中波紫外线诱导SD大鼠白内障模型的实验研究
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摘要
目的：探讨用中波紫外线照射SD大鼠建立紫外线诱导的动物白内障模型及对比三种晶状体图像采集的效果,从而为进一步研究白内障的形成,混浊程度的评定及白内障的防治提供相关实验依据。方法：1.选用3月龄的一级大白鼠20只,随机分为实验组和对照组,实验组大鼠置于20×40×20cm3的笼中,笼子上方安置中波紫外线光源,光源距离大鼠约40cm,给予每天2小时中波紫外线照射；对照组不给于任何干预处理。选择不同照射时间段大鼠(0天、5天、20天、40天)摘取眼球去除角膜后在眼前节照相系统下采集晶状体混浊程度及晶状体核的颜色变化。然后环形剪下晶状体前囊膜,细胞面向上铺于玻片上,TUNEL法检测晶状体上皮细胞凋亡情况。观察晶状体上皮细胞的形态变化。2.另选4只大白鼠,用中波紫外线照射40天,采集眼球后对8只眼球同时选用三种方法采集白内障晶状体图像,并对比分析三种方法所获图像的效果。结果：1.眼前节照相系统观察晶状体：实验组结果显示经中波紫外线照射活体SD大鼠每天2小时,在第5天时晶状体保持透明、无明显混浊,随着照射时间的延长,在20天时出现皮质轻度混浊、多集中在“Y”字缝附近,呈现前部片状、羽毛状朝向赤道部的浅层皮质混浊,颜色呈白色,未见晶状体核的透明性及颜色改变。在40天时前囊下皮质混浊程度更加明显,前部浅皮质层及深皮质层均可观察到白色混浊,范围可以扩大到近赤道部；在手术摘取前囊膜时可见多数大鼠浅层混浊的皮质与前囊膜粘连较正常组紧密。40天照射组的晶状体核未发现有明显颜色变化。对照组未发现晶状体皮质及晶状体核有明显混浊和颜色的变化,仍保持其透明性。2. TUNEL细胞凋亡检测：DNA缺口末端原位检测部分大鼠晶状体上皮细胞发现经紫外线照射活体大鼠5天时即可发现晶状体前囊膜上皮细胞出现凋亡,凋亡的细胞呈棕黄色。3.细胞形态变化：5天组的细胞形态、大小与对照组相比无明显改变。40天组部分晶状体上皮细胞出现老化现象,可见大量的晶状体纤维与晶状体上皮细胞相连。4.对同一只白内障晶状体选用不同的三种晶状体采集方法获得晶状体图像,相比而言本实验设计的晶状体托板联合眼前节照相系统法采集的图像较其它两种方法获得的图像更清晰,结果稳定。结论：1.本实验方法成功实现了紫外线辐射性白内障大鼠模型的建立。2.中波紫外线是导致白内障发生的原因之一。3.晶状体混浊程度与中波紫外线照射的时间长短呈正相关。4.中波紫外线照射活体SD大鼠眼可以造成晶状体前囊膜上皮细胞凋亡。5.三种白内障晶状体图像采集方法各有优缺点,本实验采用黑色晶状体托板联合眼前节照相系统可以获得理想的晶状体图像,与其它两种方法相比较具有明显优势。
Objectives:To set up UV-induced cataract model of SD rats and compare the effects of three kind of image acquisition on lens. Thereby provide correlated experimental basis for researching the formation, development, grades of cataract and prevention of cataract. Methods:1. three months-old first class rats (20subjects) were randomized to A or B group. Group A as experiment that put rats in the cage (20×40×20cm3) which was installed UVB lamp. The UVB lamp was 40cm from the rats. The rats was irradiated for 2 hours per day. Group B as control group that did nothing. Pick the rats'eye ball at different time:5-day,20-day,40-day, and remove corneal, and observe the degree of lens turbidity and the change of lens nucleus. Cut the anterior lens capsule roundly and pave it on the glass slide.Detect the apoptotic cells cells with TUNEL method in 20X high power field perimeter and observe the morphologic change of the lens epithelium cells.2. four other rats were selected to E grup for comparing the effects of three kind of image acquisition on lens. Results:1.Observe the lens with anterior eye segment image getting system:experimental group shows the SD rats'lens which receive UVB radiation 2 hours per day keep transparent at the 5th day. The cortex becoming feeble opacity and concentrate round anterior "Y"suture, peripheral cortex opacity as white lamellar anteriorly and featheriness toward ambitus, transparency and color change of the lens nucleus were not seen at days 20. The opacity is more obvious at anterior subcapsular cortex. white haze was seen at anterior peripheral cortex and deep cortex, and extend to ambitus at days 40. The superficial lamella cortex was adhered to anterior capsular more tightly than normal. And the 40-day's radiation shows the color change was not obvios. On control group, the lens keep transparent each time.2. TUNEL apoptosis detection:TdT-mediated X-dUTP nick end labeling reflect that anterior lens capsule epithelium had apoptosis after radiation at 5th day, the apoptosis cells present claybank.3. Morphologic change of cells:The change of shape and size for non-apoptosis lens epithelium cells were not obvious compared with control group at 5th day. The aging phenomenon on lens epithelium cells appeared at 40th day as lens fiber grew out of the epithelium cells.4. the image got by using anterior eye segment image getting system combined with lens pallets were clearer than other tow methods Conclusions:1. By using ultraviolet B radiation we set up UV-induced cataract model in SD rats successfully.2. Ultraviolet B was one of the factors that cause cataract.3. There was a positive correlation between the degree of opacity and radiation lasting time.4. Ultraviolet B radiation can cause anterior lens epithelium cells apoptosis in vivo.5. Three methods of anterior eye segment image getting system each has merits and demerits. Anterior eye segment image getting system combined with lens pallets can get ideal lens images and has obvious superiority by contrast with other tow methods in our experiment.
Objectives:To set up UV-induced cataract model of SD rats and compare the effects of three kind of image acquisition on lens. Thereby provide correlated experimental basis for researching the formation, development, grades of cataract and prevention of cataract. Methods:1. three months-old first class rats (20subjects) were randomized to A or B group. Group A as experiment that put rats in the cage (20×40×20cm3) which was installed UVB lamp. The UVB lamp was 40cm from the rats. The rats was irradiated for 2 hours per day. Group B as control group that did nothing. Pick the rats'eye ball at different time:5-day,20-day,40-day, and remove corneal, and observe the degree of lens turbidity and the change of lens nucleus. Cut the anterior lens capsule roundly and pave it on the glass slide.Detect the apoptotic cells cells with TUNEL method in 20X high power field perimeter and observe the morphologic change of the lens epithelium cells.2. four other rats were selected to E grup for comparing the effects of three kind of image acquisition on lens. Results:1.Observe the lens with anterior eye segment image getting system:experimental group shows the SD rats'lens which receive UVB radiation 2 hours per day keep transparent at the 5th day. The cortex becoming feeble opacity and concentrate round anterior "Y"suture, peripheral cortex opacity as white lamellar anteriorly and featheriness toward ambitus, transparency and color change of the lens nucleus were not seen at days 20. The opacity is more obvious at anterior subcapsular cortex. white haze was seen at anterior peripheral cortex and deep cortex, and extend to ambitus at days 40. The superficial lamella cortex was adhered to anterior capsular more tightly than normal. And the 40-day's radiation shows the color change was not obvios. On control group, the lens keep transparent each time.2. TUNEL apoptosis detection:TdT-mediated X-dUTP nick end labeling reflect that anterior lens capsule epithelium had apoptosis after radiation at 5th day, the apoptosis cells present claybank.3. Morphologic change of cells:The change of shape and size for non-apoptosis lens epithelium cells were not obvious compared with control group at 5th day. The aging phenomenon on lens epithelium cells appeared at 40th day as lens fiber grew out of the epithelium cells.4. the image got by using anterior eye segment image getting system combined with lens pallets were clearer than other tow methods Conclusions:1. By using ultraviolet B radiation we set up UV-induced cataract model in SD rats successfully.2. Ultraviolet B was one of the factors that cause cataract.3. There was a positive correlation between the degree of opacity and radiation lasting time.4. Ultraviolet B radiation can cause anterior lens epithelium cells apoptosis in vivo.5. Three methods of anterior eye segment image getting system each has merits and demerits. Anterior eye segment image getting system combined with lens pallets can get ideal lens images and has obvious superiority by contrast with other tow methods in our experiment.
引文
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3 West SK, Longstreth JD, Munoz BE, et al. Model of risk of cortical cataract in the US population with exposure to increased ultraviolet radiation due to stratospheric ozone depletion[J]. Am J Epidemiol, 2005,162(11):1080-8.
4 曾庆广,左志高,杨长年,郭宝凤,赵宏,唐秀侠,秦晓平.驻西藏高原武警官兵晶状体混浊度调查分析.武警医学.(03),2001.179-180.
5 Congdon NT, West ST, Duncan DT, et al. The effect of pantethine and ultraviolet-B radiation on the development of lenticular opacity in the emory mouse. Curr Eye Res;Current eye research.20(1). ENGLAND, 2000.17-24.
6 祝芷,童文秋,曹凯,袁志兰.紫外线照射致豚鼠白内障的动物模型.南京医学院学报.(01),1994.35-36.
7 唐建,刘谊.紫外线对晶状体上皮细胞损伤的研究进展.眼视光学杂志.(03),2003.187-189.
8 Lofgren S, Michael R, Soderberg PG. Impact of age and sex in ultraviolet radiation cataract in the rat[J]. Invest Ophthalmol Vis Sci, 2003,44(4):1629-33.
9 曹晓光,刘滨,鲍永珍,等.紫外线辐射与人类年龄相关性白内障的基础研究.中国实用眼科杂志.25(8),2007.813-818.
10 Giblin FJ, Leverenz VR, Padgaonkar VA, et al. UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects[J]. Exp Eye Res,2002,75(4):445-58.
11 Kerr JF, Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J]. Br J Cancer,1972,26(4):239-57.
12 Nickells RW, Zack DJ. Apoptosis in ocular disease:a molecular overview[J]. Ophthalmic Genet,1996,17(4):145-65.
13 Mizdrak J, Hains PG, Truscott RJ, et al. Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage[J]. Free Radic Biol Med,2008,44(6):1108-19.
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15 Georgatos SD, Marchesi VT. The binding of vimentin to human erythrocyte membranes:a model system for the study of intermediate filament-membrane interactions[J]. J Cell Biol,1985,100(6):1955-61.
16叶天才,刘东光,余敏斌,孙桂珍.糖皮质激素性白内障93例临床分析.中华眼科杂志.(06),1995.443-446.
17王建伟,严宏,王永强,哈文静,丁正华,郭勇.地塞米松诱导大鼠白内障形态学及酶活性变化.第四军医大学学报.(21),2005.24-27.
18商福,张家萍,张昌颖.亚硒酸钠诱导的晶状体蛋白质聚合作用.生物化学杂志.(04),1991.476-481.
19郝振海,刘冬玲,闫明,等.亚硒酸钠诱发大鼠白内障实验方法的改进.河北医学院学报.(03),1993.147.
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22 Mathur P, Gupta SK, Wegener AR, et al. Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro[J]. Curr Eye Res,2000,21(6):926-33.
23 Taylor A, Jahngen-Hodge J, Smith DE, et al. Dietary restriction delays cataract and reduces ascorbate levels in Emory mice[J]. Exp Eye Res, 1995,61(1):55-62.
24巢国俊,马文新.裂隙灯图像分析系统对大鼠半乳糖性白内障的动态观察和定量分级.中国医药导报.(36),2006.146-147.
25徐雯,姚克,王凯军,吴仁毅,叶娟.Calpains在过氧化氢诱发大鼠晶状体上皮细胞凋亡中的机制探讨.眼科研究.(03),2003.246-249.
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2 Dong X, Lofgren S, Ayala M, et al. Maximum tolerable dose for avoidance of cataract induced by ultraviolet radiation-B for 18 to 60 week old rats[J]. Exp Eye Res,2005,80(4):561-6.
3 West SK, Longstreth JD, Munoz BE, et al. Model of risk of cortical cataract in the US population with exposure to increased ultraviolet radiation due to stratospheric ozone depletion[J]. Am J Epidemiol, 2005,162(11):1080-8.
4 曾庆广,左志高,杨长年,郭宝凤,赵宏,唐秀侠,秦晓平.驻西藏高原武警官兵晶状体混浊度调查分析.武警医学.(03),2001.179-180.
5 Congdon NT, West ST, Duncan DT, et al. The effect of pantethine and ultraviolet-B radiation on the development of lenticular opacity in the emory mouse. Curr Eye Res;Current eye research.20(1). ENGLAND, 2000.17-24.
6 祝芷,童文秋,曹凯,袁志兰.紫外线照射致豚鼠白内障的动物模型.南京医学院学报.(01),1994.35-36.
7 唐建,刘谊.紫外线对晶状体上皮细胞损伤的研究进展.眼视光学杂志.(03),2003.187-189.
8 Lofgren S, Michael R, Soderberg PG. Impact of age and sex in ultraviolet radiation cataract in the rat[J]. Invest Ophthalmol Vis Sci, 2003,44(4):1629-33.
9 曹晓光,刘滨,鲍永珍,等.紫外线辐射与人类年龄相关性白内障的基础研究.中国实用眼科杂志.25(8),2007.813-818.
10 Giblin FJ, Leverenz VR, Padgaonkar VA, et al. UVA light in vivo reaches the nucleus of the guinea pig lens and produces deleterious, oxidative effects[J]. Exp Eye Res,2002,75(4):445-58.
11 Kerr JF, Wyllie AH, Currie AR. Apoptosis:a basic biological phenomenon with wide-ranging implications in tissue kinetics[J]. Br J Cancer,1972,26(4):239-57.
12 Nickells RW, Zack DJ. Apoptosis in ocular disease:a molecular overview[J]. Ophthalmic Genet,1996,17(4):145-65.
13 Mizdrak J, Hains PG, Truscott RJ, et al. Tryptophan-derived ultraviolet filter compounds covalently bound to lens proteins are photosensitizers of oxidative damage[J]. Free Radic Biol Med,2008,44(6):1108-19.
14周健,李燕,惠延年,等.波形纤维蛋白转基因小鼠白内障的形态学观察.第四军医大学学报.21(2),2000.200-203.
15 Georgatos SD, Marchesi VT. The binding of vimentin to human erythrocyte membranes:a model system for the study of intermediate filament-membrane interactions[J]. J Cell Biol,1985,100(6):1955-61.
16叶天才,刘东光,余敏斌,孙桂珍.糖皮质激素性白内障93例临床分析.中华眼科杂志.(06),1995.443-446.
17王建伟,严宏,王永强,哈文静,丁正华,郭勇.地塞米松诱导大鼠白内障形态学及酶活性变化.第四军医大学学报.(21),2005.24-27.
18商福,张家萍,张昌颖.亚硒酸钠诱导的晶状体蛋白质聚合作用.生物化学杂志.(04),1991.476-481.
19郝振海,刘冬玲,闫明,等.亚硒酸钠诱发大鼠白内障实验方法的改进.河北医学院学报.(03),1993.147.
20 RAKIETEN N, RAKIETEN ML, NADKARNI MR. Studies on the diabetogenic action of streptozotocin (NSC-37917)[J]. Cancer Chemother Rep,1963,29:91-8.
21 丁正华,严宏,郭勇,等.链脲佐菌素致大鼠糖尿病性白内障的发病机制.第四军医大学学报.(13),2006.1208-1210.
22 Mathur P, Gupta SK, Wegener AR, et al. Comparison of various calpain inhibitors in reduction of light scattering, protein precipitation and nuclear cataract in vitro[J]. Curr Eye Res,2000,21(6):926-33.
23 Taylor A, Jahngen-Hodge J, Smith DE, et al. Dietary restriction delays cataract and reduces ascorbate levels in Emory mice[J]. Exp Eye Res, 1995,61(1):55-62.
24巢国俊,马文新.裂隙灯图像分析系统对大鼠半乳糖性白内障的动态观察和定量分级.中国医药导报.(36),2006.146-147.
25徐雯,姚克,王凯军,吴仁毅,叶娟.Calpains在过氧化氢诱发大鼠晶状体上皮细胞凋亡中的机制探讨.眼科研究.(03),2003.246-249.
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