神经肽P物质通过N-cadherin/Wnt/β-catenin通路调控角膜缘干细胞特性的机制研究

Eye branch of trigeminal nerve composes corneal sensory nerve fibers, and its damage causes dysfunction of corneal limbal stem cells(LSCs), resulting in blinding neurotrophic keratopathy(NK). However, the molecular mechanism of how corneal sensory nerve modulates the biological characteristics of LSCs remains unclear. Our previous study found that deprived innervation of trigeminal nerve resulted in a deficiency of neuropeptide substance P(SP) and decreased expression of N-cadherin and target genes of Wnt/β-catenin, along with decreased expression of LSCs marker in corneal limbus. The percentage of proliferating and differentiating cell raised. Interfering the function of SP in vitro led to decreased expression of N-cadherin and target genes of Wnt/β-catenin in LSCs. This study employs cell culture and mouse models to confirm that neuropeptide P modulates the biological characteristics of LSCs through the N-cadherin/Wnt/β-catenin pathway in the following aspects: corneal sensory nerve fibers release neuropeptide SP to modulate the characteristics of LSCs, and SP regulates the expression of Wnt/β-catenin target gene by the means of N-cadherin in LSCs. The purpose is to reveal the molecular mechanism of how corneal sensory nerve modulates the characteristics of LSCs, which would shed a light on the researches of LSCs and limbal niche and provide theoretical basis for exploring the pathological mechanism of NK and opening a new train of therapeutic targets.

三叉神经眼支组成角膜感觉神经纤维，其损伤可引起角膜缘干细胞（LSCs）异常，导致致盲性神经营养性角膜病变（NK），但角膜感觉神经调控LSCs特性的机制尚不清楚。我们前期发现：去三叉神经支配引起角膜缘P物质（SP）缺失，N-cadherin、Wnt/β-catenin靶基因及LSCs标记表达下降，增殖分化细胞增多；干扰SP引起LSCs细胞N-cadherin和Wnt/β-catenin靶基因表达下降。本课题利用细胞及小鼠模型从以下几方面证实神经肽P物质通过N-cadherin/Wnt/β-catenin通路调控LSCs特性：角膜感觉神经纤维释放神经肽SP，调控LSCs特性；N-cadherin介导SP调控LSCs细胞Wnt/β-catenin靶基因表达。目的在于揭示角膜感觉神经调控LSCs特性的分子机制，为LSCs及其巢功能研究开启新的思路，为阐明NK病理机制及探索新的治疗靶点提供理论依据。

三叉神经眼支组成角膜感觉神经纤维，其损伤可引起角膜缘干细胞（LSCs）异常，导致致.盲性神经营养性角膜病变（NK），但角膜感觉神经调控LSCs特性的机制尚不清楚。本研究首次利用氯丙嗪球后注射构建角膜去神经模型（CCTD模型），并从模型成功率、并发症及学习曲线等方面，与传统电凝法去三叉神经模型及神经离断法去三叉神经模型相比较。结果显示：CCTD模型具有更高的成功率、更稳定的可重复性、更简易的操作方法及更短的学习曲线，该模型为本研究以及未来的角膜神经相关的研究提供一个简便易行的实验模型和强有力研究工具。接下来，我们研究证实角膜感觉神经纤来源SP调控LSCs增殖及干性；体内体外研究证实补充SP能够改变LSCs细胞增殖及干性标记的表达；通过基因测序研究发现：去神经干预导致LSCs细胞1927个基因表达上调，1485个基因表达下调。进一步分析发现，与SP-NK1信号最相关的信号通路为PI3K-AKT通路，使用PI3K-AKT通路抑制剂MK-2206，能够阻断SP对LSCs细胞Ki67和DeltaNp63 表达的影响，导致Ki67和DeltaNp63 表达下降，从而提示，角膜神经来源SP通过PI3K-AKT通路调控LSCs生物学特性。由此，我们的研究显示：角膜神经来源SP通过PI3K-AKT通路调控 LSCs 生物学特性，从而证实角膜神经纤维在调控LSCs巢环境中的功能效应，为LSCs及其巢功能研究开启新的思路，为阐明角膜神经与干细胞之间的相互作用提供理论依据，并且为阐明NK病理机制及探索新的治疗靶点提供理论依据。