博莱霉素诱导胸膜间皮细胞与肺成纤维细胞交叉对话在实验性肺纤维化发生中的作用

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease of unknown cause that typically leads to respiratory failure and death within 3-5 years of diagnosis. There are no effective pharmacological therapies yet for IPF. The pathological feature of IPF includes fibroproliferative foci, depositions of extracellular matrix and alveolar collapse. Interestingly, these histopathological alterations are predominantly located in subpleural areas. The cause for the subpleural distribution of fibrotic plague in IPF is not known. Bleomycin is widely used as an inducer in the animal models of pulmonary fibrosis. Very interestingly, lung fibrosis induced by multiple doses of bleomycin by intraperitoneal injection exhibits subpleural fibrotic distribution similar to what is seen in human IPF. Thus, it is a key question why and how bleomycin induces subpleural lung fibrosis. Recently, Mubarak reported that pleural mesothelial cells (PMCs) trafficking into lung parenchyma contributed to the subpleural fibrotic foci in idiopathic pulmonary fibrosis. Our a recent study revealed that bleomycin induced proliferation and migration of PMCs in vitro and in vivo. In our preliminary experiments, we found there is a large number of PMCs and lung fibroblasts in bleomycin-induced fibrotic lung, and the two kinds of cells are mixed interlock. Moreover, we found cultured-medium of bleomycin-treated PMCs induced proliferation of lung fibroblasts. On the other hand, cultured-medium of bleomycin-treated lung fibroblasts induced PMCs migration. Taken together, there is a crosstalk between PMCs and lung fibroblasts induced by bleomycin. Thus, we hypothesized that bleomycin-induced a crosstalk between PMCs and lung fibroblasts induces subpleural lung fibrosis which plays a pivatol role in pathogenesis of experimental pulmonary fibrosis.

因缺乏有效的治疗手段，特发性肺纤维化（IPF）患者多在明确诊断后的3至5年内死亡，探索IPF的发病机制为其防治寻找新的靶点尤为迫切。IPF的一个显著特点就是胸膜下肺纤维化，采用腹部皮下注射博莱霉素复制的小鼠模型也表现为明显的胸膜下肺纤维化，与人类IPF的表现极其类似，所以，研究博莱霉素诱导的实验性肺纤维化对破解人类IPF的发病机制具有重要的意义。他人及我们的既往研究已证实胸膜间皮细胞在肺纤维化的发生中具有重要作用；我们最近的研究证实，在体及离体条件下博莱霉素诱导胸膜间皮细胞增殖与迁移；预实验提示博莱霉素作用下，在纤维化肺内胸膜间皮细胞与肺成纤维细胞交互混杂存在、体外培养中两种细胞相互促进增殖与迁移。因此，本项目拟利用细胞、动物模型及临床标本展开深入研究，以验证“博莱霉素诱导胸膜间皮细胞与肺成纤维细胞相互作用，促进实验性肺纤维化中胸膜下纤维化的发生发展”的创新性假说，并初步探讨其临床价值。

特发性肺纤维化（IPF）是原因不明的慢性进行性肺间质纤维化性疾病。IPF的主要病理表现为胸膜下的肺纤维化。肺成纤维细胞是肺纤维化的主要细胞。最近的研究发现胸膜间皮细胞可迁移入肺，参与IPF的发病。因此，我们假设：博莱霉素诱导胸膜间皮细胞与肺成纤维细胞相互作用，促进实验性肺纤维化中胸膜下纤维化的发生发展。我们既定的研究计划是：1.胸膜间皮细胞与成纤维细胞的旁分泌作用对细胞胶原产生、增殖和迁移等的影响；2. 胸膜间皮细胞与成纤维细胞直接作用对各自功能的影响；3.干预相互作用能否减轻肺纤维化。主要研究结果为：1．博莱霉素引起的肺纤维化模型中PMCs迁移入肺，并分布在胸膜下区域的成纤维细胞周围；2．MET-5A细胞的条件培养基促进博莱霉素引起的肺成纤维细胞（HFL1）胶原和α-SMA表达上调；3．肺成纤维细胞（HFL1）的条件培养基促进博莱霉素引起的MET-5A细胞胶原和α-SMA表达上调；4．博莱霉素可促进胸膜间皮细胞（MET-5A）和大鼠肺成纤维细胞（RFL）分泌TGF-β1；5．TGF-β 受体抑制剂 SB431542抑制MET-5A条件培养基对博莱霉素引起的肺成纤维细胞p-Smad2/3和collagen-I表达上调作用；6．TGF-β 受体抑制剂 SB431542抑制肺成纤维细胞条件培养基对博莱霉素引起的胸膜间皮细胞p-Smad2/3和collagen-I表达上调作用；7．TGF-β 中和抗体在体抑制博莱霉素引起的肺纤维化；8．博莱霉素可促进胸膜间皮细胞（MET-5A）和大鼠肺成纤维细胞（RFL）Wnt/β-catenin信号通路的活化；9．Wnt/β-catenin信号通路抑制剂可减轻MET-5A条件培养基对博莱霉素引起的肺成纤维细胞collagen-I表达上调作用；10．Wnt/β-catenin信号通路抑制剂可减轻RFL条件培养基对博莱霉素引起的RPMC collagen-I表达上调作用；11．Wnt/β-catenin信号通路封闭肽在体抑制博莱霉素引起的肺纤维化；12．胸膜间皮细胞和肺内成纤维细胞的直接相互作用促进collagen-I表达。总之，我们发现，迁移入肺的胸膜间皮细胞和肺内成纤维细胞通过旁分泌以及直接作用而相互作用，促进肺纤维化的发生，TGF-β和Wnt/β-catenin信号通路参与其中。