【作者】 高荣莲；

【导师】 钱焕文；

【作者基本信息】 中国人民解放军军事医学科学院 ， 病理学与病理生理学， 2011， 博士

【摘要】 本课题旨在研究角膜烧伤的特点及发展规律,探索其损伤的机制,重点研究激光角膜损伤的分子机理及促进修复措施,同时还应用傅里叶变换红外光谱技术(FT-IR),建立了FT-IR检测角膜蛋白分子结构改变的新方法。1.目的眼部碱烧伤是临床常见而处理棘手的眼外伤,国内外相关研究报道较多。激光角膜烧伤的研究报道甚少。随着激光技术的迅速发展,激光武器已经成为用于战争且技术最成熟的一种新概念武器,各国都在进行相关研究,但由于世界各军事强国都将激光武器的开发研制列入绝密的国家高新技术武器研究计划,因此激光生物效应的研究报道寥寥无几。随着激光武器研发的深入和实战应用,激光生物学效应、损伤机制和治疗措施的研究已成为各国军事医学的重要而紧迫的研究领域。眼是激光辐射最敏感的靶器官,也是激光武器重要致伤部位之一。本课题用碱和激光建立眼烧伤模型,重点研究激光对眼角膜的损伤效应、损伤机制以及成纤维细胞生长因子-10(Fibroblast growth factor-10,FGF-10)对角膜烧伤的治疗效应。激光眼角膜损伤效应与机制研究以及FGF-10治疗效应研究,将为临床角膜烧伤提供新的治疗方案,为我国激光武器的研制和防护提供生物学依据,对国家安全和保障人民身体健康均具有重大意义。2.方法采用日本大耳白兔分别构建碱和激光角膜烧伤模型,实验分烧伤对照组、bFGF治疗对照组、FGF-10 12.5μg/ml组、FGF-10 25μg/ml组和FGF-10 50μg/ml组。通过裂隙灯显微镜与病理组织学观察、烧伤斑图像分析、角膜光密度值测定和MTT法检测损伤后角膜的恢复情况,比较治疗后7、14天各组的差异。建立FT-IR技术用于角膜研究的方法。分析正常兔角膜的化学组成。在此基础上进一步应用FT-IR技术研究激光角膜损伤的分子机制,采用解卷积、曲线拟合等方法对FT-IR图谱酰胺I带中蛋白质二级结构各吸收峰进行定量分析,比较对照组与照射组特征峰的位移与积分值差异,得出蛋白质结构的变化特征。3.结果3.1成功构建稳定的角膜碱烧伤和激光烧伤模型,在角膜中央表面造成白色圆形烧灼斑。3.2物理和化学因素虽然性质不同,但引起的角膜损伤效应相似,主要表现为角膜上皮细胞和内皮细胞的坏死脱落、基质中以中性粒细胞为主的炎细胞浸润及成纤维细胞增生等。3.3 FGF-10有利于眼烧伤修复,表现为烧伤斑面积缩小,角膜透光度恢复,且药物浓度为25μg/ml时,即可达到最大修复率,增加药物浓度并不能相应增加药效。3.4 FGF-10对角膜上皮细胞没有表现促增殖作用。3.5对FGF-10与阳性对照药bFGF的疗效进行比较,结果表明,二者疗效相当,但在改善角膜损伤后组织结构的恢复和抑制角膜新生血管(CNV)形成方面,浓度为25μg/ml的FGF-10治疗作用更显著。3.6 FGF-10可能通过缓和促进角膜上皮细胞的再生和迁移、减轻角膜烧伤后的炎症反应及抑制成纤维细胞过度增生等机制,促进碱和激光烧伤后受损角膜的修复,具有一定的治疗效应。3.7针对兔眼角膜上皮层和基质层的FT-IR研究发现,代表蛋白质酰胺Ⅰ带的红外吸收最大,表明FT-IR是研究角膜损伤分子机制的有效手段。3.8角膜受激光辐射后,发生蛋白质二级结构特征峰的位移,α-螺旋减少与β-折叠结构增加,表明蛋白质结构稳定性下降,趋向无序化。4结论4.1物理和化学因素引起的角膜损伤效应相似,主要表现为急性炎症反应、角膜上皮细胞和内皮细胞坏死脱落、以中性粒细胞为主的炎细胞浸润及成纤维细胞增生等。4.2 FGF-10可通过缓和促进角膜上皮细胞的再生和迁移、减轻角膜烧伤后的炎症反应及抑制成纤维细胞过度增生等机制,促进碱和激光烧伤后受损角膜的修复,具有一定的治疗效应。4.3在改善受损后组织结构的恢复和抑制CNV方面,浓度为25μg/ml的FGF-10治疗效应优于临床常用药bFGF。4.4 FT-IR是研究角膜分子结构组成的有效方法。4.5激光辐射使蛋白质二级结构的特征峰发生位移,构象发生改变,进而引起蛋白质生物功能的紊乱或丧失。热效应可能是激光角膜烧伤的主要机制。更多还原

【Abstract】 Features and law of development of corneal burn were investigated, and mechanisms of injury were explored, focusing on molecular mechanisms of laser induced corneal injury. During mechanism investigation of laser induced corneal injury, Fourier transform-infrared spectrum technology (FT-IR) was employed for the first time, and new protocols of detecting structural changes of corneal proteins through FT-IR were established.1. ObjectivesAlkali burn of eye is a kind of ubiquitous and refractory ocular injury which has been research focus at home and abroad. Investigations about laser induced corneal burn are rarely reported. Laser weapons is a kind of new concept weapons most likely to be used in wars with the most mature technologies, which has entered research plans in many countries. Since research and development of laser weapons has been rated as top secret national high tech weapon research plan in all military great powers, biological effects of laser are rarely reported. With the progress of research and development as well as actual combat usage of laser weapons, investigation about biological effects and injury mechanism of laser has turned into key pressing tasks of military medicine.Eye is one of the most sensitive target organs to laser radiation, and is one of the key damage positions of laser weapons. Injury effects of alkali and laser on corneal, mechanisms of injury, as well as therapeutic effects of fibroblast growth factor-10 (FGF-10) on corneal burn, were investigated in this project. It is expected that investigation on injury effects of laser on corneal, mechanisms of injury, as well as therapeutic effects of FGF-10 may provide new therapy for clinical corneal burn, provide biological basis for development and protection on laser weapons in our country, and be great importance to national security and public health.2. MethodsAlkali and laser corneal burn models were established in Japanese white rabbits. 5 groups were set up, including burn control group, bFGF therapy control group, FGF-10 12.5μg/ml group, FGF-10 25μg/ml group, and FGF-10 50μg/ml group. Corneal recovery was evaluated through slit lamp microscope and histopathological observation, image analysis of burn spot, determination of corneal OD, and MTT method. Group difference was evaluated 7 and 14 days after therapy. FT-IR technology was applied in corneal study. Chemical composition of normal rabbit cornea was investigated. Then FT-IR technology was applied to explore the molecular mechanisms of laser induced corneal injury. Quantitative analysis was performed to absorption peaks resulting from secondary structures of amideⅠband proteins on FT-IR spectrum through deconvolution and curve fitting methods. By comparing displacement and score of marker peaks between control and irradiation groups, structural changes of protein were evaluated3. Results3.1 Stable alkali and laser burn models of cornea were successfully established. White round burn spot was generated in corneal central surface.3.2 Though characteristically different between physical and chemical factors, they induced similar injury effects to cornea, mostly appearing as necrosis and desquamation of corneal epithelial and endothelial cells, matrix infiltration of inflammatory cells, and hyperplasia of fibroblasts.3.3 FGF-10 was found to facilitate reduction of burn spot area and recovery of cornea transparence. Maximum recovery rate was reached at 25μg/ml without efficacy enhancement with further concentration increase.3.4 No proliferative activity on corneal epithelial cells was observed with FGF-10.3.5 Efficacy between FGF-10 and positive control bFGF was compared in this experimental model. The results indicated comparable efficacy, but 25μg/ml FGF-10 was more potent in improving tissue recovery after corneal injury and inhibiting corneal new vasculature (CNV) formation.3.6 By slowly facilitating regeneration and migration of corneal epithelial cells, alleviating various inflammatory responses after corneal burn, and inhibiting excessive hyperplasia of fibroblast, FGF-10 was found to promote recovery of injured cornea after laser burn, thus indicating some therapeutic effects.3.7 FT-IR examination on epithelial and matrix layers of rabbit cornea revealed maximum infrared absorption in amideⅠband proteins, indicated that FT-IR is suitable for the mechanism research of cornea injury.3.8 Displacement of marker peaks in secondary protein structure, decrease inα-helix and increase inβ-sheet after laser radiation indicates decreased stability in protein structure and disorder trend.4 Conclusions4.1 Physical and chemical factors induce similar corneal injury effects, mostly appearing as acute inflammatory responses, necrosis and desquamation of corneal epithelial and endothelial cells, matrix infiltration of inflammatory cells, and hyperplasia of fibroblasts.4.2 By slowly facilitating regeneration and migration of corneal epithelial cells, alleviating various inflammatory responses after corneal burn, and inhibiting excessive hyperplasia of fibroblast, FGF-10 can promote recovery of injured cornea after laser burn, thus indicating some therapeutic effects.4.3 25μg/ml FGF-10 is more potent than commonly used bFGF in improving tissue recovery after corneal injury and CNV formation.4.4 FT-IR is an effective method for molecular structure exploration of cornea.4.5 Laser radiation can result in displacement of marker peaks in secondary protein structure, modify protein conformation, and thus result in disorder or loss of biological function of protein. Thermal effect may be the main mechanism of corneal injury after CO2 laser radiation.更多还原