Sirt1通过去乙酰化Cortactin调控角膜上皮损伤修复的分子机制研究

Sirt1, a class III deacetylase, has been demonstrated to possess multiple biological effects，including proliferation, migration, differentiation, and so on. Corneal epithelial wound healing (CEWH) is an essential pathophysiologic process to maintain corneal integrity. However, the mechanisms that Sirt1 regulates this process remain unclear. Our preliminary studies showed: Sirt1 is upregulated at both mRNA and protein levels during CEWH process; Sirt1 deficiency in mouse delays repair after corneal epithelial wounding; Cytoskeletal protein Cortactin is screened to bind to Sirt1 by mass spectrometry. All above imply Sirt1 promotes corneal epithelial wound healing by deacetylating Cortactin. Based on these data, Sirt1 loss or gain-of-function is firstly generated on human corneal epithelial cells (HCECs) to determine its effects on proliferation and migration. Cortactin is further confirmed to be deacetylated by Sirt1 in vivo and vitro by co-immunofluorescence and co-immunoprecipitation technologies. Lastly, the effects of the status of acetylated-Cortactin on wound healing are investigated in vivo and vitro and the mechanism underlying it is explored. This study will clarify the molecular mechanisms that Sirt1 regulates corneal epithelial wound healing by deacetylating Cortactin, as well as provide an effective therapeutic strategy to enhance corneal wound healing in clinic.

Sirt1是第III类组蛋白去乙酰化酶，对细胞增殖、迁移、分化等生物学行为发挥重要的作用。角膜上皮损伤修复是维持角膜完整性的重要生理病理过程，Sirt1对其调控机制尚未十分明了。本课题前期发现：损伤修复的角膜上皮中Sirt1显著升高；Sirt1敲除明显延缓角膜上皮损伤修复；Sirt1和细胞迁移相关蛋白Cortactin相互作用，提示Sirt1介导的Cortactin去乙酰化促进角膜上皮损伤修复。在此基础上，以人角膜上皮细胞（HCEC）为模型，通过基因干扰和过表达技术明确Sirt1对细胞增殖和迁移的影响。进一步通过免疫荧光染色和免疫沉淀技术阐明Sirt1在体内和HCEC中去乙酰化Cortactin。最后阐明乙酰化Cortactin对角膜上皮损伤修复的影响及其影响细胞迁移的机制。本课题旨在阐明Sirt1通过去乙酰化Cortactin调控角膜上皮损伤修复的分子机制，为临床治疗提供新的策略和思路。

Sirt1是第III类组蛋白去乙酰化酶，对细胞增殖、迁移、分化等生物学行为发挥重要的作用。角膜上皮损伤修复是维持角膜完整性的重要生理病理过程，Sirt1对其调控机制尚未十分明了。本项目研究发现：1、损伤修复的角膜上皮中Sirt1显著升高；2、角膜上皮细胞中Sirt1敲除小鼠中角膜上皮损伤修复明显延缓。在此基础上，以人角膜上皮细胞（HCEC）为模型，通过基因干扰和过SIRT1抑制剂明确Sirt1表达减少或者活性减弱明显抑制角膜上皮细胞的迁移。进一步通过免疫荧光染色和免疫沉淀技术阐明Sirt1在体内和HCEC中去乙酰化Cortactin。最后阐明乙酰化Cortactin对角膜上皮损伤修复的影响及其影响细胞迁移的机制。本课题揭示了Sirt1通过去乙酰化Cortactin调控角膜上皮损伤修复的分子机制，为临床治疗提供新的策略和思路。