角膜感觉神经TRPV1在干眼病发病中的机制研究

As one of the most common disorders in the ophthalmic practice, dry eye disease is affecting millions of people worldwide. Its pathogenesis is complicated and effective therapeutic tools for this disease is limited. It is currently accepted that high osmolarity and inflammation play pivotal roles in the pathogenesis of dry eye disease,while new investigation found that transient receptor potential vanilloid 1(TRPV1) on neurons is activated by high osmolarity and takes part in inflammation. Its activation induces an action potential which is read out as "burning pain" by the central nervous system.And we research found that TRPV1 of cornea了stroma is enhaced in dry eye mouse model induced by hypertonic saline,thus we estimate that TRPV1 of corneal nerves may take a pivotal role in the pathogenesis of dry eye disease. Our research intends to investigate this topic at clinical, animal model and in vitro studys by using in vivo confocal microscopy, immunofluorescence labeling, patch clamp, real time-PCR, western blot and so on. The research content includes the following experiment. The first one is effects of severity of dry eye disease on corneal nerve morphology and function, expression of tear neurotrophic factor and corneal epithelial cell TRPV1. The second one is relationship between corneal nerve TRPV1 and MAPK pathway proteins in dry eye mouse model. The third one is the effects of hypertonic stress on TRPV1 electrophysiology, MAPK pathway and inflammation mediators of trigeminal ganglia neuron. Through the above experiments, we want to clarify the role of corneal nerve TRPV1 in the pathogenesis of dry eye disease, and provide theoretical basis and new drug targets for treatment of this disease.

干眼病是眼科常见病，发病机制复杂，目前有效治疗手段甚少。目前，认为炎症和渗透压是该病的重要发病因素，新研究发现神经细胞TRPV1参与感知高渗环境和调节炎症通路，并向中枢神经系统发出被解读为"烧灼"感的动作电位。我们发现角膜基质TRPV1在高渗盐水诱导的干眼病模型中表达明显增高，故推测其在该病的发病机制中可能扮演重要角色。本研究拟从临床、动物模型和离体实验三个方面，应用角膜共聚焦显微镜、免疫荧光标记、膜片钳、rt-PCR、western blot等方法进一步研究：1干眼病严重程度对角膜神经、泪液神经营养因子和角膜上皮细胞TRPV1表达的影响；2干眼病角膜感觉神经TRPV1的表达与MAPK炎症通路的关系；3高渗环境对三叉神经节细胞TRPV1电生理、MAPK通路蛋白和炎症介质的影响。通过以上实验，以期初步阐明角膜感觉神经TRPV1在干眼病发病机制中的作用，为该病的治疗提供新的理论依据和药物靶点。

干眼病是眼科常见病，发病机制复杂，目前有效治疗手段甚少。该病是泪液和眼球表面的多因素疾病，引起眼表不适、泪膜不稳定，造成眼球表面损害和视觉障碍，伴有泪膜渗透压的改变和眼表炎症。目前，认为炎症和渗透压是该病的重要发病因素，新研究发现神经细胞TRPV1参与感知高渗环境和调节炎症通路，并向中枢神经系统发出被解读为“烧灼”感的动作电位。因此，我们研究了TRPV1在干眼发病中的作用机制中的作用。.课题组在眼科收集干眼病患者32例，其中轻度干眼患者11例，中度10例，重度11例，通过角膜共焦显微镜观察角膜神经形态；通过印迹细胞学方法测定眼表TRPV1的表达；检测患者泪液中NGF， CGRP以及SP的表达。并选择了12例正常人作为正常对照。我们发现随着干眼病情的加重，角膜TRPV1表达明显增加，而角膜神经纤维密度降低、排列紊乱，泪液NGF和SP表达增加，而CGRP表达量降低。证明TRPV1参与干眼病的发病机制。.将健康大鼠60只，随机分为正常对照组、模型对照组和钌红(2µM，TRPV1抑制剂)干预组，每组20只。模型对照组大鼠双眼采用高渗盐水（500mOsM）点双眼，每日5次；钌红干预组大鼠双眼采用高渗盐水（500mOsM）点眼，每日5次，并采用钌红滴眼，每日3次；正常对照组大鼠双眼每日点生理盐水（295 mOsM），每日5次。结果发现与正常对照组相比，模型对照组和钌红干预组大鼠出现明显的角膜荧光素染色。角膜共聚焦显微镜的检查发现，与正常对照组相比，模型对照组和钌红干预组大鼠角膜上皮细胞境界不清，有炎症细胞浸润。组织免疫荧光发现模型对照组大鼠角膜TRPV1表达增高，而钌红干预组较模型对照组TRPV1表达略有降低，但在JNK、评8和ERK的表达上两无明显差异。.我们的研究发现眼表高渗环境能够使角膜TRPV1的表达增高，而该通道的表达增高可能与干眼病患者眼部烧灼感不适症状相关，但采用TRPV1抑制剂虽降低角膜TRPV1的表达，但未能降低角膜的炎症和MAPK通路蛋白的表达，提示干眼病作为一种多因素疾病，角膜神经TRPV1只是治疗干眼病的潜在靶点之一，该病的治疗还需采用综合的治疗手段。