DNA去甲基化酶TET2在特发性肺纤维化发生中的作用及其调控机制研究

Idiopathic pulmonary fibrosis (IPF) is a fatal interstitial lung disease with unclear pathogenesis and lack of effective therapeutic drugs. In recent years, the role of DNA methylation in the development of pulmonary fibrosis has been paid attention, but the mechanism of the change of DNA methylation level in the lung and the downstream pathway are far from clear. Based on previous studies that we first found the expression of DNA demethylase TET2 was significantly decreased in human and mouse pulmonary fibrosis lesions and the profibrotic factor transforming growth factor (TGF)-β1 significantly downregulated the expression of TET2 protein and autophagy in fibroblasts, we propose to use TET2 as a key link to explore the signaling pathway that TGF-β1 downregulates TET2 and the effect of TET2 downregulation on the downstream methylation of Beclin1 and fibroblast autophagy, proliferation, migration, and differentiation into myofibroblast. This research project firstly observes the change of TET2 in pulmonary fibrosis at the overall level, and then constructs overexpression and knockdown of lentivirus to elucidate the role of TET2 in the development of pulmonary fibrosis, and further reveals the upstream and downstream signaling mechanism of TET2 change in pulmonary fibrosis development at the cellular level, for the first time to clarify the role of TET2 in IPF and its regulatory mechanism, providing a new target for the prevention and treatment of IPF.

特发性肺纤维化（IPF）是一种致死性间质性肺疾病，发病机制不明，缺乏有效治疗药物。近年来DNA甲基化修饰在肺纤维化发生中的作用受到重视，但肺内DNA甲基化水平变化的机制及下游途径远未阐明。我们基于前期研究首次发现人和小鼠的肺纤维化病变中DNA去甲基化酶TET2表达明显降低，促纤维化因子转化生长因子（TGF）-β1显著下调成纤维细胞TET2蛋白表达和自噬水平的初步结果，提出以TET2为关键环节，探讨TGF-β1下调TET2表达的信号通路，以及TET2表达下调对下游基因Beclin1的甲基化和成纤维细胞自噬、增殖、迁移和向肌成纤维细胞分化的影响。本项目首先在整体观察肺纤维化时TET2的变化，再构建过表达和敲低慢病毒阐明TET2在肺纤维化发生中的作用，并进一步在细胞水平揭示肺纤维化发生中TET2变化的上游和下游信号机制，首次阐明TET2在IPF中的作用及其调控机制，为IPF的防治提供新的靶点。

DNA甲基化修饰在肺纤维化发生中的作用受到重视，但肺内DNA甲基化水平变化的机制及下游途径远未阐明。目的：探讨DNA去甲基化酶TET2在肺纤维化发生发展中的作用并初步研究其调控机制，为肺纤维化的防治提供新的理论依据。方法：(1)动物实验：气管注射博来霉素(BLM)复制小鼠肺纤维化模型，检测肺组织TET2和IDAX的表达。TET2过表达病毒在肺组织表达30 d天后，复制纤维化模型。造模7d时检测急性肺损伤的病变情况；造模21 d时检测肺纤维化的病变情况。(2)细胞实验：2 ng/ml TGF-β1处理HFL1细胞48 h 后，检测TET2和IDAX的蛋白表达。1×107TU/ml的TET2过表达或敲减慢病毒分别感染 HFL1细胞72 h后，再用2 ng/ml TGF-β1处理HFL1细胞48 h，Western blot检测α-SMA、Col Ⅰ、Col Ⅲ和FN的蛋白表达。2 ng/ml TGF-β1处理HFL1细胞15 min 后，Western blot 检测Smad3磷酸化水平。20 μm的Smad3抑制剂（SIS3）预处理HFL1细胞18 h，再用2 ng/ml TGF-β1处理HFL1细胞48 h，Western blot 检测TET2和IDAX蛋白表达。结果：(1)动物实验：造模后7 d和21 d肺组织中TET2的mRNA和蛋白表达降低、IDAX的mRNA和蛋白表达升高。过表达TET2后，急性肺损伤得到缓解，TNF-α和IL-1β的表达下降，IL-10的表达升高，白细胞数量和肺泡灌洗液中的蛋白浓度也下降。过表达TET2后，肺纤维化时期的肺组织损伤得到缓解，胶原沉积减少，α-SMA、Col Ⅰ和Col Ⅲ的表达均明显降低，免疫组化显示，α-SMA和vimentin的阳性区域减少。(2)细胞实验：TGF-β1处理的HFL1细胞中TET2蛋白表达降低、IDAX蛋白表达升高。过表达TET2后，TGF-β1诱导的Col Ⅰ和Col Ⅲ表达均明显下降。敲减 TET2后，TGF-β1诱导的Col Ⅰ、Col Ⅲ和 FN的表达均明显升高。结论：过表达TET2可减轻BLM诱导的肺组织炎症反应和纤维化病变；TGF-β1下调成纤维细胞TET2表达，且受TGF-β1/Smad3-IDAX信号通路介导，TET2的改变可影响TGF-β1诱导的成纤维细胞转分化。