α-MSH对大鼠干眼模型眼表的保护作用和机制研究

发布时间：2018-04-20 12:59

本文选题：α-MSH + 干眼　；参考：《天津医科大学》2017年博士论文

【摘要】：目的干眼是一种伴随泪液渗透压增高和眼表炎症的泪液和眼表多因素疾病,通常引起眼部不适、视觉障碍和泪膜不稳定,影响患者视力及生活质量。本研究旨在建立氢溴酸东莨菪碱诱导的泪液分泌不足型大鼠干眼模型,探索α-MSH对大鼠干眼眼表的保护作用及信号转导机制,运用QAH-DED-1蛋白芯片技术检测角结膜组织中与干眼相关的因子在蛋白水平上的变化。在角膜上皮细胞中确定α-MSH是否对苯扎氯铵刺激角膜上皮细胞具有保护作用和其机制。进一步了解α-MSH在不同生理作用下的个性特点以及共性通路,明确其对干眼眼表保护的作用机制,为研究干眼的发病机制和药物作用的靶点、设计和研制药物或干预措施提供理论依据。方法1、腹腔注射氢溴酸东莨菪碱建立泪液分泌不足型大鼠干眼模型。运用α-MSH给予局部治疗,通过各项临床指标的检测、组织病理学检测等方法,探索α-MSH对大鼠干眼眼表的保护作用。并与临床常用干眼治疗药物羧甲基纤维素钠联合,观察、检测及对比两者对干眼眼表的保护作用。2、运用H89、PD98059分别阻断PKA-CREB和MEK-Erk两条信号通路,再局部应用α-MSH,结合各时间点干眼临床指标,并通过组织石蜡切片染色、Western Blot等实验,进一步深入研究α-MSH在改善干眼症状的作用中的信号转导机制。3、运用QAH-DED-1蛋白芯片技术检测角结膜组织中的42个与干眼相关因子的蛋白水平,针对变化最为显著的EGFR因子进行细胞实验。运用苯扎氯铵刺激人角膜上皮细胞,模拟干眼眼表的炎症及氧化应激状态。并运用α-MSH处理细胞,通过检测细胞活性、细胞凋亡及细胞迁移能力,确定α-MSH是否对苯扎氯铵刺激角膜上皮细胞有保护作用。并运用EGFR抑制剂阻断EGF/EGFR信号通路,确定α-MSH是否仍具有对细胞的保护作用,明确α-MSH对角膜细胞保护作用的机制。结果1、腹部皮下注射氢溴酸东莨菪碱可成功引起大鼠眼表泪膜破裂时间缩短、泪液分泌量下降及角膜上皮完整性的破坏,同时引起角膜上皮增生、水肿,结膜杯状细胞萎缩、数量减少等干眼症状。α-MSH的局部应用可有效防止干眼大鼠泪液分泌量的减少,延长泪膜破裂时间,维持泪膜稳定性;保护角膜上皮完整性,改善角膜各层细胞结构,减轻角膜水肿情况;对结膜杯状细胞具有保护作用,防止杯状细胞的萎缩和减少。与羧甲基纤维素钠相比,α-MSH在泪膜稳定性和角膜上皮完整性方面凸显出一定的优势。2、α-MSH在氢溴酸东莨菪碱诱导的大鼠干眼模型中具有改善眼表功能、抗炎、形态维持、抗凋亡和细胞保护的作用。同时在保护眼表的过程中,α-MSH激活了大鼠干眼眼表组织中的PKA-CREB和MEK-Erk两条信号通路。通过特异性药理学阻断剂阻断任一途径均可消除α-MSH对眼表的保护作用。其中,PKA-CREB信号通路为主要途径,MEK-Erk为辅助性信号通路。两条信号通路的相互作用,共同支持α-MSH对干眼眼表的保护作用。3、EGFR的蛋白含量在干眼状态下明显下调,而α-MSH提高了 EGFR的蛋白水平。同时,α-MSH能够保护HCE细胞免于苯扎氯铵的破坏,具有维持HCE细胞活性、迁移能力和抗细胞凋亡的作用。用EGFR抑制剂Erlotinib-HCl阻断HCE细胞EGFR通路后,清除了 α-MSH对HCE细胞的保护作用,说明了 α-MSH对角膜上皮细胞的保护作用是通过EGF/EGFR系统得以实现。结论α-MSH可有效保护乙酰胆碱受体阻断剂氢溴酸东莨菪碱诱导的泪液分泌不足型大鼠干眼模型的眼表状态,增加泪液分泌量,延长泪膜破裂时间,维持泪膜稳定性和角膜上皮完整性;改善角膜各层细胞结构,减轻角膜水肿情况;防止结膜杯状细胞的萎缩和减少。与羧甲基纤维素钠相比,α-MSH在泪膜稳定性和角膜上皮完整性方面凸显出一定的优势。α-MSH激活了大鼠干眼眼表组织中的PKA-CREB和MEK-Erk两条信号通路,并上调了 EGFR的蛋白水平。两条信号通路的直接和间接相互作用,共同支持α-MSH对干眼眼表的保护作用。同时,α-MSH具有维持HCE细胞活性、迁移能力和抗细胞凋亡的作用,并且该作用是通过EGF/EGFR系统得以实现。
[Abstract]:Objective dry eye is a multifactor disease of tear and eye surface associated with increased lacrimal osmotic pressure and ocular surface inflammation, which usually causes eye discomfort, visual impairment and tear film instability, and affects the visual acuity and life quality of the patients. The aim of this study was to establish a dry eye model in rats induced by scopolamine hydrobromide, and to explore the effect of alpha -MSH on rats The protective effect of dry eye ocular surface and the mechanism of signal transduction, QAH-DED-1 protein chip technique was used to detect the changes in the protein level associated with the dry eye in the conjunctiva. In corneal epithelial cells, it was determined that alpha -MSH has protective effect and mechanism on the stimulation of corneal epithelial cells by alpha chloro ammonium chloride, and further understand that alpha -MSH is not. In order to study the mechanism of dry eye eye surface protection and provide a theoretical basis for the study of the pathogenesis of dry eye and the target of drug effect, and the design and development of drugs or intervention measures, 1, intraperitoneal injection of scopolamine hydrobromide to establish a dry eye model of tear secretory rats. The protective effect of alpha -MSH on the eye surface of the dry eye of rats was explored through the detection of various clinical indexes and histopathological detection by alpha -MSH, and the protective effect of.2 on dry eye eye surface was observed, detected and compared with the common dry eye drug carboxymethyl cellulose sodium, and H89 and PD98059 were used to block PKA respectively. Two signal pathways of -CREB and MEK-Erk, and then local application of alpha -MSH, combined with the clinical indexes of dry eyes at each time point, and by tissue paraffin staining and Western Blot, further study the signal transduction mechanism of alpha -MSH in improving dry eye symptoms, and use QAH-DED-1 protein chip technology to detect 42 in the angular conjunctiva. With the protein level of dry eye related factors, cell experiments were conducted to stimulate the most significant change of EGFR factor. Using benzalkonium chloride to stimulate human corneal epithelial cells to simulate the inflammatory and oxidative stress state of dry eye eye surface. The cells were treated with alpha -MSH, and the cell activity, cell apoptosis and cell migration ability were detected to determine whether alpha -MSH was benzene. The protective effect of chloro ammonium chloride on corneal epithelial cells was stimulated. And EGFR inhibitors were used to block the EGF/EGFR signaling pathway to determine whether alpha -MSH still had protective effects on the cells, and the mechanism of alpha -MSH to the protection of corneal cells was clearly defined. Results 1, subcutaneous injection of scopolamine hydrobromide could lead to a shortened tear film time in the ocular surface of the rat. The decrease of tear secretion and the destruction of corneal epithelial integrity, and the corneal epithelial hyperplasia, edema, conjunctival goblet cell atrophy, decrease in quantity and other dry eye symptoms. The local application of alpha -MSH can effectively prevent the decrease of tear secretion in dry eye rats, prolong tear film rupture time, maintain tear film stability, protect corneal epithelium integrity, change The cell structure of all layers of the cornea can reduce the corneal edema, protect the conjunctival goblet cells and prevent the atrophy and decrease of goblet cells. Compared with sodium carboxymethyl cellulose, alpha -MSH shows a certain dominant.2 in tear film stability and corneal epithelial integrity, and alpha -MSH is in the dry eye model of rats induced by scopolamine hydrobromide It can improve eye surface function, anti-inflammatory, morphologic maintenance, anti apoptosis and cell protection. In the process of protecting eye surface, alpha -MSH activates two signal pathways of PKA-CREB and MEK-Erk in the eye surface tissue of rats. The protective effect of alpha -MSH on eye surface can be eliminated by blocking any path through specific pharmacological blockers. PKA-CREB signaling pathway is the main pathway and MEK-Erk is an auxiliary signaling pathway. The interaction of two signaling pathways supports the protective effect of alpha -MSH on dry eye eye surface (.3). The protein content of EGFR is obviously down under dry eye, and alpha -MSH improves the protein level of EGFR. At the same time, alpha -MSH can protect HCE cells from benzalkonium chloride. Destruction has the role of maintaining HCE cell activity, migration ability and anti apoptosis. After blocking the EGFR pathway of HCE cells with the EGFR inhibitor Erlotinib-HCl, the protective effect of alpha -MSH on HCE cells is cleared. It shows that the protective effect of alpha -MSH on corneal epithelial cells is realized through the EGF/EGFR system. Conclusion alpha -MSH can protect acetyl effectively. The eye surface state of the dry eye model rats induced by the cholinergic receptor blocker was induced by scopolamine hydrobromide, which increased tear secretion, prolonged tear film rupture time, maintained tear film stability and corneal epithelial integrity, improved the cell structure of all layers of the cornea, alleviated corneal swelling, and prevented the atrophy and decrease of conjunctival goblet cells. Compared with sodium carboxymethyl cellulose, alpha -MSH highlights some advantages in tear film stability and corneal epithelial integrity. Alpha -MSH activates two signal pathways of PKA-CREB and MEK-Erk in the eye surface tissue of rats and up regulation of the protein level of EGFR. Direct and indirect interactions of two signal pathways together support alpha -MSH to the stem. At the same time, alpha -MSH has the role of maintaining HCE cell activity, migration ability and anti apoptosis, and the effect is realized through the EGF/EGFR system.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R777.34;R-332

【参考文献】