PPARγ拮抗Egr-1对增生性瘢痕TGF-β1促纤维化信号的作用及机制

The hypertrophic scar is currently one of the extremely difficult and important problems to be treated effectually in the tissue repair and wound healing management, due to its high incidence, undefined pathogenetic mechanism, and unsatisfactory outcome in the patients suffering from skin burn or trauma. The peroxisome proliferator activated receptor gamma (PPARγ) agonist has been demonstrated effective against the pathological fibrogenesis in related diseases, attracting more attention with promising novel therapeutical strategy. We has previously found that PPARγ activation could inhibit the TGF-β1-induced phenotype transformation of dermal myofibroblasts. However, the molecular regulation mechanism between PPARγ and TGF-β1 signal pathways, "cross-talk", is remaining unclear. The present project is designed to investigate the potential antagonistic effect of PPARγ activation on the relevant moleculs in TGF-β1 signal pathway of the dermal fibroblasts derived from the burn-caused hypertrophic scar. Furthermore, according to the Smads nucleus-enterring translocation of the downstream moleculs in TGF-β1 signaling pathway and the role of Smad3/4 interacting with PPARγ, the nuclear transcription factor Egr-1 will be focused as a target molecule to be observed for possible PPARγ blockage role in the fibrogenic gene transcription. The electrophoresis assay, DNA affinity precipitation, up- and down-regulation of egr-1 by gene transfection, egr-1 gene siRNA, and egr-1 gene knock-out mouse are going to be implemented in the whole experiment for elucidation of the regulation mechanism between PPARγ and Egr-1. The data from this project will add novel molecular target for further exploration in both pathogenic mechanism and effective therapy of hypertrophic scar.

皮肤烧（创）伤后的增生性瘢痕发病率高，相关机制尚不清楚，临床疗效不确定，为组织修复领域亟待解决的难题之一。过氧化物酶体增殖物激活受体γ(PPARγ)激动剂抗纤维增生性疾病的有效作用逐渐引起人们的关注。我们前期实验发现PPARγ激活后具有抑制TGF-β1诱导皮肤肌成纤维细胞表型转化作用，然而对PPARγ和TGF-β1信号通路间的分子调控（cross-talk）机制目前尚不清楚。本课题拟以烧伤后增生性瘢痕为研究对象，检测激活PPARγ对成纤维细胞TGF-β1通路的相关分子的拮抗作用。进而依据核转录中Smads入核分子Smad3/4同PPARγ可能的作用分子为筛选靶分子，以转录因子Egr-1为交叉点，通过电泳迁移率分析、DNA亲和沉淀、基因转染过表达Egr-1与siRNA沉默、基因敲除动物等方法，揭示PPARγ同Egr-1间的调控机制。为增生性瘢痕机制研究和寻找有效治疗途径，提供新靶点。

皮肤纤维化是由细胞外基质蛋白进行性沉积引起，并导致在皮肤中形成异常的瘢痕。不正常的瘢痕引起诸多严重的临床问题，如皮肤功能改变，关节活动障碍和美容问题等。我们的研究已经确定，核受体过氧化物酶体增殖物激活受体-γ（PPAR-γ）激动剂能够激活PPAR-γ来调节ECM蛋白的表达。首先，我们利用高通量芯片筛选，发现在增生性瘢痕成纤维细胞中，miR-181c和miR-10a表达异常，通过生物信息学和实验分析，证实分别表达并靶向uPA和PAI-1。其次，我们在体外模型中研究增生性瘢痕成纤维细胞的基本分子机制和基因相互作用。这些发现表明PPAR-γ-miR-145-Smad3轴在调控增生性瘢痕成纤维细胞的胶原合成中发挥关键作用。第三，根据高通量芯片数据研究PPAR-γ激动剂曲格列酮治疗瘢痕疙瘩成纤维细胞的基本分子机制。我们进一步揭示曲格列酮治疗瘢痕疙瘩成纤维细胞下调早期生长反应-1（Egr1）和胶原蛋白-1的表达。这部分表明PPAR-γ激动剂介导的miR-543和Egr1信号通路在抑制瘢痕疙瘩成纤维细胞胶原合成中发挥重要作用。第四，根据蛋白质芯片数据，我们证明了PPAR-γ诱导的miR-92b表达抑制Axl表达，进而降低KF中TGF-β1和下游基因的表达，表明靶向这种新的基因途径可能是有用的用于治疗纤维化或瘢痕疙瘩。最后，我们的工作得到国际同行的认可， Chemo Open Access邀请我们发表述评。我们简要总结了课题组的工作，我们认为miRNA调节PPAR-γ激动剂曲格列酮抑制胶原合成和下游靶点的能力。本项目的研究成果，解释了miRNA在PPAR-γ激动剂治疗瘢痕中的分子机制，这些miRNA有望转化为靶向治疗瘢痕的药物。