机械牵张力调控皮肤再生关键基因JAG1表达及组蛋白甲基化机制研究

Skin tissue expansion has been demonstrated to induce skin regeneration through mechanical stretching. However, the mechanism of skin tissue expansion is largely unknown. Transcription factor YAP has been considered as one of the most important mediators in mechanotransduction and regulates the proliferation of epidermal stem cells. Our previous work found that short-term mechanical stretch could promote the proliferation of epidermal stem cells by inducing nuclear translocation of YAP and up-regulating JAG1 expression. However, long-term mechanical stretch caused upregulated expression of inhibitory EZH2, depressed expression of JAG1 and epidermal stem cells proliferation, which explained that skin expansion induced significant skin regeneration in short-term but limited in long-term (Cell Mol Life Sci 2015). So, we speculate that the mechanism which regulates the JAG1 expression may be the main reason for limited skin regeneration in long-term skin expansion. By mechanical stretch model of epidermal stem cells in vitro and skin expansion model of mice in vivo, the target genes of YAP under mechanical stretch will be identified by the combination of chromatin immunoprecipitation (ChIP) sequencing and microarrays. The function of JAG1 will be studied by PCR, Western blot and 3D organic culture experiments. Then, the statues of histone methylation of JAG1 can be achieved by ChIP-PCR, to investigate the epigenetic mechanism of temporal mechanical stretch regulated skin regeneration. Collectively, our finds will reveal the mechanisms of mechanical stretch regulated skin regeneration and provide a novel strategy for promoting skin regeneration.

皮肤扩张术利用机械牵张力刺激皮肤再生，但相关机制不明。现有研究表明转录因子YAP是细胞内生物力学信号传导的重要分子，可调控表皮干细胞增殖。申请人前期研究首次发现短期牵张力诱导YAP及其靶基因JAG1表达升高，表皮干细胞增殖活跃。但是长期牵张后，抑制JAG1表达的组蛋白甲基化酶EZH2表达升高，表皮干细胞增殖能力下降，较好地解释了临床早期扩张皮肤再生明显而后期再生受限的现象（Cell Mol Life Sci 2015）。据此提出JAG1表达改变机制可能是扩张皮肤再生能力有限主要原因的假说。为此，本课题拟在前期基础上通过细胞牵张模型，采用染色体免疫共沉淀测序技术，研究牵张力是否通过YAP介导激活JAG1表达，并明确JAG1与皮肤再生的关系；然后通过ChIP-PCR法及EZH2敲除鼠，研究牵张力对JAG1组蛋白甲基化修饰的影响，探讨牵张力调控皮肤再生的生物学机制，为拓展皮肤扩张治疗提供新思路。

皮肤软组织扩张术通过对皮肤施加机械力学的作用来刺激皮肤组织再生，同时，我们发现在皮肤组织扩张到后期往往出现皮肤再生能力的下降。对于机械力学是如何激活皮肤组织再生的具体机制能缺乏完整的认识。研究其中的分子生物学机制即有可能成为促进扩张皮肤再生的全新治疗靶点。我们通过体外细胞机械牵张力学刺激，通过联合使用Chip-on-chip和表达谱芯片的方法，研究发现在体外对人表皮干细胞进行周期性机械牵张力下细胞增殖加快。机械牵张力促进转录因子YAP的表达并且可以诱导YAP转入细胞核。通过Chip-on-chip和表达谱芯片的联合分析，我们找到了机械牵张力作用下YAP入核后激活转录表达的靶基因JAG1。在体外我们验证了YAP可以结合在靶基因JAG1的启动子区域，而且JAG1与表皮干细胞增殖呈正相关。然而长期力学作用下，YAP-JAG1通路表达下降到正常水平。体外的表皮干细胞及扩张人皮肤样本中的表皮基底层细胞中组蛋白甲基化H3K9/27me3修饰水平都显著性地升高。我们的研究发现了机械牵张力可以调控皮肤再生的现象，并发现了其中的表观遗传学机制。