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应用精密切割肺切片技术研究DDR2在肺纤维化中的作用

黄锡惠¹, 程涛², 牟棱¹, 卜歆³, 魏新茹^1,4,*

¹呼吸疾病全国重点实验室，粤港澳呼吸系统传染病联合实验室，广州医科大学附属第一医院，广州 510000；²上饶市人民医院，上饶 334000；³空军军医大学基础医学院生物化学与分子生物学教研室，西安 710032；⁴深圳霁因生物医药转化研究院，深圳 518000

摘要

肺纤维化是一类以肺组织结构破坏、成纤维细胞过度活化增生并分泌和累积大量细胞外基质(extracellular matrix, ECM)、肺功能受损为特征的肺间质疾病。由于该疾病的复杂性，目前还未找到合适的模拟人肺纤维化的动物模型。精密切割肺切片(precision-cut lung slice, PCLS)是近年来广泛应用的一种体外研究肺生理和发病机制的方法。该方法是介于器官与细胞水平之间的体外培养技术，因其可保存完好的肺组织结构和肺组织内各种类型的呼吸道细胞，在一定程度上模拟了体内肺环境，有利于观察各类型细胞和ECM的相互作用，可弥补其他模型如细胞培养的局限性。为进一步探讨盘状结构域受体2 (discoidin domain receptor 2, DDR2)在肺纤维化中的作用，本研究成功构建Ddr2flox/flox小鼠，利用Cre-LoxP系统和PCLS技术在小鼠PCLS中成功敲降DDR2蛋白的表达。小鼠PCLS可被转化生长因子-β1 (transforming growth factor β1, TGF-β1)在体外成功诱导纤维化，且能够很好地模拟体内环境；而在敲降DDR2表达的小鼠PCLS体外TGF-β1诱导纤维化模型中，其各纤维化相关因子表达显著降低，提示敲降DDR2可有效改善纤维化。本文的研究方法和结果为DDR2作为肺纤维化治疗靶点的临床前研究提供了新的视角。

关键词： 精密切割肺切片; 肺纤维化; DDR2; Cre-LoxP系统

Application of precision-cut lung slice technology to study the role of DDR2 in pulmonary fibrosis

HUANG Xi-Hui¹, CHENG Tao², MOU Ling¹, BO Xin³, WEI Xin-Ru^1,4,*

¹State Key Laboratory of Respiratory Disease, Guangdong-Hong Kong-Macao Joint Laboratory of Respiratory Infectious Disease, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510000, China；²Shangrao People’s Hospital, Shangrao 334000, China；³Department of Biochemistry and Molecular Biology, School of Basic Medicine, Air Force Military Medical University, Xi’an 710032, China；⁴Shenzhen International Institute for Biomedical Research, Shenzhen 518000, China

Abstract

Pulmonary fibrosis is a severe lung interstitial disease characterized by the destruction of lung tissue structure, excessive activation and proliferation of fibroblasts, secretion and accumulation of a large amount of extracellular matrix (ECM), and impaired lung function. Due to the complexity of the disease, a suitable animal model to mimic human pulmonary fibrosis has not yet been established. Precision-cut lung slice (PCLS) has been a widely used in vitro method to study lung physiology and pathogenesis in recent years. This method is an in vitro culture technology at the level between organs and cells, because it can preserve the lung tissue structure and various types of airway cells in the lung tissue, simulate the in vivo lung environment, and conduct the observation of various interactions between cells and ECM. Therefore, PCLS can compensate for the limitations of other models such as cell culture. In order to explore the role of discoidin domain receptor 2 (DDR2) in pulmonary fibrosis, Ddr2flox/flox mice were successfully constructed. The Cre-LoxP system and PCLS technology were used to verify the deletion or knockdown of DDR2 in mouse PCLS. Transforming growth factor β1 (TGF-β1) can induce fibrosis of mouse PCLS in vitro, which can simulate the in vivo environment of pulmonary fibrosis. In the DDR2 knock down-PCLS in vitro model, the expression of various fibrosis-related factors induced by TGF-β1 was significantly reduced, suggesting that knocking down DDR2 can inhibit the formation of pulmonary fibrosis. The results provide a new perspective for the clinical study of DDR2 as a therapeutic target in pulmonary fibrosis.

Key words: precision-cut lung slice; pulmonary fibrosis; DDR2; Cre-LoxP system

收稿日期：　　录用日期：

通讯作者：魏新茹　　E-mail: weixinru2012@163.com

引用本文：

黄锡惠, 程涛, 牟棱, 卜歆, 魏新茹. 应用精密切割肺切片技术研究DDR2在肺纤维化中的作用[J]. 生理学报 2023; 75 (4): 512-520.

HUANG Xi-Hui, CHENG Tao, MOU Ling, BO Xin, WEI Xin-Ru. Application of precision-cut lung slice technology to study the role of DDR2 in pulmonary fibrosis. Acta Physiol Sin 2023; 75 (4): 512-520 (in Chinese with English abstract).