【摘要】 目的 探讨Egr-1在低频闪烁光（FL）诱导的小鼠近视中的表达及与细胞外基质重塑通路可能的调控机制。方法 实验研究。将28日龄健康C57BL/6J（B6）小鼠（100只）随机分为正常对照组和FL组。闪烁光组光照频率为2 Hz，明暗周期为50%。分别于实验前，实验后1 h、1 d、1周、2周、恢复1周（FL诱导2周后撤除FL，于正常光照下继续饲养1周），使用红外偏心验光仪和动物专用A超测定仪测量所有小鼠右眼的屈光度及眼轴。于实验后每个时间点每组各提取10只小鼠右眼的视网膜组织进行RT-PCR检测Egr-1、膜型基质金属蛋白酶1（MT1-MMP）、金属基质金属蛋白酶2（MMP-2）、组织抑制金属蛋白酶2（TIMP-2）的动态表达，Western Blot及免疫组化、免疫荧光观察Egr-1与MT1-MMP蛋白表达及定位。FL组与对照组各指标组内均数行ANOVA分析，组间均值行独立样本t检验比较分析。结果 FL光照2周后，小鼠的屈光度相比对照组发生了近视改变[（0.32±0.14）D vs. （3.42±0.31）D，t=29.08，P<0.01]，眼轴也显著增长[（2.97±0.01）mm vs. （2.93±0.01）mm，t=6.914，P<0.01]。FL光照1 h后Egr-1 mRNA和蛋白快速下调，且随诱导时间持续低于正常，MT1-MMP mRNA和蛋白水平快速持续上调，实验1 d MMP-2 mRNA水平开始上调，趋势与MT1-MMP相同，TIMP-2 mRNA在实验1周和2周的时候明显低于正常组。恢复1周后，Egr-1和TIMP-2明显上调，而MT1-MMP和MMP-2表达下调，Egr-1与MT1-MMP呈现负相关关系（r2=0.6478，P<0.05）。免疫荧光双标示Egr-1与MT1-MMP在视网膜神经节细胞存在共表达。结论 视网膜中Egr-1可能通过调控MT1-MMP的表达来影响MMP-2的活化，进而参与闪烁光诱导的小鼠近视的发生及恢复过程。
【关键词】 近视； Egr-1； 闪烁光； 基质金属蛋白酶类，膜相关； 基质金属蛋白酶2； 模型，动物
DOI：10.3760/cma.j.issn.1674-845X.2014.06.003
作者单位：226001 南通医院眼科（李曼现在济南市明水眼科医院）
通信作者：陈辉，Email：chenhuieye@126.com
The modulation of retinal Egr-1 and the extracellular matrix remodeling pathway in flickering light-induced myopia in mice Li Man， Yu Ying， Guan Huaijin， Chen Hui. Department of Ophthalmology， Affiliated Hospital of Nantong University， Nantong 226001， China
Corresponding author：Chen Hui，Email：chenhuieye@126.com
【Abstract】 Objective To investigate the expression of early growth response protein-1 （Egr-1） in the retina of myopic mice induced by a low-frequency flickering light （FL） and its underlying regulation mechanism in the extracellular matrix remodeling pathway. Methods C57BL/6 mice （n=100） 28 days of age were randomly assigned to two groups： a normal control （n=50） and an FL stimulation group （n=50）. The FL group were raised under illumination with a duty cycle of 50% at a flash rate of 2 Hz. Refractive state and axial length （AL） of the right eyes were measured by a murine-specific eccentric infrared photorefraction and A-scan ultrasonography， respectively. The data were collected at pre-treatment and at 1 hour， 1 day， 1 week， 2 weeks， and after 1 week of recovery. The FL group were removed after 2 weeks of FL stimulation， and the mice were returned to normal light conditions as in the control group. Retinal tissue was collected at each time point to measure the levels of mRNA in Egr-1， membrane type 1 matrix metalloproteinase （MT1-MMP）， matrix metalloproteinase-2 （MMP-2）， and the tissue inhibitor of metalloproteinase-2 （TIMP-2） by quantitative real-time PCR. In addition， the level and location of Egr-1 and MT1-MMP proteins were analyzed by Western Blot， immunohistochemistry and immunofluorescence. A one-way analysis of variance was used to compare the data from the FL and control groups. An independent samples t test was used to compare indexes between the FL group and control group. Results After 2 weeks of FL stimulation， refraction became more myopic compared with the control group （0.32±0.14 D vs. 3.42±0.31 D， t=29.08， P<0.01）， and AL increased faster （2.97±0.01 mm vs. 2.93±0.01 mm， t=6.914， P<0.01）. During the FL treatment phase， Egr-1 mRNA and the protein levels in the treated eyes were rapidly down-regulated after 1 hour， and persistently down-regulated in the retina during treatment. MT1-MMP and MMP-2 levels were up-regulated while TIMP-2 levels were down-regulated in the treated eyes only at time points of 1 or 2 weeks of treatment. During the recovery phase， after 1 week， Egr-1 and TIMP-2 were up-regulated， while MT1-MMP and MMP-2 were down-regulated. Egr-1 and MT1-MMP or MMP-2 were negatively correlated. Immunofluorescence double staining showed Egr-1 immunoreactivity co-localization with MT1-MMP in retinal ganglion cells. Conclusion Retinal Egr-1 may have an effect on the activation of MMP-2 by regulating the expression of MT1-MMP in FL-induced mice myopia and during the recovery process.
【Key words】 Myopia； Egr-1； Flickering light； Matrix metalloproteinases，membrane-associated； Matrix metalloproteinase 2； Models，animal
