紫外线诱导的大鼠晶状体氧化损伤和防御机制的实验研究

发布时间：2018-06-13 18:11

本文选题：光散射 + 晶状体　；参考：《山东大学》2010年博士论文

【摘要】：临床意义白内障是世界首位致盲眼病,晶状体的氧化损伤是白内障形成的主要机制。大量临床和流行病学研究已经证实,紫外线辐射(ultraviolet radiation, UVR)是人类白内障形成和发展最重要的危险因素之一。近年来,气候污染造成的大气臭氧层破坏更增加了紫外线对地球的辐射,不可避免地危害人类健康,大大增加了白内障形成的危害性。同时,随着世界人口的不断增长和人口老龄化的加剧,白内障的发病率会持续升高。白内障造成的公共健康问题和所带来的经济负担日益加重,对我们研究紫外线辐射导致白内障形成和发展机制、探讨晶状体内自身抗氧化防御体系的保护作用提出了新的要求；同时对研究和开发非手术性预防和延迟白内障的新方法提出了更高的目标。因此WHO提出防护紫外线辐射,研究开发低成本的抗氧化剂是预防和延迟白内障的首要目标。本课题的研究目的：通过建立近阈值量紫外线(UVR-300nm)诱导的大鼠白内障模型,采用测量晶状体光散射强度的方法,对紫外线照射后晶状体的混浊程度进行定量研究和动态观察；分光光度法对紫外线照射后不同时间晶状体内谷胱甘肽氧化还原体系中的相应生化指标进行定量分析和动态研究,进一步探讨紫外线导致白内障形成和发展的机制。同时研究和探讨抗氧化剂α-生育酚(维生素E)对紫外线诱导大鼠白内障的保护作用与其剂量之间的量效关系；以及α-生育酚对紫外线照射后晶状体内谷胱甘肽氧化还原体系的影响和相互作用。为今后进行α-生育酚防护紫外线辐射白内障的研究提供详实的实验数据,更好地开发和利用维生素E的重要生物功能,为临床合理应用维生素E预防和延迟白内障的发展提供可循的实验依据。实验一：紫外线诱导的大鼠白内障晶状体内谷胱甘肽氧化还原平衡体系的变化目的：观察近阈值量紫外线诱导的大鼠白内障随时间变化的特点；研究紫外线照射后不同时间晶状体内谷胱甘肽氧化还原平衡体系的动态变化；探讨紫外线诱导白内障形成和发展的机制。方法：40只6周龄的albino Spraque Dawley雌性大鼠随机分为4组,每组10只大鼠,实验组大鼠单侧眼行紫外线照射(UVR-300 nm,8 kJ/m2)15分钟；另一组未行紫外线照射的大鼠作为正常对照组。3个实验组大鼠分别于紫外线照射后1、3和7天处死,摘除双侧眼球,分离晶状体,测量晶状体光散射强度,定量分析晶状体混浊度,暗背景光晶状体照像,分光光度法测定晶状体内还原型(reduced glutathione, GSH)和氧化型(oxidized glutathione, GSSG)谷胱甘肽浓度及谷胱甘肽还原酶(glutathione reductase, GR)和谷胱甘肽过氧化物酶(glutathione peroxidase, GPx)的活性。正常对照组大鼠,提供大鼠正常晶状体各项实验参数的基线值。紫外线照射眼晶状体与对侧未照射眼晶状体各项实验参数差值的平均值作为统计学分析的原始数据。结果：紫外线照射眼的晶状体混浊程度随紫外线照射后时间的增加而逐渐加重；而正常对照组大鼠晶状体和实验组大鼠的对侧未照射眼晶状体,均未发现明显混浊；紫外线照射眼与对侧未照射眼晶状体光散射强度的差值,与正常对照组大鼠的晶状体比较,差异有统计学意义。紫外线照射后第1天,晶状体内GSH浓度出现一过性升高,随后GSH浓度随照射后时间的增加而逐渐下降,至紫外线照射后第7天GSH浓度仍未恢复到正常水平；紫外线照射后第1天,紫外线照射眼与对侧未照射眼晶状体GSH浓度的差值,与紫外线照射后第3天和第7天比较,差异有统计学意义。紫外线照射后第1天,晶状体内GPx活性出现一过性增强,随后GPx活性随照射后时间的增加而逐渐下降,至紫外线照射后第7天GPx活性已恢复到正常水平；紫外线照射后第1天,紫外线照射眼与对侧未照射眼的晶状体内GPx活性的差值,与紫外线照射后第3天和第7天比较,差异有统计学意义。紫外线照射后1-7天晶状体内GSSG浓度和GR活性均无显著性变化。结论：近阈值量紫外线照射(UVR-300 nm)可以改变大鼠晶状体内谷胱甘肽氧化还原平衡状态,并启动晶状体细胞内适应性抗氧化反应；晶状体混浊度随紫外线照射后时间增加逐渐加重；近阈值量紫外线照射后1周晶状体形态学和细胞内氧化还原体系均未恢复到正常状态。实验二： α-生育酚对紫外线诱导的大鼠白内障保护作用量效关系的实验研究目的：探讨α-生育酚对紫外线诱导的大鼠白内障的保护作用与口服α-生育酚剂量之间的量效关系；阐述α-生育酚对紫外线照射后晶状体内谷胱甘肽氧化还原体系的影响及晶状体内不同抗氧化剂之间的相互作用。方法：100只6周龄的albino Spraque Dawley雌性大鼠随机分为5组,每组20只。4个实验组大鼠每日给予α-生育酚喂养,剂量分别为5、25、50和100 IU/天；另一组无α-生育酚喂养的大鼠为对照组。喂养4周后,所有大鼠单侧眼行紫外线照射(UVR-300 nm,8 kJ/m2)15分钟。大鼠分别于紫外线照射后1周处死,摘除双侧眼球,分离晶状体,测量晶状体光散射强度,定量分析晶状体混浊度,暗背景光晶状体照像,分光光度法测定晶状体内GSH/GSSG浓度及GR/GPx的活性。紫外线照射眼晶状体与对侧未照射眼晶状体各项实验参数差值的平均值作为统计学分析的原始数据。结果：α-生育酚喂养组大鼠紫外线照射眼仅出现表浅和轻度的晶状体混浊；对照组大鼠紫外线照射眼则出现明显的皮质性和致密的赤道部晶状体混浊。所有大鼠紫外线照射眼的晶状体光散射强度均高于对侧未照射眼：紫外线照射眼与对侧未照射眼晶状体光散射强度的差值,随口服α-生育酚剂量增加而逐渐下降(速率常数为4.5),当α-生育酚剂量超过25 IU/天以上后,晶状体光散射强度的变化趋于平稳；各实验组大鼠与对照组大鼠晶状体光散射强度差值比较,差异均有统计学意义。紫外线照射后大鼠晶状体内GSH浓度明显下降；GSH的下降量随口服α-生育酚剂量的增加而逐渐减小(速率常数为52),当α-生育酚剂量超过25 IU/天以上后,晶状体内GSH的下降量渐趋平稳；对照组和小剂量(5IU/天)α-生育酚喂养组大鼠,与大剂量(25-100 IU/天)α-生育酚喂养组大鼠比较,紫外线照射后晶状体内GSH的下降量,差异有统计学意义。α-生育酚喂养组大鼠与对照组大鼠紫外线照射后晶状体内GSSG浓度及GR/GPx活性均无显著性变化。结论：α-生育酚对紫外线诱导的大鼠白内障的保护作用与口服α-生育酚的剂量呈剂量依赖关系。α-生育酚能够降低紫外线照射后晶状体的混浊程度,但不能完全阻止白内障的发生。α-生育酚能够减少紫外线照射后晶状体内GSH的消耗,其保护作用与口服α-生育酚的剂量呈剂量依赖关系。紫外线照射导致的大鼠白内障只有在晶状体内GSH的下降量超出其阈值时才会发生。口服α-生育酚不能改变紫外线照射后晶状体内GSSG的浓度及GR/GPx的活性。
[Abstract]:Clinical significance
Cataract is the first blind eye disease in the world, and the oxidative damage of the lens is the main mechanism of cataract formation. A large number of clinical and epidemiological studies have confirmed that ultraviolet radiation (UVR) is one of the most important risk factors for the formation and development of human cataracts. It also increases the radiation of ultraviolet radiation to the earth, inevitably endangers human health and greatly increases the dangers of cataract formation. At the same time, the incidence of cataracts will continue to rise as the world population grows and population aging, and the public health problems and economic burdens caused by cataracts are increasing. It is important for us to study the mechanism of cataract formation and development by ultraviolet radiation, to explore the new requirements for the protection of the antioxidation defense system in the crystalline body, and to put forward higher targets for the new methods for the study and development of non operative prevention and delayed cataract. Therefore, WHO proposes to protect ultraviolet radiation and study it. Low cost antioxidants are the primary goal of preventing and delaying cataract.
The purpose of this study is to establish a rat model of cataract induced by near threshold ultraviolet (UVR-300nm). Using the method of measuring the intensity of light scattering of the lens, the degree of turbidity in the lens after ultraviolet radiation is quantitatively studied and dynamic observed. The quantitative analysis and dynamic study of the corresponding biochemical indexes in the peptide redox system were carried out to further explore the mechanism of ultraviolet induced cataract formation and development. At the same time, the quantitative relationship between the protective effect of alpha tocopherol (vitamin E) on ultraviolet induced cataract in rats and its dose, and the alpha birth were studied and discussed. The effect and interaction of phenol on the glutathione redox system in the crystalline body after ultraviolet irradiation, provide detailed experimental data for the future research on alpha tocopherol protection against ultraviolet radiation cataract, and better develop and utilize the important biological functions of vitamin E, for the clinical application of vitamin E to prevent and delay cataract The development provides the basis for the experiment.
Experiment 1:
Changes of glutathione redox balance in rat cataract induced by ultraviolet light
Objective: To observe the changes of rat cataracts induced by near threshold ultraviolet (UVB) and to study the dynamic changes in the system of GSH redox balance at different times after ultraviolet radiation, and to explore the mechanism of ultraviolet induced cataract formation and development.
Methods: 40 6 weeks old albino Spraque Dawley female rats were randomly divided into 4 groups, with 10 rats in each group. The experimental group was irradiated with ultraviolet radiation (UVR-300 nm, 8 kJ/m2) for 15 minutes. The other group of rats without ultraviolet radiation was killed in 1,3 and 7 days after ultraviolet radiation in the normal control group, and the two groups were removed. Side eyeball, lens separation, measurement of light scattering intensity of lens, quantitative analysis of lens turbidity, dark background photo lens, spectrophotometric determination of reduced glutathione, GSH and oxidized glutathione, GSSG Valley caspin concentration and glutathione reductase (glutathione reductase, GR) and valley The activity of glutathione peroxidase (GPx) in the normal control group, provided the baseline values of the experimental parameters of the normal lens of the rat. The mean value of the difference between the lens of the ultraviolet light and the experimental parameters of the unirradiated eye lens was used as the original data of the analysis.
Results: the degree of lens opacity increased gradually with the increase of the time after ultraviolet radiation, while there was no obvious turbidity in the lens of the normal control group and the lens of the experimental group, and the difference between the ultraviolet light and the light scattering intensity of the unirradiated eye was normal. The difference was statistically significant in the lens of the rats. The concentration of GSH in the crystalline body increased at first days after ultraviolet radiation, and then the concentration of GSH decreased gradually with the increase of the time after irradiation, and the concentration of GSH was still not recovered to the normal level at seventh days after ultraviolet radiation, and the ultraviolet rays irradiated the eye first days after ultraviolet radiation. The difference in the concentration of GSH in the unirradiated eyes was statistically significant compared with the third and seventh days after ultraviolet irradiation. The activity of GPx in the crystalline body appeared to be enhanced at first days after ultraviolet radiation, and then the activity of GPx decreased gradually with the increase of the irradiation time, and the activity of GPx was restored to normal after the ultraviolet radiation. The difference of GPx activity between ultraviolet light and unirradiated eye first days after ultraviolet radiation was statistically significant compared with the third days and seventh days after ultraviolet radiation. There was no significant change in the concentration of GSSG and the activity of GR in the crystalline body 1-7 days after ultraviolet irradiation.
Conclusion: near threshold ultraviolet radiation (UVR-300 nm) can change the redox equilibrium state of glutathione in the rat crystalline body and initiate the adaptive antioxidant reaction in the lens cells, and the lens opacity increases gradually with the increase of the time after ultraviolet radiation, and the morphology and cell of the lens are 1 weeks after the near threshold ultraviolet radiation. The redox system did not recover to the normal state.
Experiment two:
Experimental study on dose effect relationship of alpha tocopherol on ultraviolet induced cataract in rats
Objective: To investigate the relationship between the protective effect of alpha tocopherol on ultraviolet induced cataract in rats and the dose effect of oral alpha tocopherol, and the effect of alpha tocopherol on the redox system of glutathione in crystalline body after ultraviolet irradiation and the interaction between different antioxidants in the crystalline body.
Methods: 100 6 weeks old albino Spraque Dawley female rats were randomly divided into 5 groups. Each group of 20 rats in each group was given alpha tocopherol every day with a dose of 5,25,50 and 100 IU/ days, and the other group without alpha tocopherol was fed as the control group. After feeding for 4 weeks, the unilateral eyes of all rats were irradiated with ultraviolet radiation (UVR-300 nm, 8 kJ/m2). ) 15 minutes. The rats were killed at 1 weeks after the ultraviolet radiation, the lens was removed, the lens was separated, the light scattering intensity of the lens was measured, the lens opacity, the dark background light lens were quantified, the concentration of GSH/GSSG in the crystalline body and the viability of the GR/GPx were measured by spectrophotometry. The lens of the ultraviolet light and the unirradiated eye in the opposite side The average value of the experimental parameters difference is the original data of statistical analysis.
Results: only light and mild lens opacities were found in the ultraviolet light of the rats in the alpha tocopherol feeding group, and the ultraviolet light in the control group showed obvious cortical and dense equatorial lens opacities. The intensity of the light scattering of the ultraviolet light in all rats was higher than that in the unirradiated eye: ultraviolet radiation. The difference between the light scattering intensity of the eye and the contralateral unirradiated eyes gradually decreased with the increase of the dose of alpha tocopherol (rate constant 4.5). When the dose of alpha tocopherol exceeded 25 IU/ days, the change of light scattering intensity of the lens tended to be stable; the difference between the experimental group and the lens light scattering intensity difference between the experimental group and the rats was poor. The concentration of GSH decreased significantly in the crystalline body of rats after ultraviolet radiation, and the decrease of GSH gradually decreased with the increase of oral alpha tocopherol dose (rate constant was 52). When alpha tocopherol dose exceeded 25 IU/ days, the decrease of GSH in the crystalline body gradually became stable; the control group and small dose (5IU/ days) alpha birth Compared with the large dose (25-100 IU/ days) alpha tocopherol feeding rats, the decrease of GSH in the crystalline body after ultraviolet irradiation was statistically significant. There was no significant change in the concentration of GSSG and the activity of GR/GPx in the crystalline body of the rats of the alpha tocopherol feeding group and the control group.
Conclusion: the protective effect of alpha tocopherol on ultraviolet induced cataract in rats is dependent on the dose of oral alpha tocopherol. Alpha tocopherol can reduce the turbidity of the lens after ultraviolet radiation, but can not completely prevent the occurrence of cataract. Alpha tocopherol can reduce the consumption of GSH in the crystalline body after ultraviolet radiation. The protective effect is in a dose-dependent manner with the dose of oral alpha tocopherol. The cataract induced by ultraviolet radiation occurs only when the decrease of GSH in the crystalline body exceeds its threshold. The oral alpha tocopherol can not change the concentration of GSSG in the crystalline body and the activity of GR/GPx in the crystalline body after ultraviolet radiation.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R776.1

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