中华眼视光学与视觉科学杂志
中華眼視光學與視覺科學雜誌
중화안시광학여시각과학잡지
CHINESE JOURNAL OF OPTOMETRY OPHTHALMOLOGY AND VISUAL SCIENCE
2014年
6期
328-334
,共7页
李曼%俞莹%管怀进%陈辉
李曼%俞瑩%管懷進%陳輝
리만%유형%관부진%진휘
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
近視%Egr-1%閃爍光%基質金屬蛋白酶類,膜相關%基質金屬蛋白酶2%模型,動物
근시%Egr-1%섬삭광%기질금속단백매류,막상관%기질금속단백매2%모형,동물
Myopia%Egr-1%Flickering light%Matrix metalloproteinases,membrane-associated%Matrix metalloproteinase 2%Models,animal
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1h、1d、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1h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.
目的 探討Egr-1在低頻閃爍光(FL)誘導的小鼠近視中的錶達及與細胞外基質重塑通路可能的調控機製.方法 實驗研究.將28日齡健康C57BL/6J(B6)小鼠(100隻)隨機分為正常對照組和FL組.閃爍光組光照頻率為2 Hz,明暗週期為50%.分彆于實驗前,實驗後1h、1d、1週、2週、恢複1週(FL誘導2週後撤除FL,于正常光照下繼續飼養1週),使用紅外偏心驗光儀和動物專用A超測定儀測量所有小鼠右眼的屈光度及眼軸.于實驗後每箇時間點每組各提取10隻小鼠右眼的視網膜組織進行RT-PCR檢測Egr-1、膜型基質金屬蛋白酶1(MT1-MMP)、金屬基質金屬蛋白酶2(MMP-2)、組織抑製金屬蛋白酶2(TIMP-2)的動態錶達,Western Blot及免疫組化、免疫熒光觀察Egr-1與MT1-MMP蛋白錶達及定位.FL組與對照組各指標組內均數行ANOVA分析,組間均值行獨立樣本t檢驗比較分析.結果 FL光照2週後,小鼠的屈光度相比對照組髮生瞭近視改變[(0.32±0.14)D vs.(3.42±0.31)D,t=29.08,P<0.01],眼軸也顯著增長[(2.97±0.01)mm vs.(2.93±0.01)mm,t=6.914,P<0.01].FL光照1h後Egr-1 mRNA和蛋白快速下調,且隨誘導時間持續低于正常,MT1-MMP mRNA和蛋白水平快速持續上調,實驗1 d MMP-2 mRNA水平開始上調,趨勢與MT1-MMP相同,TIMP-2 mRNA在實驗1週和2週的時候明顯低于正常組.恢複1週後,Egr-1和TIMP-2明顯上調,而MT1-MMP和MMP-2錶達下調,Egr-1與MT1-MMP呈現負相關關繫(r2=0.6478,P<0.05).免疫熒光雙標示Egr-1與MT1-MMP在視網膜神經節細胞存在共錶達.結論 視網膜中Egr-1可能通過調控MT1-MMP的錶達來影響MMP-2的活化,進而參與閃爍光誘導的小鼠近視的髮生及恢複過程.
목적 탐토Egr-1재저빈섬삭광(FL)유도적소서근시중적표체급여세포외기질중소통로가능적조공궤제.방법 실험연구.장28일령건강C57BL/6J(B6)소서(100지)수궤분위정상대조조화FL조.섬삭광조광조빈솔위2 Hz,명암주기위50%.분별우실험전,실험후1h、1d、1주、2주、회복1주(FL유도2주후철제FL,우정상광조하계속사양1주),사용홍외편심험광의화동물전용A초측정의측량소유소서우안적굴광도급안축.우실험후매개시간점매조각제취10지소서우안적시망막조직진행RT-PCR검측Egr-1、막형기질금속단백매1(MT1-MMP)、금속기질금속단백매2(MMP-2)、조직억제금속단백매2(TIMP-2)적동태표체,Western Blot급면역조화、면역형광관찰Egr-1여MT1-MMP단백표체급정위.FL조여대조조각지표조내균수행ANOVA분석,조간균치행독립양본t검험비교분석.결과 FL광조2주후,소서적굴광도상비대조조발생료근시개변[(0.32±0.14)D vs.(3.42±0.31)D,t=29.08,P<0.01],안축야현저증장[(2.97±0.01)mm vs.(2.93±0.01)mm,t=6.914,P<0.01].FL광조1h후Egr-1 mRNA화단백쾌속하조,차수유도시간지속저우정상,MT1-MMP mRNA화단백수평쾌속지속상조,실험1 d MMP-2 mRNA수평개시상조,추세여MT1-MMP상동,TIMP-2 mRNA재실험1주화2주적시후명현저우정상조.회복1주후,Egr-1화TIMP-2명현상조,이MT1-MMP화MMP-2표체하조,Egr-1여MT1-MMP정현부상관관계(r2=0.6478,P<0.05).면역형광쌍표시Egr-1여MT1-MMP재시망막신경절세포존재공표체.결론 시망막중Egr-1가능통과조공MT1-MMP적표체래영향MMP-2적활화,진이삼여섬삭광유도적소서근시적발생급회복과정.
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.