磷脂酰乙醇胺结合蛋白1（PEBP1）对角膜缘干细胞自噬的调控及分子机制研究

The abnormality of homeostasis and survival in limbal stem cells (LSCs) would cause limbal stem cells deficiency (LSCD), which is extremely hard to cure and will lead to vision lost eventually. Recent studies provide plenty of evidence that autophagy, as a high-efficiency cellular catabolic and recycling pathway, involved in several key functions in multiple adult stem cells, including differentiation, anti-apoptoic and anti-senescence. However, whether and how autophagy participates physiologic processes in LSCs and then affects LCSD is still unclear, and the underling mechanism of autophagy regulation also need to be unveiled. Our previous RNA microarray result showed that the autophagy regulator gene phosphatidylethanolamine-binding protein 1 (PEBP1) expressed obviously higher in LSCs comparing to differentiated corneal epithelial cells (CECs), and some autophagy related genes have lower expression level in LSCs than CECs. Based on these results, we hypothesized that PEBP1 could negatively regulate autophagy and impact physiologic processes like differentiation, apoptosis or proliferation in LSCs. We will apply multiple methods to manipulate and examine genes expression level, autophagy level, and physiological functions in LSCs in 3D culture system in vitro and mouse corneal injury model in vivo. Theoretically, the future result will not only provide sufficient evidence to elucidate the relation and functions of PEBP1 and autophagy in LCSs, but also delineate the underling mechanisms of autophagy regulation. Besides the physiological functions and regulation mechanisms of autophagy in corneal health, our finding will also give insight to the treatment and prevention of LSCD by possible autophagy-related therapy.

角膜缘干细胞的生存与生理状态异常会导致角膜缘干细胞缺乏，而角膜缘干细胞缺乏属难治性致盲性角膜病。新近研究显示，自噬作为高效的细胞内降解循环途径，参与多种成体干细胞的分化、抗凋亡和拮抗老化等生理过程。然而，自噬是否及如何参与角膜缘干细胞的哪些生理过程，从而影响角膜缘干细胞缺乏还不清楚，自噬调控机制更不明确。预实验发现，角膜缘干细胞与角膜上皮细胞相比自噬基因表达显著降低，自噬负调控基因PEBP1则升高。因此，我们提出假说：PEBP1通过调控负自噬影响角膜缘干细胞分化、凋亡或增殖等生理过程。本研究采用3D培养分化和小鼠角膜损伤模型，结合基因表达干预和多种自噬干预检测技术，获得PEBP1和自噬在角膜缘干细胞中影响各项生理过程的直接证据，进一步揭示PEBP1调控自噬的具体分子机制。研究结果将阐明自噬在角膜缘干细胞中的变化趋势和具体生理功能，为通过调控自噬治疗和预防角膜缘干细胞缺乏提供理论支持。

角膜缘干细胞是角膜上皮细胞自我更新的唯一来源，角膜缘干细胞缺乏症造成角膜上皮异常直至视功能丧失。角膜缘干细胞的生理状态与角膜缘干细胞缺乏直接相关，而自噬是干细胞生理状态维持的重要影响因素，在角膜缘干细胞中自噬的变化、功能及调控机制未得到充分研究。干性老年黄斑变性（dry-AMD）是导致老龄群体视力障碍的主要原因，自噬相关的视网膜色素细胞（RPE）氧化损伤是dry-AMD重要病因。迄今，dry-AMD仍缺乏有效治疗手段。. 本项目进行了角膜缘干细胞生理及老化过程中自噬的水平变化研究，并验证PEBP1对角膜缘干细胞的自噬调控作用。完成不同年龄人源角膜缘干细胞体外衰老模拟及相应本底和应激自噬能力检测，获得角膜缘干细胞在人类衰老过程中自噬改变信息。研究了PEBP1对角膜缘干细胞的自噬调控作用。在完成本项目任务目标基础上，重点扩展了自噬及PEBP1抑制剂D609在角膜上皮的干眼模型以及视网膜色素上皮的dry-AMD模型中研究。. 本项目发现：角膜缘干细胞传代老化后丧失干细胞形态伴随自噬小体及底物P62增多，细胞降解自噬小体能力下降。老化角膜缘干细胞对饥饿诱导自噬的敏感度明显下降，自噬能力伴随年龄增长呈现降低趋势。PEBP1的血管内皮抑制剂D609在体内体外均能有效保护碘酸钠诱导的视网膜色素上皮细胞氧化损伤。保护作用通过增加金属硫蛋白表达实现。高渗干眼模型中角膜上皮细胞自噬流受到损伤，DDIT4参与此过程。. 阐明自噬在角膜缘干细胞衰老中的变化，为进一步研究调控作用和分子机制提供重要指向性，为治疗和预防角膜缘干细胞缺乏提供新思路，为角膜缘干细胞移植过程中如何通过调控自噬实现细胞保护这一问题提供理论支持和方向指引。本项目发现D609可以在体内和体外作为RPE的新型抗氧化保护剂，有治疗预防dry-AMD的潜在应用价值。.