PTEN介导肺泡上皮细胞衰老参与特发性肺纤维化的机制研究

The incidence of idiopathic pulmonary fibrosis (IPF) increases with age, suggesting that IPF is an age-related disease. Alveolar epithelial cell senescence have been observed in the lung tissue obtained from the pulmonary fibrotic animal and IPF patients, but the specific molecular mechanisms led to senescence of alveolar epithelial cell have not been fully elucidated. PTEN is a tumor suppressor. It negatively regulates the phosphatidylinositol 3 kinase/Akt (PI3K/Akt) signaling pathway and thus controls cell growth, proliferation, apoptosis and adhesion. We previously investigated that increased expression of senescent-markers p16INK4a, decreased expression of PTEN in mice, and activated phosphorylation of Akt were observed in the lung tissue both form pulmonary fibrotic animal and IPF patients. Therefore, we hypothesized that by negatively regulating PI3K/Akt pathway, PTEN controls epithelial senescence and further activates the activity of NF-κB which mediates senescence associated secretory phenotype (senescence associated-secretory phenotype, SASP) and thus promotes the development of lung fibrosis. In this study, by using siRNA and specific inhibitor both in animal model and cell experiments to explore the role of PTEN as to regulate the senescence of alveolar epithelial cell during the development of pulmonary fibrosis and interpret the related molecular mechanisms. This study will provide the theoretical support for targeted-therapy and intervention in future.

特发性肺纤维化（IPF）的发病率随着年龄增加而明显升高，提示IPF是一种衰老相关性疾病。在肺纤维化动物模型和IPF肺组织标本中观察到肺泡上皮细胞衰老现象，但导致肺泡上皮细胞衰老分子机制尚未完全阐明。PTEN是抑癌基因，主要通过负性调控PI3K/Akt信号通路，影响细胞生长、增殖、凋亡及黏附等。我们前期研究发现，小鼠肺纤维化和IPF患者肺组织的细胞衰老分子标记物p16ink4a表达增加，肺泡细胞上皮的PTEN表达下降，但PTEN/PI3K/Akt信号通路中的 pAkt表达上调。我们推测，PTEN可能通过PI3K/Akt调控上皮细胞衰老及激活NF-κB介导的衰老相关性分泌表型参与了肺纤维化的发生和发展。本课题拟采用siRNA及特异性通路抑制剂等方法在动物及细胞水平探讨，PTEN调控肺泡上皮细胞衰老在肺纤维化发生和发展中的作用及其分子机制，为将来针对上皮细胞衰老的靶点治疗和干预提供理论依据。

特发性肺纤维化（IPF）是一种特殊形式的慢性、进行性、纤维化间质性肺炎，其特点是大量纤维化瘢痕组织的形成和肺实质的破坏，导致气体交换异常和呼吸衰竭。IPF的发病率随着年龄增加而明显升高，提示 IPF 是一种衰老相关性疾病，但导致肺泡上皮细胞衰老分子机制尚未完全阐明。. 我们在项目支持下，首先研究了IPF患者肺组织PTEN/NF‐κB与细胞衰老之间关系；我们用免疫组织化学，免疫荧光共定位，SA-β-gal，WB等方法，证明在IPF患者肺组织及肺泡上皮细胞（AECs）衰老，NF-κB激活，PTEN表达下降。在博莱霉素诱导衰老细胞实验中，证明AECs衰老，SASP，NF-κB激活，PTEN表达下降。敲减PTEN，IκB, IKK, NF-κB激活，加速AECs衰老；而敲减NF-κB及用特异性抑制剂（BMS-345541）阻断NF‐κB通路，可逆转AECs衰老。将衰老AECs上清液，与成纤维细胞共培养，增加成纤维细胞胶原合成；相反，敲减NF-κB 后AECs上清液，与成纤维细胞共培养，成纤维细胞胶原合成减少趋势。我们以上结果证明，PTEN通过NF-κB通路，调控上皮细胞衰老及SASP参与了肺纤维化的发生和发展。以上研究结果已经发表在Aging cell（IF=7.64）。. 在PTEN是否通过 PI3K/Akt调控肺泡上皮细胞衰老研究中，发现PTEN敲减后，Akt通路被激活， SA-β-Gal及衰老相关标记P21WAF1增加。分别敲减Akt1或Akt2，发现敲减Akt2显著降低博莱霉素刺激的AEC衰老。用特异性抑制剂（LY294002）抑制Akt激活后，AEC衰老的标记P21WAF1明显降低。在博莱霉素诱导小鼠肺纤维化模型中，用特异性抑制剂抑制Akt通路，肺组织P21WAF1表达降低，可减轻小鼠肺纤维化程度。以上结果提示PTEN的缺失，激活Akt通路，导致AEC衰老；敲减Akt2或抑制Akt活化可部分逆转AEC衰老。以上研究结果，证实我们项目的提出科学假设，PTEN通过 PI3K/Akt 调控上皮细胞衰老及激活 NF-κB 介导的衰老相关性分泌表型（SASP）参与了肺纤维化的发生和发展，我们的研究结果为将来针对肺泡上皮细胞衰老的靶点治疗和干预提供理论依据。

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