| 目的：研究外源性非特异性基质金属蛋白酶抑制剂GM6001对小鸡形觉剥夺性近视眼（FDM）形成的影响，以确证MMPs，特别是MMP-2在小鸡FDM后极部巩膜细胞外基质（ECM）主动重塑中的关键作用及其酶活性调节的可能分子机制。
方法：150只1 d龄来亨雏鸡采用半透明塑料眼罩单眼遮盖制备FDM动物模型，在遮盖同时进行干预，按干预因素不同随机分为GM6001组、PBS组与阴性对照组。GM6001组分别球后注射GM6001 10μM/50μl、50μM/50μl及100μM/50μl，PBS组注射PBS 50ul，每日1次，分别注射4 d、14 d。阴性对照组进行单纯遮盖而不予GM6001或PBS处理。另随机选取150只同龄、未遮盖的正常小鸡进行上述相同干预处理作为各组的正常对照组。上述各实验组每组15只小鸡。4 d、14 d后测量各组小鸡眼轴长度及屈光度变化，取后极部巩膜，连续冰冻切片，常规HE染色法进行组织病理学观察，免疫组织化学法检测后极部巩膜MMP-2蛋白质表达，明胶酶谱法检测小鸡后极部巩膜基质金属蛋白酶酶活性表达水平，RT-PCR一步法检测MMP-2 mRNA表达。
结果：① GM6001能明显抑制遮盖4 d小鸡眼眼轴过度延长与近视性屈光度增加，与PBS组、阴性对照组比较差别均有统计学意义（P<0.01），而对遮盖14 d小鸡眼GM6001仅起部分抑制作用；不同剂量GM6001对FDM眼轴长度及屈光度影响无明显差别；GM6001对正常小鸡眼轴长度及屈光度变化无明显作用。② 组织病理学结果表明，经GM6001干预的遮盖4 d组小鸡后极部巩膜与正常小鸡眼基本相似，而GM6001干预的遮盖14 d组小鸡后极部巩膜与FDM眼基本相似，表现为纤维层明显变薄与纤维化，软骨层相对增厚，软骨细胞明显空泡变性，双核细胞与不规则细胞增多。③免疫组化结果显示，正常眼后极部巩膜仅软骨层少量软骨细胞膜和软骨囊、纤维层少数成纤维细胞胞质呈淡黄染色，而FDM后极部巩膜阳性表达细胞明显增多，且细胞周围ECM亦存阳性表达；经GM6001干预的遮盖4 d组小鸡免疫组化特征与正常眼基本相似，而经GM6001处理的遮盖 14 d组小鸡后极部巩膜仅少量软骨细胞膜及软骨囊及纤维层少数成纤维细胞呈阳性表达。④明胶酶谱法结果发现，与正常小鸡比较，FDM组小鸡眼后极部巩膜仅MMP-2酶活性（约62 kDa）明显增高（P<0.01），且呈时间依赖性；GM6001干预组明胶酶谱图未检测到透亮带。⑤ GM6001对正常小鸡与FDM后极部巩膜MMP-2 mRNA表达均无明显影响。
结论：① GM6001能明显抑制早期FDM形成，而对中后期FDM形成作用微弱；② MMP-2酶活性增高极可能为启动小鸡FDM后极部巩膜ECM重塑的关键因子之一；③ TIMPs抑制在早期小鸡FDM后极部巩膜MMP-2酶活性调控中起主导作用；
关键词： 形觉剥夺性近视眼；GM6001；基质金属蛋白酶；小鸡
Inhibitory effects of tissue inhibitor of matrix metalloproteinase Galardin injected retoocularly on the development of form-deprivation myopia of chick
Objective: To investigate the inhibitory effects of tissue inhibitor of matrix metalloproteinase Glardin injected retroocularly on the development of form-deprivation myopia, which would help determine whether elevation of matrix metalloproteinase 2 (MMP-2) activity in the posterior sclera was the key to trigger the active scleral remodeling process happened in deprived chick eye.
Methods: 150 chicks covered monocularly with semi-translucent plastic goggle for 4 days or 14 days to establish an animal model of form-deprivation myopia were divided randomly into GM6001 group, PBS group and negative control group according to interference received. Chicks in GM6001 group were further divided randomly into three groups which were injected Galardin 10μM/50μl, 50μM/50μland 100μM/50μl respectively. Chicks in PBS group received sterile PBS 50μl retro-ocularly, and those in negative control group were covered only. And meanwhile another 150 1-day-old normal chicks non-covered were also treated as above to serve as control for each groups. Each groups has 15 chicks. After 4 or 14 days having been measured the axial length and refractive degree of experimental eyes by A ultrasonography and retinoscopy respectively, chicks were killed to get posterior sclera. Finally changes in scleral tisse pathology were examined using H.E dying, and MMP-2 expression was evaluated immuno-histochemistically, and expression level of messenger RNA was measured by one step reverse transcriptiontase-polymerase chain reaction (RT-PCR) using special primers for MMP-2 gene, and enzyme activity of MMP-2 was measured by gelatin enzymography.
Results: With the time of form-deprivation increasing the axial length became longer and the myopic refractive degree more serious, which would be almost suppressed in eyes deprived for 4 days(MD 4d groups ) by whenever injecting retroocularly Galardin 10μM/50μl, 50μM/50μor 100μM/50μl, and however the inhibitory effect was depressed partly in MD 14d group and there existed little effects in normal control groups. Tissue pathologic studies have shown that with the time increase of form deprivation fibril sclera in MD group became more fibrosis and thinner, and while the cartilage layer became thicker relatively and chondro-cells became more distinct vacuole denaturalized and the number of binucleus and irregular cells increased. These changes would nearly not happen when retroocularly injected Galardin in MD 4d group and that would be lessened much more in MD 14d group. Immunohistochemistry study has showed that both in normal non-deprived eyes and in MD 4d group interferenced with Galardin a few membrane of chondrocytes and cartilage vesicles presented positive dye, and while both in MD groups and in MD 14d group received Galardin the number of positive cells increased remarkably and further more some extracellular matrix was dying positively. Gelatin enzymography research has showed that the levels of MMP-2 activity in MD groups were much higher than in normal control groups, and with the time increase of monocular deprivation these changes were more significant. However in groups treated with Galardin there were no positive bands appeared on the map of gelatin enzymography. One step RT-PCR revealed that no effects of Galardin on MMP-2 mRNA expression existed both in normal control group and in MD groups.
Conclusion: That Galardin could almost only inhibit the early development of form-deprivation myopia suggested that increase of MMP-2 enzyme activity on the posterior sclera be probably an key step to trigger early active sclera ECM remodeling process, which to some extent indicated that tissue inhibitor of matrix metalloproteinases should take an essential role in modulating MMP-2 enzyme activity in the early development of form-deprivation myopia in chicks.
Key words: Form-deprivation myopia; GM6001; Matrix metalloproteinases;Chick.
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