Kenney M C, Chwa M, Opbroek A J, Brown D J
Department of Surgery, Los Angeles Medical School Affiliate 90048.
Cornea. 1994 Mar;13(2):114-24. doi: 10.1097/00003226-199403000-00003.
Keratoconus is a noninflammatory corneal disorder characterized by gradual stromal thinning and astigmatism. Altered degradation of corneal extracellular matrix is a suggested etiology for this disorder. In the present study we established keratocyte cultures from normal and keratoconus corneas and investigated the roles that matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMP, TIMP-2) may play. After chemical modification (reduction and alkylation) to remove the inhibitor and activation of enzyme with p-aminophenylmercuric acetate (APMA), keratoconus-conditioned media displayed a significant increase (p < 0.05) in the total potential gelatinolytic activity when compared with normal culture media treated in a similar manner. Basal levels of gelatinolytic activity in keratoconus culture media (no reduction, alkylation, or APMA treatment), determined by two different assay methods, tended to be about twice that of normal cell cultures. By zymography, both keratoconus and normal cultures showed identical enzyme patterns, which represented MMP-2 (72 kDa) in its proform and, depending on the treatment of the media, varying amounts of activated MMP-2 (65 kDa). This suggests that the increased gelatinolytic activity in keratoconus was not correlated with an increased appearance of either the 65-kDa-activated form of MMP-2 or a new MMP species. In addition, no differences in the amount of MMP-2 were detected that could account for the increased activities in keratoconus cultures. However, a relative decline in the detectable TIMP levels in keratoconus cultures resulted in an apparent three-fold increase in the ratio of MMP-2/TIMP. Northern blots showed no significant changes in mRNA levels for MMP-1, MMP-2, MMP-3, TIMP, or TIMP-2. These data suggest that a possible alteration in the interaction between MMP-2 and TIMP may play a role in the increased gelatinolytic activity seen in keratoconus tissues.
圆锥角膜是一种非炎症性角膜疾病，其特征为角膜基质逐渐变薄和散光。角膜细胞外基质降解改变是该疾病的一种推测病因。在本研究中，我们从正常角膜和圆锥角膜建立了角膜细胞培养物，并研究了基质金属蛋白酶（MMPs）和金属蛋白酶组织抑制剂（TIMP，TIMP-2）可能发挥的作用。在进行化学修饰（还原和烷基化）以去除抑制剂并用对氨基苯基汞乙酸盐（APMA）激活酶后，与以类似方式处理的正常培养基相比，圆锥角膜条件培养基的总潜在明胶分解活性显著增加（p < 0.05）。通过两种不同的测定方法确定，圆锥角膜培养基中的基础明胶分解活性水平（未进行还原、烷基化或APMA处理）往往约为正常细胞培养物的两倍。通过酶谱分析，圆锥角膜和正常培养物均显示出相同的酶谱模式，其代表前体形式的MMP-2（72 kDa），并且根据培养基的处理方式，有不同量的活化MMP-2（65 kDa）。这表明圆锥角膜中增加的明胶分解活性与MMP-2的65 kDa活化形式或新的MMP种类的增加出现无关。此外，未检测到MMP-2量的差异可解释圆锥角膜培养物中增加的活性。然而，圆锥角膜培养物中可检测到的TIMP水平相对下降导致MMP-2/TIMP比值明显增加三倍。Northern印迹显示MMP-1、MMP-2、MMP-3、TIMP或TIMP-2的mRNA水平无显著变化。这些数据表明，MMP-2与TIMP之间相互作用的可能改变可能在圆锥角膜组织中所见的增加的明胶分解活性中起作用。
