晶状体再生修复过程中TGF-β通过EGFR通路调控EMT的分子机制

Maintaining transparency is crucial to the normal physiological function of the lens. Epithelial-Mesenchymal Transition (EMT) is a common pathological phenomenon during tissue repair, and is also the bottleneck for the functional repair of the lens. We have established a minimally invasive surgery to activate the lens endogenous stem cells, and achieved in-situ regeneration to repair the lens (Nature.2016). However, the EMT at the site of micro-capsulorhexis causes scar repair and local opacity postoperatively, affecting the transparency and refractive function of the regenerative lens. Transforming growth factor (TGF-β) plays a critical role in the process of EMT. We found in the lens regeneration model that TGF-β induced an increase in the ligand of epidermal growth factor receptor (EGFR) after minimally invasive surgery, and the extent of the increase was associated with EMT. We speculate that TGF-β regulates EMT through the EGFR pathway during the process of lens regeneration and repair, but the molecular mechanism of this process has not been demonstrated. Therefore, our project aims to: 1. At the cellular level, investigate the molecular mechanism of TGF-β mediated EMT via EGFR pathway by modulating the key factors of the signaling pathway; 2. Quantitatively evaluate the effects of regulating EGFR pathway on TGF-β induced EMT using the established lens models in vitro; 3. Investigate the effects of regulating EGFR pathway on the EMT at the site of micro-capsulorhexis and the surrounding tissues in animal lens regeneration model using gene expression chips, and to provide a theoretical basis for improving the function of the regenerative lens.

晶状体是重要屈光组织，透明性是发挥生理功能的关键。上皮间质转分化(EMT)是修复过程常见病理现象，也是晶状体功能性修复的瓶颈。课题组创建晶状体微创手术，成功激活内源性干细胞再生修复晶状体(Nature.2016)。然而，术后微撕囊口EMT引起局部混浊，影响再生晶状体透明性和屈光功能。转化生长因子(TGF-β)是诱导EMT的关键因子。我们在再生模型中发现，微创术后TGF-β诱导表皮生长因子受体(EGFR)的配体升高，且与EMT程度相关。我们推测晶状体再生过程中TGF-β通过EGFR通路调控EMT。本项目拟：1.在细胞水平调控信号通路关键因子，研究TGF-β通过EGFR通路调控EMT的分子机制；2.利用离体晶状体模型定量评估调控EGFR通路对TGF-β诱导的EMT的影响；3.在兔晶状体再生模型中利用基因芯片研究调控EGFR通路对微撕囊口EMT及周围组织的影响，为优化再生晶状体提供理论基础。

晶状体微创手术后微撕囊口上皮间质转分化(EMT)引起局部混浊，影响再生晶状体透明性和屈光功能是再生晶状体功能性修复的瓶颈。.转化生长因子(TGF-β)是诱导EMT的关键因子，课题组前期研究发现，微创术后TGF-β诱导表皮生长因子受体(EGFR)的配体升高，且与EMT程度相关，提示晶状体再生过程中TGF-β通过EGFR通路调控EMT。本项目通过在细胞水平从体外和体内研究TGF-β通过EGFR调控EMT的机制，发现：1.再生晶体中表皮生长因子受体通路发生改变，包括了EGF和HBEGF表达水平依次出现明显的升高。用TGF-β刺激晶状体上皮细胞可诱导HB-EGF水平升高以及EMT的发生，这一作用可被PCK抑制剂阻断，且EGFR抑制剂可阻断TGF-β诱导的EMT。TGF-β在EMT中起作用的过程涉及PLC、PKC和ras/MAPK等信号分子。2.在细胞外的改变中，ROS诱导的EVs处理细胞可激活LECs的EMT。EVs参与了ROS诱导的晶状体EMT，使EVs成为晶状体再生修复的潜在靶点。3.再生晶状体细胞中UPP的损伤可能导致EGFR途径的改变，从而扰乱晶状体再生的过程。综合上述结果，晶状体再生过程中TGF-β激活下游HB-EGF，进一步激活ERK诱导LECs的EMT，同时晶状体损伤后的微环境刺激氧化应激下的LECs产生胞外EVs也可激活EMT，为优化再生晶状体提供理论基础与干预靶点。项目资助发表论文22篇，其中SCI论文16篇。项目投入经费25.0000万元，支出20.0587万元，各项支出基本与预算相符，剩余经费4.9413万元。剩余经费计划用于本项目研究后续支出。