Smad7在皮肤纤维化发生中的作用及靶分子的鉴定

Cutaneous fibrosis including scleroderma and keloid is common and detrimental disease as lacking of efficient therapy. Activation of TGF-β and Wnt signaling pathway mainly contribute to the pathogenesis of fibrosis. As an antagonist of TGF-β signaling, Smad7 has reduced expression in cutaneous fibrotic diseases. Studies from animal model have suggested that overexpression of Smad7 in epidermis promote degradation of β-catenin and also inhibits expression of collagen by fibroblasts during wound healing through epidermal-dermal interaction. We hypothesize that Smad7 from both keratinocytes and fibroblasts may play anti-fibrotic role in different mechanism or using different targets. In current application, we propose to evaluate the role of endogenous epidermal and fibroblast Smad7 in the pathogenesis of cutaneous fibrosis. Furthermore, high throughput sequencing will be performed to screen Smad7 target genes using different Smad7 transgenic or knockout cells. For top competitive Smad7 target genes, we will perform immunologic and molecular analysis to determine their association with Smad7 and also validate their clinical significance in human cutaneous fibrotic diseases. The study will advance our current understanding for the mechanism of cutaneous fibrosis and might provide novel potential targets for treating skin fibrosis.

皮肤纤维化如硬皮病及瘢痕疙瘩是危害人类身心健康的一类常见疾病，目前缺乏有效疗法。TGF-β及Wnt信号通路的异常活化与组织纤维化的发生密切相关。Smad7作为TGF-β信号通路的重要抑制因子，在皮肤纤维化组织中表达下调。动物实验提示表皮细胞Smad7可促进β-catenin的降解而抑制Wnt信号通路、并通过表皮-真皮相互作用而抑制创伤愈合时纤维细胞胶原蛋白的表达，因而我们推测皮肤表皮细胞和纤维细胞Smad7可能通过不同的靶基因或机制发生抗纤维化作用。为证实这一设想，本申请拟应用条件型基因敲除动物分别解析表皮细胞及纤维细胞源性Smad7在纤维化发生中的作用，进一步以Smad7转基因或基因敲出的表皮或纤维细胞为材料，应用高通量基因测序技术、免疫学及分子生物学方法筛选鉴定介导Smad7抗纤维化的靶基因及其临床意义。研究结果将推进皮肤纤维化作用机制的研究并可能提供治疗纤维化疾病的潜在新靶点。

为评价Smad7在皮肤纤维化发生中的作用，课题利用基因工程技术分别敲除Smad7在小鼠表皮、成纤维细胞或组织中的表达，分析Smad7低表达状态下皮肤表型及病理特征的改变，对内源性Smad7在皮肤纤维化中的作用进行了评估。以TGFβ1皮肤转基因小鼠(K5.TGFβ1)为纤维化模型，通过与不同Smad7基因工程型小鼠杂交，进一步观察内源性Smad7表达低下在TGFβ1诱导纤维化发生中的促进作用，并对Smad7蛋白抗纤维化的作用进行了探讨。主要结果与结论包括：①单纯表皮细胞、成纤维细胞或组织Smad7水平降低不足以引起皮肤纤维化的发生。②Smad7下降的基础上，外源性刺激如升高TGFβ1水平则加快皮肤纤维化的发生。提示内源性Smad7低下联合外源性刺激是导致纤维化发生的因素。③Smad7表达低下及TGFβ1表达升高的皮肤组织，成纤维细胞β-catenin核表达升高，显示Smad7的抗纤维化效应可能通过调节Wnt信号通路发生作用。④以皮肤TGFβ1转基因小鼠为模型，证实Smad7蛋白皮下注射可缓解皮肤炎症和纤维化的表型及病理特征，提示Smad7蛋白抗纤维化的潜在临床应用价值。⑤利用Smad7高表达小鼠皮肤组织的基因芯片分析结果未能鉴定出Smad7特异靶基因，目前进行中的利用新型技术直接寻找人成纤维细胞Smad7的靶基因可能更具临床相关性。⑥该课题资助的研究生相关课题显示竹红菌素B联合黄光照射的光动力疗法可诱导瘢痕疙瘩成纤维细胞凋亡而具有潜在的临床应用价值。