USP15靶向调控TGFβ/Smad信号通路在增生性瘢痕形成中的作用与分子机制

Changes of the TGF-β/Smad signaling pathway play a key role in the formation and transformation of hypertrophic scars, but the mechanisms of regulation and degradation remain unclear. Our previous study found that ubiquitin-specific protease 15 (USP15) and Smad7 were highly expressed in hypertrophic scars and negatively correlated with low expression of transforming growth factor receptor (TβRI),and USP15 can be used as a "thermostat" to activate TβRI through Smad7 recruitment to regulate TGF-β activity. Therefore, we hypothesized that USP15 may be involved in the formation and outcome of hypertrophic scars by regulating the TGF-β / Smad signaling pathway. To verify this hypothesis, we will focus on USP15 interacting with TGF-β/Smad signal pathway in this study. The impact of USP15 on the TGF-β/Smad signal pathway and the biological behavior of fibroblasts were designed to investigate by over expressing and silencing USP15. To analyze the function of USP15 in TGF-β/Smad signal pathway, we will use the methods of RNAi, RT-PCR, Western-blotting, co-immunoprecipitation to investigate USP15, TGF-β/Smad signaling pathway and their interaction. In vivo experiments using USP15 knockout animal model to verify the above results to explore USP15 in scar fibrosis and collagen metabolism changes in the interaction and its molecular mechanisms, in order to provide a new theoretical basis for the formation of hypertrophic scars and prevention and control strategies.

TGF-β/Smad信号通路变化在增生性瘢痕形成与转化中发挥了关键作用，但其调节和降解机制仍不清楚。我们前期研究发现泛素特异性蛋白酶15(USP15)和Smad7在增生性瘢痕中高表达，与转化生长因子受体(TβRI)低表达呈负相关；而USP15可作为“温控器”通过 Smad7募集来激活 TβRI干预TGF-β活性。因此我们推测USP15可能通过调控TGF-β/Smad信号通路转导从而参与增生性瘢痕的形成和转归。本研究拟以USP15与TGF-β/Smad信号通路为主线，应用基因过表达、RNAi观察USP15对Smad信号通路及成纤维细胞生物学行为的影响，通过免疫共沉淀检测USP15与Smad信号分子的相互作用，采用基因敲除动物模型体内实验验证上述结果，探索USP15靶向调控TGFβ/Smad在瘢痕纤维化与胶原代谢改变中的作用及其分子机制，以期对增生性瘢痕的形成与防治策略提供新的思路和理论基础。

TGF-β/Smad信号通路变化在增生性瘢痕形成与转化中发挥了关键作用，但其调节和降解机制仍不清楚。本项目在比较观察人增生性瘢痕与正常皮肤组织 USP15与TGF-β/Smad 信号通路相关分子差异表达基础上，构建携 USP15 干扰和过表达序列的慢病毒，筛选稳转细胞株，检测USP15对Smad信号通路及瘢痕成纤维细胞生物学行为的影响；采用蛋白酶体抑制实验检测TβRI蛋白降解情况，明确TβRI是否经泛素-蛋白酶体通路降解，通过免疫共沉淀、泛素化实验等技术检测USP15与TβRI相互结合以及对该分子靶点泛素化修饰水平的调节；进一步采用人增生性瘢痕裸鼠动物模型，观察USP15 shRNA重组质粒对瘢痕形成的体内干预作用。取得了以下主要成果：1. 提出并证实USP15在增生性瘢痕中高表达，且可明显促进增生性瘢痕成纤维细胞增殖、迁移和侵袭；2. 提出并证实增生性瘢痕成纤维细胞中USP15可通过去泛素化TβRI 并抑制其降解从而稳定TβRI蛋白水平、增强TGF-β/Smad信号转导；3. 提出并证实干扰USP15可阻止增生性瘢痕纤维化进程，可作为增生性瘢痕治疗的新靶点。该项目研究提出了USP15 靶向TGF-β/Smad信号通路促进增生性瘢痕形成的新机制，为深入探讨其作为防治靶点的可能性奠定了理论依据和实验基础。