本文選題：α-MSH + 干眼�。� 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：目的干眼是一種伴隨淚液滲透壓增高和眼表炎癥的淚液和眼表多因素疾病,通常引起眼部不適、視覺障礙和淚膜不穩(wěn)定,影響患者視力及生活質(zhì)量。本研究旨在建立氫溴酸東莨菪堿誘導(dǎo)的淚液分泌不足型大鼠干眼模型,探索α-MSH對大鼠干眼眼表的保護作用及信號轉(zhuǎn)導(dǎo)機制,運用QAH-DED-1蛋白芯片技術(shù)檢測角結(jié)膜組織中與干眼相關(guān)的因子在蛋白水平上的變化。在角膜上皮細(xì)胞中確定α-MSH是否對苯扎氯銨刺激角膜上皮細(xì)胞具有保護作用和其機制。進一步了解α-MSH在不同生理作用下的個性特點以及共性通路,明確其對干眼眼表保護的作用機制,為研究干眼的發(fā)病機制和藥物作用的靶點、設(shè)計和研制藥物或干預(yù)措施提供理論依據(jù)。方法1、腹腔注射氫溴酸東莨菪堿建立淚液分泌不足型大鼠干眼模型。運用α-MSH給予局部治療,通過各項臨床指標(biāo)的檢測、組織病理學(xué)檢測等方法,探索α-MSH對大鼠干眼眼表的保護作用。并與臨床常用干眼治療藥物羧甲基纖維素鈉聯(lián)合,觀察、檢測及對比兩者對干眼眼表的保護作用。2、運用H89、PD98059分別阻斷PKA-CREB和MEK-Erk兩條信號通路,再局部應(yīng)用α-MSH,結(jié)合各時間點干眼臨床指標(biāo),并通過組織石蠟切片染色、Western Blot等實驗,進一步深入研究α-MSH在改善干眼癥狀的作用中的信號轉(zhuǎn)導(dǎo)機制。3、運用QAH-DED-1蛋白芯片技術(shù)檢測角結(jié)膜組織中的42個與干眼相關(guān)因子的蛋白水平,針對變化最為顯著的EGFR因子進行細(xì)胞實驗。運用苯扎氯銨刺激人角膜上皮細(xì)胞,模擬干眼眼表的炎癥及氧化應(yīng)激狀態(tài)。并運用α-MSH處理細(xì)胞,通過檢測細(xì)胞活性、細(xì)胞凋亡及細(xì)胞遷移能力,確定α-MSH是否對苯扎氯銨刺激角膜上皮細(xì)胞有保護作用。并運用EGFR抑制劑阻斷EGF/EGFR信號通路,確定α-MSH是否仍具有對細(xì)胞的保護作用,明確α-MSH對角膜細(xì)胞保護作用的機制。結(jié)果1、腹部皮下注射氫溴酸東莨菪堿可成功引起大鼠眼表淚膜破裂時間縮短、淚液分泌量下降及角膜上皮完整性的破壞,同時引起角膜上皮增生、水腫,結(jié)膜杯狀細(xì)胞萎縮、數(shù)量減少等干眼癥狀。α-MSH的局部應(yīng)用可有效防止干眼大鼠淚液分泌量的減少,延長淚膜破裂時間,維持淚膜穩(wěn)定性;保護角膜上皮完整性,改善角膜各層細(xì)胞結(jié)構(gòu),減輕角膜水腫情況;對結(jié)膜杯狀細(xì)胞具有保護作用,防止杯狀細(xì)胞的萎縮和減少。與羧甲基纖維素鈉相比,α-MSH在淚膜穩(wěn)定性和角膜上皮完整性方面凸顯出一定的優(yōu)勢。2、α-MSH在氫溴酸東莨菪堿誘導(dǎo)的大鼠干眼模型中具有改善眼表功能、抗炎、形態(tài)維持、抗凋亡和細(xì)胞保護的作用。同時在保護眼表的過程中,α-MSH激活了大鼠干眼眼表組織中的PKA-CREB和MEK-Erk兩條信號通路。通過特異性藥理學(xué)阻斷劑阻斷任一途徑均可消除α-MSH對眼表的保護作用。其中,PKA-CREB信號通路為主要途徑,MEK-Erk為輔助性信號通路。兩條信號通路的相互作用,共同支持α-MSH對干眼眼表的保護作用。3、EGFR的蛋白含量在干眼狀態(tài)下明顯下調(diào),而α-MSH提高了 EGFR的蛋白水平。同時,α-MSH能夠保護HCE細(xì)胞免于苯扎氯銨的破壞,具有維持HCE細(xì)胞活性、遷移能力和抗細(xì)胞凋亡的作用。用EGFR抑制劑Erlotinib-HCl阻斷HCE細(xì)胞EGFR通路后,清除了 α-MSH對HCE細(xì)胞的保護作用,說明了 α-MSH對角膜上皮細(xì)胞的保護作用是通過EGF/EGFR系統(tǒng)得以實現(xiàn)。結(jié)論α-MSH可有效保護乙酰膽堿受體阻斷劑氫溴酸東莨菪堿誘導(dǎo)的淚液分泌不足型大鼠干眼模型的眼表狀態(tài),增加淚液分泌量,延長淚膜破裂時間,維持淚膜穩(wěn)定性和角膜上皮完整性;改善角膜各層細(xì)胞結(jié)構(gòu),減輕角膜水腫情況;防止結(jié)膜杯狀細(xì)胞的萎縮和減少。與羧甲基纖維素鈉相比,α-MSH在淚膜穩(wěn)定性和角膜上皮完整性方面凸顯出一定的優(yōu)勢。α-MSH激活了大鼠干眼眼表組織中的PKA-CREB和MEK-Erk兩條信號通路,并上調(diào)了 EGFR的蛋白水平。兩條信號通路的直接和間接相互作用,共同支持α-MSH對干眼眼表的保護作用。同時,α-MSH具有維持HCE細(xì)胞活性、遷移能力和抗細(xì)胞凋亡的作用,并且該作用是通過EGF/EGFR系統(tǒng)得以實現(xiàn)。
[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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R777.34;R-332

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本文編號：1777845