bFGF调节GSK3β磷酸化的机制及β-catenin在成纤维细胞迁移中所起作用的研究

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. RNA-deep sequencing and bioinformatics analyses against the bFGF-stimulated human foreskin fibroblast cells identified that the Wnt canonical pathway genes are significantly altered upon bFGF stimulation. In addition, application of LiCl and IWR-1, typical inhibitors of GSK3β and Wnt pathway respectively, promoted and inhibited cell migration. Also, bFGF treatment accumulated more phospho-GSK3β and nuclear β-catenin. Therefore, it suggests that bFGF may modulate GSK3β phosphorylation to reduce the GSK3β activity, which may results in reduction of β-catenin degradation. The objective of this project is to understand the regulatory mechanism of bFGF-mediated GSK3β phosphorylation and the role of β-catenin in cell migration process. This study will provide a theoretical basis for bFGF medication and new targets for drug that skin repairs wound.

皮肤创面修复是临床上亟需解决的难题之一。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RNA深度测序及生物信息学分析结果表明，Wnt信号通路关键基因在bFGF刺激下有显著性变化。细胞迁移实验结果显示，LiCl刺激 (GSK3β抑制剂，激活Wnt信号通路) 促细胞迁移，相反IWR-1刺激 (Wnt信号通路抑制剂) 抑制细胞迁移。而且bFGF处理上调GSK3β磷酸化及核内的β-catenin水平，提示bFGF可能通过调节GSK3β磷酸化水平，抑制GSK3β活性，降低β-catenin降解，促细胞迁移。本课题旨在前期研究基础上进一步探索bFGF调控GSK3β磷酸化的机制及β-catenin在细胞迁移过程中的作用。本项研究结果不仅能在理论上指导临床，而且可以为开发皮肤创面修复药物提供新靶点。

本研究旨在探讨bFGF调控的基因库并且进一步确定在人成纤维细胞中参与细胞迁移的靶分子。.我们采用RNA-Seq转录组学技术鉴定出在bFGF处理后，有显著性差异的基因和许多信号通路，并且采用qRT-PCR进一步验证。之后，我们采用Western blot实验检测受bFGF和Wnt通路调控的关键蛋白，用大鼠皮肤创伤模型检测Wnt信号通路在皮肤愈合中所起的作用。运用小干扰RNA实验抑制内源性的β-catenin，同时进一步采用RNA-Seq转录组学技术鉴定出在β-catenin干扰后，总的转录组的变化情况。.RNA测序实验结果显示在bFGF处理后，Wnt信号通路的基因发生了改变，包括Wnt2b，Wnt3，Wnt11，T-cell factor 7（TCF7）和Frizzled 8（FZD8）。并且，在LiCl（Wnt通路激活剂）与IWR-1（Wnt通路抑制剂）处理后，前者能促进成纤维细胞的迁移速率，而后者相反。此外，在bFGF处理后，GSK3β在ser9位点的磷酸化和核内的β-catenin均能上升。分子和生化实验提示PI3K信号能通过JNK激活GSK3β/β-catenin/Wnt信号通路，说明PI3K和JNK处于β-catenin的上游。β-catenin干扰后，细胞迁移速率降低。此外，RNA-Seq转录组学技术鉴定出β-catenin干扰后bFGF和FGF21的表达也下降。并且，Western blot实验表明FGF21与bFGF作用相似，能激活AKT和JNK，促进细胞迁移速率。.综上所述，本研究结果显示，在皮肤创伤愈合过程中，FGF与Wnt信号通路通过β-catenin形成反馈调节，促进创面愈合。