domain of matrilin 2 that have distinct roles in regulating oligomerization and proteolysis, both of which have an impact on the homeostasis of the matrilin filamentous network of the extracellular matrix. Radiation pulomonary injury is caused by the accumulation of extracellular matrix proteins in the alveolar interstitial space, resulting in fibrosis. Although the matrilin 2 null mice showed no gross abnormalities during embryonic or adult development,the accumulating evidence showed that matrilin 2 has been involved in reorganization of tissue architecture in liver cirrhosis and hepatocellular carcinoma, the balance of keratinocyte and fibroblast in response to wounding, participation in peripheral nerve regeneration,down reglated in early phase of muscle injury and then increased in its late phase. There exist seven putative Smad-binding sites with human matrilin 2 promoter as well as exon I. The role in the accumulation of extracellular matrix proteins is of upmost significance in understanding fibrosis of radiation pulmonary injury. Recently, we found pulmonary fibroblast cell line express two alternative splicing isoforms of matrilin 2. The radiation treated cells showed higher level of matrilin 2 mRNA and protein expression than the controls. We hypothesis the homeostasis of the matrilin 2 filamentous network plays an essential role in the pathogenesis of radiation pulmonary injury. In this project, we will investigate the distinct roles of the two alternative splicing isoforms of matrilin 2 in the synthesis of major extracellular matrix protein in vitro using pulmonary fibroblast line and lung cancer cell line, and in vivo using radiation pulmonary injury models. Identifying homeostasis of the matrilin 2 filamentous network may provide new clues in understanding the mechanisms of radiation pulmonary injury and in the development of specific treatments.

放射性肺损伤的主要形态学改变是肺泡间隙处细胞外基质过度聚集。基质素2作为新近发现在体内广泛分布的一种细胞外基质蛋白。它能与胶原蛋白或其它基质蛋白形成胶原依赖性和胶原非依赖性的网络结构。我们首次报道基质素2独特功能域19个氨基酸不同的剪切影响该蛋白多聚体形成和酶切，从而影响基质素2网络在细胞外基质网络中的稳定性。我们最近的初步结果还发现放射线处理小鼠使其mRNA 和蛋白都增高，两种基质素2比例亦发生变化。据此我们提出了基质素2的剪切及其酶切产物在肺泡间隙处细胞外基质过度聚集和纤维化过程中扮演重要作用的设想。为证实此假说，本项目拟体外观察两种不同剪接方式的基质素2及其酶切产物对肺成纤维细胞合成和降解细胞外基质的影响，同时研究放射性肺损伤动物模型基质素2与其它基质蛋白的合成降解的相互关系。旨在阐明基质素2分子结构稳定性对自身及与其它细胞外基质蛋白稳态的调节作用，从而为放射性肺损伤防治提供新的思路

放射性肺损伤是核辐射灾难事故、核恐怖事件、胸部肿瘤治疗和骨髓移植预处理的常见并发症。放射性肺损伤发生率较高，缺少有效治疗方法，已经成为临床上一大重要难题。放射性肺损伤的发病机理是受照射部位的正常肺组织无菌性、特异性炎症，肺泡上皮细胞持续性损伤及肺泡间隔处细胞外基质的反复损伤（破坏），修复，重建，从而使该处细胞外基质合成与降解的平衡破坏，最终导致此处细胞外基质过度聚集。肺泡间隔处细胞外基质合成与降解是受多种因素相互影响综合调控的复杂过程。.我们通过动物实验和离体细胞培养首次报道发射处理的肺组织和 HPAEpiC 细胞存在高水平的基质素2表达。质粒，慢病毒，MTT增殖实验和流式细胞分析实验发现基质素2促进HPAEpiC细胞周期相关基因的转录调节,从而保护发射性肺损伤。小鼠系膜细胞细胞模型发现高糖诱导基质素-2蛋白质和mRNA的表达。TGF-β1和Smad3抑制剂明显抑制了基质素-2的表达，且呈时间依赖性。TGF-β1/Smad3抑制剂可以抑制TGF-β1诱导基质素-2的表达。基质素2可以预防TNF-α诱导的WB-F344细胞死亡，且通过JNK信号通路抑制TNFα诱导的WB-F344细胞。我们的结果首次报道放射损伤与非胶原网络稳定性有关，并且初步探索其发病机理。