Rada J A, Brenza H L
Department of Ophthalmology, University of Pittsburgh School of Medicine, Pennsylvania, USA.
Invest Ophthalmol Vis Sci. 1995 Jul;36(8):1555-65.
Gelatinase activity was measured in the normal chick sclera and in sclera of form-deprived (myopic) eyes to assess the role of this metalloproteinase in ocular elongation associated with experimental myopia.
Gelatinases were extracted from anterior and posterior regions of normal chick sclera and sclera from eyes that had been form-vision deprived for 11 days. Gelatinase activity in the extracts was determined by measuring the digestion of 3H-gelatin after incubation with the extracts in the absence or presence of 1 mM aminophenylmercuric acetate (APMA) to activate latent gelatinases. Scleral gelatinases were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gelatin zymography and immunoprecipitation analyses.
No significant differences were detected in gelatinase activity between normal and deprived posterior sclera in the absence of APMA. However, when scleral extracts were incubated with APMA, extracts from the posterior sclera of deprived eyes contained significantly more gelatinase activity than paired controls (+127%, P = 0.0105). In contrast, no differences in active or latent gelatinase activity were detected in extracts from the anterior sclera. Removal of tissue inhibitors of metalloproteinases (TIMP) from control scleral extracts by reduction and alkylation resulted in a 222% increase in gelatinolytic activity after APMA-activation (P < or = 0.001), whereas similar treatment of deprived scleral extracts resulted in only a 76% increase in gelatinolytic activity (P < or = 0.001). A 65/58-kd doublet was the major gelatinolytic species from control and deprived posterior sclera that represent the proenzyme and active forms of the 72-kd gelatinase (MMP-2).
These data indicate that visual deprivation is associated with an increased amount of the 72-kd progelatinase and a decreased amount of TIMP within the posterior sclera. Therefore, an imbalance between the levels of 72-kd progelatinase and its inhibitor may play a role in the remodeling processes of the posterior sclera during the development of form-deprivation myopia.
检测正常雏鸡巩膜及形觉剥夺（近视）眼巩膜中的明胶酶活性，以评估这种金属蛋白酶在与实验性近视相关的眼轴伸长中的作用。
从正常雏鸡巩膜的前部和后部区域以及形觉剥夺11天的眼的巩膜中提取明胶酶。通过在不存在或存在1 mM对氨基苯基汞乙酸盐（APMA）以激活潜在明胶酶的情况下，将提取物与3H - 明胶孵育后测量其消化情况，来测定提取物中的明胶酶活性。通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳明胶酶谱法和免疫沉淀分析对巩膜明胶酶进行表征。
在不存在APMA的情况下，正常和剥夺后巩膜之间的明胶酶活性未检测到显著差异。然而，当巩膜提取物与APMA孵育时，剥夺眼后巩膜的提取物中明胶酶活性明显高于配对对照组（增加127%，P = 0.0105）。相比之下，在前部巩膜提取物中未检测到活性或潜在明胶酶活性的差异。通过还原和烷基化从对照巩膜提取物中去除金属蛋白酶组织抑制剂（TIMP），在APMA激活后明胶分解活性增加了222%（P≤0.001），而对剥夺巩膜提取物进行类似处理仅导致明胶分解活性增加76%（P≤0.001）。65/58-kd双峰是对照和剥夺后巩膜中的主要明胶分解成分，代表72-kd明胶酶（MMP-2）的酶原和活性形式。
这些数据表明，视觉剥夺与后巩膜中72-kd前明胶酶量的增加和TIMP量的减少有关。因此，72-kd前明胶酶及其抑制剂水平之间的失衡可能在形觉剥夺性近视发展过程中后巩膜的重塑过程中起作用。
