bFGF通过β-catenin调控Smoothened和Gli1转录促皮肤创面愈合的机制研究

Skin wound healing has become one of the difficulties in clinical therapy. Skin wound healing is a complex process that requires action of many cell types, and fibroblast cell migration plays important role in skin wound closure. Efficacy of bFGF on fibroblast cell migration is well known, however, the underlying mechanism by which bFGF regulates cell migration remains unclear. Previous studies found that bFGF regulated the expression of Hh pathway key factors SMO and Gli1, and the activation of Hh pathway could promote fibroblast migration and wound healing. Meanwhile, we had already revealed that bFGF facilitated the accumulation of β-catenin into nucleus to accelerate fibroblast migration. Further, analyses of β-catenin knock-down cells indicated that SMO and Gli1 are under the control of β-catenin. These results suggested that bFGF regulate the transcription of SMO and Gli1 via β-catenin mediation to accelerate fibroblast migration and wound repairing. The objective of this project is to confirm the influence of SMO and Gli1 on fibroblast migration and wound healing, to clarify the relationship between SMO/Gli1 and β-catenin, and to explore the connection between SMO/Gli1 and the downstream factors of bFGF. This study will provide new targets for drugs that skin repairs wound.

皮肤创面修复是临床上亟待解决的难题之一。皮肤创面修复过程需要多种活动细胞的参与，其中成纤维细胞的迁移对皮肤创面修复具有重要意义。bFGF促成纤维细胞迁移的作用已经明确，但其调控机制尚不清楚。前期研究发现，bFGF能调控Hh通路关键因子（SMO和Gli1）的表达，且外源性激活Hh通路可促进细胞迁移和创面愈合。同时，前期我们已经揭示bFGF能通过促β-catenin入核，加速细胞迁移。进一步通过β-catenin干扰分析发现，β-catenin可参与SMO和Gli1的转录调控，提示bFGF可能通过介导β-catenin调控SMO和Gli1的转录，促进成纤维细胞迁移，进而加速创面愈合。本项目旨在前期研究基础上，进一步验证SMO、Gli1对细胞迁移和创面愈合的影响，阐明SMO、Gli1与β-catenin的关系，并探究其与已知bFGF下游因子的关系。本项目将为开发皮肤创面修复药物提供新靶点。

本研究旨在探讨Hedgehog通路在bFGF促成纤维细胞迁移过程中的重要作用及潜在的分子机制。.我们采用细胞划痕实验，3D球状体侵袭实验，大鼠皮肤创伤模型来检测成纤维细胞迁移，侵袭和皮肤创伤愈合的速度。通过Western blot实验检测受bFGF和Hh通路调控的相关蛋白的表达水平。运用siRNA实验抑制内源性的Smo，Gli1和β-catenin转录表达，同时进一步采用RNA深度测序、转录组学和qRT-PCR分析等技术检测在β-catenin干扰后，总转录组的变化情况及对Hh通路相关基因表达的影响。.提取bFGF处理后的成纤维细胞中的RNA进行RNA测序。测序实验结果显示，在bFGF处理后，Hh通路关键基因（包括SMO，Patch1，Gli1，Gli2，Gli3）的表达水平都发生了改变。并且发现用SAG（Smo的激动剂，激活Hh通路）能促进成纤维细胞的迁移，侵袭和皮肤的创伤愈合，而Cyc（Cyclopamine，Smo的抑制剂，抑制Hh通路）使成纤维细胞的迁移，侵袭和皮肤创伤愈合的速度都明显减缓。GANT61（Gli1/Gli2的选择性抑制剂）也能明显抑制细胞的迁移，侵袭和皮肤的创伤愈合的速度。Western blot和siRNA转染实验结果显示，SAG能上调AKT和JNK的磷酸化水平及β-catenin在细胞核内的积累量，证明Smo位于PI3K-JNK-β-catenin的上游，进而促进成纤维细胞的迁移。此外，RNA测序和qRT-PCR分析结果显示，β-catenin调控Hh通路关键基因的转录表达（包括SMO，Patch1，Gli1，Gli2，Gli3），进而影响成纤维细胞的迁移。.综上所述，本实验结果显示，Hh通路参与bFGF促成纤维细胞迁移的调控过程，bFGF下游的β-catenin通过调控Smo和Gli1的转录，反馈调节成纤维细胞的迁移。