肺泡上皮细胞端粒调控机制在IPF发病中的作用研究

Idiopathic pulmonary fibrosis (IPF) is the most common and clinically severest type of idiopathic interstitial pneumonias. It is characterized by progressive dyspnea and restriction on pulmonary function testing. The average survival is about 3 years from the diagnosis of IPF. Cellular senescence was believed to play a crucial role in the development of IPF since about 97% of IPF patients had shortened telomeres. Wnt signaling, induced by insufficient AECII-AECI differentiation and abnormal epithelial repairing, triggers the pathogenesis of pulmonary fibrosis via a key process called epithelial mesenchymal transition (EMT). The disruption of Wnt signaling, therefore, could be beneficial in IPF treatment. However, Wnt pathway is also important in prompting telomerase gene expression in cells. The inhibition of Wnt pathway may accelerate the process of telomere shortening and AEC senescence. Recent studies indicated that the AEC EMT induced by ectopic Wnt signaling is likely via β-Catenin/CBP pathway, whereas the telomerase expression effect of Wnt is related to β-Catenin/KLF4. To further delineate the mechanism, in the present study, we will investigate the molecular mechanisms of Wnt regulating TERT gene expression. Meanwhile, we will also investigate whether TERT expression will influence the course of EMT, the in-vitro cell models and TERT gene over expression or silencing approach will be applied to answer these questions. We anticipate that our study can help to find a clue to explain the pathogenesis of IPF, which will open a new avenue for IPF therapy.

特发性肺纤维化（IPF）是以慢性、进展性肺间质纤维化为特征的致死性疾病, 为一种增龄相关性疾病。端粒及端粒酶在细胞衰老中发挥关键作用。我们的前期工作表明，外周血粒细胞端粒长度缩短是IPF发生及预后的独立危险因素。已知外周血粒细胞与肺泡上皮细胞端粒长度呈正相关，提示肺泡上皮细胞衰老机制参与IPF的发生发展。Wnt信号过度激活在IPF病理形成中起重要作用。我们前期发现，阻断Wnt信号通路可抑制成纤维细胞向肌成纤维细胞转化，减轻动物模型的肺纤维化程度。但是，Wnt信号通路是否参与对肺泡上皮细胞端粒长度的调控，影响其老化过程尚不清楚。本研究在前期工作基础上，拟探寻Wnt通路对肺泡上皮细胞端粒酶表达的调控机制，并进一步探讨特异性抑制Wnt通路促纤维生长的同时，维持肺泡上皮细胞端粒稳定的可能性。此研究的开展将为解释IPF是增龄性疾病提供理论依据，为通过干预细胞衰老机制的治疗提供靶点。

特发性肺纤维化（IPF）是增龄相关疾病。文献及前期研究表明，肺泡上皮细胞衰老是IPF发病的始动触发环节，与细胞衰老密切相关的端粒长度缩短是IPF发病及预后的分子危险因素。然而，调控肺泡上皮细胞衰老及衰老相关端粒酶表达的分子机制，以及细胞衰老与成纤维细胞活化增生的关联尚待阐述。在本项目的资助下，申请人带领的研究团队开展了系列研究，首先在IPF肺组织标本验证，衰老标记P16、P21表达升高，WNT/β-catenin信号通路活化分子β-catenin的下调，转录因子TCF4、KLF4下调，端粒酶逆转录酶（TERT）表达下调，提示肺泡上皮细胞WNT信号通路活性受到抑制，细胞提前老化。采用经典的博来霉素刺激小鼠肺泡上皮细胞系MLE12，构建细胞衰老模型，细胞实验显示衰老的肺泡上皮细胞β-catenin、TCF4、KLF4均表达下调，TERT的表达下调，端粒酶活性减低；采用WNT通路激动剂处理衰老肺泡上皮细胞或TCF4、KLF4过表达，TERT表达稳定或增加，衰老标记下调。染色质免疫共沉淀结果表明，WNT/β-catenin信号通路对TERT基因表达的调控是通过β-catenin与TCF4、KLF4结合至TERT基因启动子区实现的。采用博来霉素气管雾化给药诱导的肺纤维化小鼠模型，利用慢病毒载体过表达TCF4以及KLF4，可上调TERT的表达，稳定端粒酶活性，减轻肺泡上皮细胞衰老，实验动物的肺纤维化程度减轻。进一步构建衰老肺泡上皮细胞与成纤维细胞共培养体系，该体系内成纤维细胞激活的WNT信号通路可上调干性因子NANOG的表达，进而促进成纤维细胞活化增生。本研究结果证实肺泡上皮细胞受损老化是肺纤维化发病机制的核心环节，首次系统阐述WNT/β-catenin信号通路对肺泡上皮细胞端粒酶基因的表达及端粒长度的细胞特异性稳定作用，为针对肺泡上皮细胞的修复和再生能力作为新的治疗干预策略提供了理论基础。