Expression of degradative enzymes and protease inhibitors in corneas with keratoconus.
Expression of degradative enzymes and protease inhibitors in corneas with keratoconus.
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DOI：
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发表时间：
1998-06
影响因子：
4.4
通讯作者：
Lili Zhou;S. Sawaguchi;S. Twining;J. Sugar;R. Feder;B. J. Yue
中科院分区：
文献类型：
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作者：
Lili Zhou;S. Sawaguchi;S. Twining;J. Sugar;R. Feder;B. J. Yue
圆锥角膜的特征是角膜中央区域变薄和瘢痕形成。先前的研究表明，在从圆锥角膜患者获得的角膜中，溶酶体酶活性升高，而蛋白酶抑制剂如α 1-蛋白酶抑制剂的水平降低。进行这项研究，以进一步检查一系列蛋白水解酶和蛋白酶抑制剂的表达。方法收集圆锥角膜患者、健康人和其他角膜疾病患者的角膜扣。石蜡切片进行免疫组织化学染色。对组织蛋白酶B和G进行酶法测定和蛋白质印迹分析。此外，在原位酶谱程序被用来检查明胶和酪蛋白消化的圆锥角膜的活动。结果抗组织蛋白酶B和G抗体染色增强。酶法和蛋白质印迹法证实，这两种酶的水平在圆锥角膜角膜升高。尽管原位酶谱确实表明圆锥角膜的净明胶和酪蛋白消化活性增加，但基质金属蛋白酶（MMP）家族成员和其他酶及抑制剂均未观察到变化。丝氨酸和半胱氨酸蛋白酶的抑制剂，这些活动大多被废除，但不是由那些基质金属蛋白酶和天冬氨酸蛋白酶。结论：圆锥角膜组织蛋白酶B和G水平升高。这些酶可能有助于提高原位明胶和酪蛋白消化活性，导致圆锥角膜异常。
PURPOSE Keratoconus is characterized by thinning and scarring of the central region of the cornea. Previous research showed that, in corneas obtained from patients with keratoconus, lysosomal enzyme activities are elevated, whereas levels of protease inhibitors such as alpha1-proteinase inhibitor are reduced. This study was undertaken to examine further the expression of a spectrum of proteolytic enzymes and protease inhibitors. METHODS Corneal buttons were collected from patients with keratoconus, healthy subjects, and patients with other corneal diseases. Immunohistochemical staining was performed on paraffin sections. Enzymatic assays and western blot analysis were carried out for cathepsins B and G. In addition, an in situ zymography procedure was used to examine the gelatin- and casein-digesting activities in corneas with keratoconus. RESULTS An enhanced staining was found with antibodies to cathepsins B and G. Enzymatic assays and western blotting confirmed that the levels of these two enzymes were elevated in corneas with keratoconus. No alteration was noted with any of the matrix metalloproteinase (MMP) family members and other enzymes and inhibitors examined, although in situ zymography did indicate an increase in net gelatin- and casein-digesting activities in corneas with keratoconus. These activities were mostly abolished by inhibitors for serine and cysteine proteinases, but not by those for MMPs and aspartic proteinases. CONCLUSIONS Levels of cathepsins B and G are increased in corneas with keratoconus. These enzymes may contribute to the heightened in situ gelatin- and casein-digesting activities, leading to abnormalities in keratoconus.