信号素3A 对氧诱导视网膜病变大鼠视网膜细胞凋亡的影响  

The effect of semaphorin 3A in the process of apoptosis in oxygen induced retinopathy in rats

作　　者：华宁[1] 刘宏伟[1] 钱学翰[1] 东莉洁[1] 武晶晶[1] 李筱荣[1] 

机构地区：[1]天津医科大学眼科医院,300384

出　　处：《中华眼科杂志》2014年第6期440-447,共8页Chinese Journal of Ophthalmology

基　　金：天津市卫生局科技基金（2011KZ104）

摘　　要：目的：探讨信号素3A（ SEMA3A）在大鼠氧诱导视网膜病变（ OIR）模型中的表达变化及其对视网膜组织损伤的影响。方法实验研究。采用随机数字表法将新生大鼠48只分为4组，每组12只（12眼）。正常对照组于正常空气环境下饲养，余3个组隔天交替吸入氧含量为50％和10％的氮氧混合气体，制备OIR模型。 OIR＋SEMA3A抑制组于出生后7 d给予单眼玻璃体腔注射兔抗大鼠SEMA3A多克隆抗体（40 mg/L，2μl）；OIR＋IgG组出生后7 d给予单眼玻璃体腔注射等量兔IgG （40 mg/L，2μl）；OIR组不进行玻璃体腔注射。各组大鼠出生后18 d取材，HE染色检测视网膜厚度、神经节细胞层（ RGCL）细胞数和突破内界膜内皮细胞数，TUNEL染色检测视网膜细胞凋亡并计算凋亡指数（ AI）；免疫印迹法检测视网膜SEMA3A和Caspase-3蛋白p17亚基表达；免疫荧光染色定位检测SEMA3A表达。各组大鼠视网膜厚度、RGCL细胞数、突破内界膜内皮细胞数，视网膜AI，SEMA3A和Caspase-3蛋白p17亚基相对表达量的比较采用单因素方差分析，进一步的两两比较采用LSD-t检验。结果 HE染色结果显示，正常对照组视网膜厚度为（117.07±8.13）μm，OIR组为（70.93±5.68）μm，OIR＋SEMA3A抑制组为（91.28±4.58）μm，OIR＋IgG组为（67.27±10.15）μm，差异有统计学意义（F＝13.222，P＝0.002）。正常对照组RGCL细胞数为42.7±3.6，OIR组为24.3±3.1， OIR＋SEMA3A抑制组为35.0±6.2，OIR＋IgG组为22.8±4.3，差异有统计学意义（ F＝22.537，P＝0.000）。正常对照组突破内界膜的内皮细胞数为1.0±0.3，OIR组为14.2±3.2，OIR＋SEMA3Aab组为9.6±1.1， OIR ＋IgG 组为10.8±1.6，差异有统计学意义（ F ＝14.478， P ＝0.002）。 OIR ＋SEMA3A抑制组视网膜厚度、RGCL细胞数均低于正常对照组（P＜0.01），但均较OIR组和OIR＋IgG组明显增高（P＜0.01）；OIR＋SEMA3A抑制组突破内界膜的内皮细胞�Objective To observe the change of semaphorin 3A（SEMA3A）expression in the retina of oxygen induced retinopathy （ OIR ） in rats and to investigate its influence on retinal degeneration in OIR.Methods Experimental study.Forty-eight newborn pups were randomly classified into four groups and only one eye was examined in each pup.Thirty-six pups was induced into OIR model through aspirating 50%and 10% oxygen every 24 hours alternatively while the control group （ 12 pups ） was raised under normal atmosphere.OIR＋SEMA3Aab group accepted intravitreous injection of rabbit anti-rat SEMA3A antibody （ Abcam Co.LTD, 40 mg/L, 2μl） while OIR＋IgG group was injected the same amount of non-active rabbit IgG at postnatal day 7.And the OIR group had no injections.All the pups were executed at postnatal day 18.Histological changes of retinas were examined through HE stain while Immunoflurecence staining and TUNEL procedure were used to detect focal expression of SEMA 3A and apoptosis respectively.Total tissue protein was extracted and the expression of SEMA 3A and Caspase-3 p17subunit were examined by Western blot.The data including retinal thickness, cell counting of retinal ganglion cells layers （RGCL）, endothelia outside inner limiting membrane, retinal apoptosis index （AI）, the relative expression of SEMA3A and Caspase3 p17 subunit were analyzed by One-way ANOVA and followed LSD-t test to compare group differences.Results There were significant differences of retinal thickness , cell counting of RGCL and endothelia outside inner limiting membrane among each groups respectively （ F=13.222,F=22.537,F=14.478;P〈0.01 ）.The retinal thickness was （ 117.07 ±8.13 ） μm in normal control group , （ 70.93 ± 5.68） μm in OIR group, （91.28 ±4.58） μm in OIR＋SEMA3Aab group, and （67.27 ±10.15） μm in OIR＋IgG group;the cell counting of RGCL was 42.7 ±3.6 in normal control group , 24.3 ±3.1 in OIR group, 35.0 ±6.2 in OIR＋SEMA3Aab group, and 22.8 ±4.3 in OIR＋IgG group while the endot

关 键 词：视网膜病 早产儿 细胞凋亡 疾病模型 动物 

分 类 号：R774.1[医药卫生—眼科]