小鼠视网膜下注射淀粉样蛋白β（1-42）后视网膜组织的变化

Alterations of retinal tissue induced by amyloid β（1-42） subretinal injection in mice

目的 探讨小鼠视网膜下注射淀粉样蛋白β（1-42）寡聚体（OAβ1-42）后视网膜组织的变化.方法 实验研究.120只8周龄C57BL/6N小鼠单纯随机抽样法分为双蒸水组和OAβ（1-42）组,分别在视网膜下腔注射双蒸水和浓度为0.312 5mmol/L的OAβ（1-42）.注射后于不同时间点对实验眼进行观察,包括眼底照相及自发荧光成像、视网膜电图检查、视网膜组织切片HE染色、神经视网膜铺片β-半乳糖苷酶染色、视网膜色素上皮/脉络膜铺片老化标志物免疫荧光染色、视网膜组织中老化相关因子的免疫印迹法及荧光定量PCR检测.实验数据采用独立样本t检验进行统计学分析.结果 与对照组比较,注射后1周时实验组小鼠眼底照相可见视网膜组织萎缩伴自发荧光增强;暗适应视网膜电图显示a波振幅均值下降为（39.94±7.75）μV[对照组（225.27±28.94）μV,t=12.45,P＜0.001],b波振幅均值下降为（185.55±4.62）μV[对照组（873.78±43.80）μV,t=27.06, P＜0.001];HE染色可见视网膜色素上皮细胞色素分布紊乱,视网膜厚度显著变薄（t=75.13,P＜0.001）;β半乳糖苷酶染色及免疫荧光染色老化标志物表达明显增高;以上表现持续到注射后8周.同时分子生物学检测提示,注射后1周时实验组小鼠视网膜组织老化相关因子较对照组表达显著增高.结论 OAβ（1-42）在小鼠视网膜下聚集后可导致视网膜组织老化,使其视觉功能下降,该表现与AMD临床表现类似.

Objective To investigate the alterations of retinal tissue induced by OAβ（1-42） subretinal injection in normal C57BL/6N mice.Methods Experimental study.One hundred and twenty C57BL/6N mice aged 8 weeks participated in this study.Among them, 60 eyes received subretinal injection of OAβ（1-42）;another 60 received double distilled water as control.The concentration of OAβ（1-42） was 0.312 5 mmol/L.We took fundus photograph and autofluorescence, electroretinogram（ERG）, haematoxylineosin staining of retinal paraffin sections, β-galactosidase staining of neural retinal flatmounts, immunofluorescence of senescence marker P16INK4a of RPE/choroidal flatmounts, Westeru blot and RT-PCR of senescence relative cytokines IL-6, TGF-β1 before and after the injection.The data were analyzed by independent sample t-test.Results Comparing with controls, 1 week post injection, the experimental group eyes had atrophied area with high autofluorescence site, and showed evident decrease in amplitudes of a wave （39.94±7.75）μV comparing with the controls （225.27±28.94）μV（t=12.45, P〈0.001） in scotopia ERG, so did the amplitude of b wave （185.55±4.62）μV comparing with the controls （873.78±43.80）μV（t=27.06, P〈0.001）, retinal structures appeared significant loss of retinal thickness （t=75.13, P〈0.001） along with hypo-or hyperpigmentation;the experimental eyes had high frequency of blue-green deposits in retinal β-gal staining;the green fluorescence sites of RPE/choroidal flatmounts were clear and bright, indicating higher expression of P16INK4a in the nucleus;these results were the same till 8 weeks post operation.Western blot and RT-PCR vertified conspicuous increases in expression of TGF-β1, IL-6 in retinas of experimental eyes compared with the control ones 1 week post injection.Conclusions OAβ（1-42） subretinal injection could induce visual function deficiency and retinal tissue senescence.All the manifestations were AMD-like retinopathy, which implied that Aβ（1-42） might be an essential clue in human AMD pathology.