Nogo-B调控循环纤维细胞迁移能力及其在肺纤维化形成中的作用

The bone marrow-derived circulating fibrocytes (CF) play an important role in the development of pulmonary fibrosis. In preliminary study, we successfully established pulmonary fibrosis model in mice by intratracheal injection of bleomycin. The CF was isolated and we found that both of number of CF and the expression of Nogo-B in CF were significantly increased than that from normal mice. What’s more, the results of Transwell experiment showed that the migration of Nogo-B knock-out CF which was isolated from the established Nogo-B knock-out mice in our previous study, was significantly decreased compare with wild type. Based on these findings, we plan to use the techniques of gene knock-out, and bone marrow transplantation to observe the influence of Nogo-B on CF migration in vivo and the development of pulmonary fibrosis. Furthermore, we are going to use the techniques of lentiviral transfection and RNA interference to explore the influence of Nogo-B on CF cytoskeleton, cell adhesion, migration and transendothelial migration, in vitro. The mechanism of the effect of Nogo-B is going to be explored as well. This study may not only provide important information for the investigations focusing on the mechanism of CF migration, but also may provide a new insight to the prevention and treatment of pulmonary fibrosis.

骨髓来源的循环纤维细胞（CF）迁移在肺纤维化的发生、发展中起重要作用，深入研究其调控机制具有重要意义。前期研究显示，小鼠肺纤维化组织中CF比例显著增加，且CF高表达Nogo-B；从Nogo-B基因敲除小鼠中分离的CF，其体外迁移能力显著降低。据此，本研究拟以循环纤维细胞为研究对象，采用基因敲除和骨髓移植等手段，观察Nogo-B表达变化对CF体内迁移能力及小鼠肺纤维化形成的影响；在此基础上，采用慢病毒转染及RNA干扰等手段，进一步于体外观察Nogo-B表达变化对CF细胞骨架、黏附、移行及跨内皮迁移能力的影响，并探讨其分子机制。以求证实Nogo-B促进CF迁移的作用，并力求阐明其在肺纤维化形成中的作用及地位，以期为肺纤维化的预防和救治提供新的思路。

特发性肺纤维化是一类临床常见的间质性肺疾病，其发病机制尚未完全阐明。骨髓来源的循环纤维细胞迁移和肺泡上皮细胞间质转化在肺纤维化的发生、发展中起重要作用，深入研究其调控机制具有重要意义。为此，本研究主要探索了Nogo-B，一种网状蛋白家族4的主要成员，调控循环纤维细胞的肺部迁移能力而参与肺纤维化的作用及机制，不仅如此，我们还延伸研究了Nogo-B促进肺泡上皮细胞间质转化而参与肺纤维化的作用及机制。研究结果①显示肺纤维化小鼠模型中的循环纤维细胞高表达Nogo-B，且肺组织中循环纤维细胞的数量显著增多；分别于野生型小鼠和Nogo-B敲除小鼠的外周血中分离循环纤维细胞，体外实验显示Nogo-B -/- 循环纤维细胞的迁移能力较前者显著降低，而Nogo-B 敲除小鼠的肺组织中循环纤维细胞显著减少，其肺纤维化程度降低，胶原沉积减少；同时我们发现Nogo-B-/-循环纤维细胞中TGF-β通路被抑制，恢复TGF-β的表达，可逆转Nogo-B -/-循环纤维细胞的迁移能力，这提示Nogo-B可通过TGF-β信号通路调控循环纤维细胞迁移而参与肺纤维化的形成。结果②显示肺纤维化小鼠的肺泡上皮细胞亦高表达Nogo-B，过表达Nogo-B可促进肺泡上皮细胞株A549的上皮间质转化（EMT），通过生物信息学工具，我们分析了过表达和正常表达Nogo-B细胞株之间的基因表达差异，发现MMP14可能介导了Nogo-B促进肺泡上皮细胞EMT的作用，上调和下调A549中Nogo-B的表达水平，可分别增加和降低MMP14的表达，而恢复Nogo-B-/- A549细胞中MMP14的表达，可逆转其EMT能力，此外我们还发现MMP14可促进TGF-β1从蛋白前体中释放，这提示Nogo-B可能通过MMP14调控TGF-β1的形成而促进肺泡上皮细胞EMT而参与肺纤维化的形成。综上所述：本项目证实了Nogo-b不仅可以通过调控循环纤维细胞的肺部迁移能力促进肺纤维化的形成，还可通过调节肺泡上皮细胞间质转化而参与肺纤维化，同时我们较为深入地探索了其分子机制，为肺纤维化的诊治研究提供了新的思路和理论依据。