金纳多和萝卜硫素对小鼠视网膜神经细胞凋亡的保护作用与机制研究
发布时间：2018-07-25 12:16
【摘要】：目的：光照可引起视网膜细胞凋亡,致视网膜外核层损伤。本实验通过对视网膜光损伤小鼠进行金纳多(GBE)和萝卜硫素(SF)心腔注射,探讨GBE和SF对光照引起的视网膜细胞凋亡是否具有预防保护作用及其机制。 方法： 1.建立小鼠光损伤模型：Bal b/c小鼠暗室饲养24小时,次日置于光照箱内,分别于照射24h、48h和72h处死,取眼球制作石蜡切片。HE染色进行视网膜形态学观察；进行外核层厚度测量和外核层层数统计,分析不同光照长度对视细胞造成的损伤;DNA梯度电泳及TUNEL法检测视细胞凋亡。 2. Bal b/c小鼠随机分为6组即正常对照组,实验对照组(生理盐水),GBE组(GBE-Ⅰ组100 ul/二次；GBE-Ⅱ组为50μl/一次,浓度为3.5mg/ml),SF组(SF-Ⅰ组为60μl/二次；SF-Ⅱ组为30μl/一次,浓度为50mg/ml),每组5只,心脏注射给药(替代静脉注射),除正常对照组之外的5组小鼠均在自制光照箱内光照72h后断颈处死,常规制备眼球石蜡切片,HE染色后形态学观察并测量外核层厚度;RT-PCR检测细胞色素C和Bak-1的表达；Caspase活性试剂盒检测Caspase-3活性。使用SPSS13.0统计软件,单因素方差分析法进行分析。 结果： 1.小鼠光损伤模型建立：HE染色形态学观察发现正常组小鼠视网膜结构层次清楚；光照各组外核层明显变薄,视网膜视细胞内、外节排列明显紊乱,分界不清,出现不规则细胞核。凋亡检测及定位：DNA电泳结果显示,光照各组均出现DNA梯状条带,其中光照72h组DNA梯状条带最为显著；TUNEL法检测显示细胞凋亡位于外核层。 2.GBE和SF对视网膜光损伤的预防保护作用：形态学观察发现,实验对照组视网膜细胞排列稀疏,内、外节排列明紊乱、肿胀、破碎,外核层明显变薄；给药组及正常对照组的视网膜结构均优于实验对照组。外核层厚度测量结果,正常对照组49.23±2.32μm,实验对照组40.36±2.17μm; GBE-Ⅰ组、GBE-Ⅱ组外核层厚度分别为42.0770±0.8999μm和40.9411±0.706μm; SF-Ⅰ组、SF-Ⅱ组外核层厚度分别为48.2352±2.0447μm和46.8004±0.6014μm。对结果进行统计学分析表明,GBE-Ⅰ组、GBE-Ⅱ组、SF-Ⅰ组、SF-Ⅱ组与实验对照组比较均有显著差异(P0.001)。 3.GBE和SF对视网膜光损伤的预防保护机制：GBE-Ⅰ组、GBE-Ⅱ组、SF-Ⅰ组、SF-Ⅱ组与实验对照组比较,RT-PCR检测结果显示细胞色素C和Bak-1的表达均不同程度下调。GBE-Ⅰ组、GBE-Ⅱ组、SF-Ⅰ组、SF-Ⅱ组mRNA水平表达与对照组比较有显著性差异(P0.01,P0.05); Caspase-3活性检测结果：GBE-Ⅰ组、GBE-Ⅱ组、SF-Ⅰ组、SF-Ⅱ组与实验对照组比较,Caspase-3活性均降低。 结论： 1.可以采用定时光照建立小鼠视网膜光损伤模型； 2.GBE和SF对视网膜光损伤具有预防保护作用。 3.GBE和SF对视网膜光损伤的预防保护机制均与抑制视网膜细胞凋亡及相关蛋白表达下调有关。
[Abstract]:Objective: light can induce apoptosis of retina cells and damage the outer nuclear layer of retina. By injecting GBE and sulforaphin (SF) into the heart of mice with retinal light injury, this experiment is to explore whether GBE and SF have protective protective effect and mechanism on retinal cell apoptosis induced by light.
Method:
1. the mouse light damage model was established: the Bal b/c mice were kept in the dark room for 24 hours, and the next day was placed in the light box. They were irradiated with 24h, 48h and 72h respectively. The eyeball was taken to make the retinal morphology observation with.HE staining of paraffin section; the thickness of outer nuclear layer and the number of outer layer layers were counted, and the damage caused by different light length to visual cells was analyzed. DNA gradient electrophoresis and TUNEL assay were used to detect the apoptosis of the optic cells.
2. Bal b/c mice were randomly divided into 6 groups: normal control group, experimental control group (physiological saline), group GBE (group GBE- I, 100 ul/ two times, GBE- II group 50 mu l/, 3.5mg/ml), group SF (SF- I group 60 mu l/ two, SF- II group 30 micron, concentration for concentration), 5 in each group, with cardiac injection (instead of intravenous injection), except normal pair 5 groups of mice outside the group were killed in the self-made light box after light 72h, and the eyeball paraffin section was routinely prepared. After HE staining, morphological observation and measuring the thickness of outer nuclear layer; RT-PCR to detect the expression of cytochrome C and Bak-1; Caspase active kit to detect Caspase-3 activity. SPSS13.0 statistical software, single factor variance analysis method was used. Line analysis.
Result:
1. the model of light damage in mice was established: HE staining morphological observation found that the structure of retina in the normal group was clear; the outer nuclear layers were obviously thinner in each group, the outer segments of the retina were obviously disorganized, the boundary was disorderly, the irregular nuclei were unclear, the apoptosis detection and location were found. The results of DNA electrophoresis showed that the DNA ladder appeared in all groups of light. TUNEL assay showed that apoptosis was located in the outer nuclear layer.
The preventive and protective effects of 2.GBE and SF on retinal light damage: morphological observation showed that the retinal cells in the experimental control group were arranged sparsely, the outer segments were arranged in disorder, swelling, broken, and the outer core layer was thinner; the retina structure of the drug group and the normal control group were superior to those in the test control group. The outer nuclear thickness measurement results, the normal control group 49 .23 + 2.32 mu m, experimental control group 40.36 + 2.17 micron m, GBE- I group, GBE- II Group outer nucleus thickness was 42.0770 + 0.8999 mu m and 40.9411 + 0.706 mu m, SF- I group, SF- II Group outer core layer thickness of 48.2352 + 2.0447 mu m and 46.8004 + 0.6014 M. on the results of statistical analysis, GBE- I, group II, group I, group II and experiment There were significant differences between the control group and the control group (P0.001).
The prevention and protection mechanism of retinal light damage by 3.GBE and SF: group GBE- I, group GBE- II, group SF- I, group SF- II and experimental control group. The results of RT-PCR detection showed that the expression of cytochrome C and Bak-1 were all down regulated in the.GBE- I group, GBE- II group and SF- group. Caspase-3 activity test results: GBE-I group, GBE-II group, SF-I group, SF-II group compared with the experimental control group, Caspase-3 activity decreased.
Conclusion:
1. The model of retinal light damage in mice can be established by fixed-time irradiation.
2. GBE and SF have preventive and protective effects on retinal light damage.
3. The protective mechanism of GBE and SF on retinal light injury is related to inhibition of retinal cell apoptosis and down-regulation of related protein expression.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1
本文编号：2143794
[Abstract]:Objective: light can induce apoptosis of retina cells and damage the outer nuclear layer of retina. By injecting GBE and sulforaphin (SF) into the heart of mice with retinal light injury, this experiment is to explore whether GBE and SF have protective protective effect and mechanism on retinal cell apoptosis induced by light.
Method:
1. the mouse light damage model was established: the Bal b/c mice were kept in the dark room for 24 hours, and the next day was placed in the light box. They were irradiated with 24h, 48h and 72h respectively. The eyeball was taken to make the retinal morphology observation with.HE staining of paraffin section; the thickness of outer nuclear layer and the number of outer layer layers were counted, and the damage caused by different light length to visual cells was analyzed. DNA gradient electrophoresis and TUNEL assay were used to detect the apoptosis of the optic cells.
2. Bal b/c mice were randomly divided into 6 groups: normal control group, experimental control group (physiological saline), group GBE (group GBE- I, 100 ul/ two times, GBE- II group 50 mu l/, 3.5mg/ml), group SF (SF- I group 60 mu l/ two, SF- II group 30 micron, concentration for concentration), 5 in each group, with cardiac injection (instead of intravenous injection), except normal pair 5 groups of mice outside the group were killed in the self-made light box after light 72h, and the eyeball paraffin section was routinely prepared. After HE staining, morphological observation and measuring the thickness of outer nuclear layer; RT-PCR to detect the expression of cytochrome C and Bak-1; Caspase active kit to detect Caspase-3 activity. SPSS13.0 statistical software, single factor variance analysis method was used. Line analysis.
Result:
1. the model of light damage in mice was established: HE staining morphological observation found that the structure of retina in the normal group was clear; the outer nuclear layers were obviously thinner in each group, the outer segments of the retina were obviously disorganized, the boundary was disorderly, the irregular nuclei were unclear, the apoptosis detection and location were found. The results of DNA electrophoresis showed that the DNA ladder appeared in all groups of light. TUNEL assay showed that apoptosis was located in the outer nuclear layer.
The preventive and protective effects of 2.GBE and SF on retinal light damage: morphological observation showed that the retinal cells in the experimental control group were arranged sparsely, the outer segments were arranged in disorder, swelling, broken, and the outer core layer was thinner; the retina structure of the drug group and the normal control group were superior to those in the test control group. The outer nuclear thickness measurement results, the normal control group 49 .23 + 2.32 mu m, experimental control group 40.36 + 2.17 micron m, GBE- I group, GBE- II Group outer nucleus thickness was 42.0770 + 0.8999 mu m and 40.9411 + 0.706 mu m, SF- I group, SF- II Group outer core layer thickness of 48.2352 + 2.0447 mu m and 46.8004 + 0.6014 M. on the results of statistical analysis, GBE- I, group II, group I, group II and experiment There were significant differences between the control group and the control group (P0.001).
The prevention and protection mechanism of retinal light damage by 3.GBE and SF: group GBE- I, group GBE- II, group SF- I, group SF- II and experimental control group. The results of RT-PCR detection showed that the expression of cytochrome C and Bak-1 were all down regulated in the.GBE- I group, GBE- II group and SF- group. Caspase-3 activity test results: GBE-I group, GBE-II group, SF-I group, SF-II group compared with the experimental control group, Caspase-3 activity decreased.
Conclusion:
1. The model of retinal light damage in mice can be established by fixed-time irradiation.
2. GBE and SF have preventive and protective effects on retinal light damage.
3. The protective mechanism of GBE and SF on retinal light injury is related to inhibition of retinal cell apoptosis and down-regulation of related protein expression.
【学位授予单位】：大连医科大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R774.1
【参考文献】
相关期刊论文 前10条
1 陶涛;迟焕芳;曹文强;;金纳多对RCS鼠视网膜色素变性神经元保护作用[J];青岛大学医学院学报;2007年02期
2 段鹏;匡洪宇;马丽丽;朱雪磊;江红;康英英;;高糖诱导视网膜血管周细胞凋亡中线粒体细胞色素C的变化[J];国际眼科杂志;2007年04期
3 李楠;黄秀蓉;邓小红;冯振娥;;手术显微镜致大鼠视网膜光损伤中Caspase-3的表达[J];甘肃中医;2009年12期
4 陈为亨;张惠蓉;;眼光性损伤的自由基机理[J];国外医学.眼科学分册;1992年04期
5 赵海山,符静;金纳多对大鼠心肌缺血再灌注损伤的保护作用[J];实用药物与临床;2005年04期
6 范俊萍,彭清;维生素C、维生素E对视网膜光损伤防护作用的研究[J];山西医科大学学报;2001年02期
7 彭清;顾国贞;邓明辉;任佩贤;;不同日龄视网膜变性大鼠眼底组织结构学观察[J];山西医科大学学报;2007年05期
8 何志巍;Caspases与细胞凋亡研究进展[J];国外医学(生理、病理科学与临床分册);1999年01期
9 刘爱琴,路雪婧,廖品正;视网膜光化学损伤机制及其防治的研究进展[J];中国中医眼科杂志;2003年04期
10 李光玲,朱秀安;大鼠实验性视网膜光损伤中的视细胞凋亡[J];中华眼底病杂志;1999年03期
，本文编号：2143794
本文链接：https://www.wllwen.com/yixuelunwen/yank/2143794.html
最近更新
教材专著
