MATRIX METALLOPROTEINASE IN INFECTIOUS KERATITIS

目的：假性角膜炎常导致严重的角膜溃疡和穿孔，视力预后较差。角膜病变可能是由于感染期间角膜中释放的蛋白水解酶，包括基质金属蛋白酶。然而，关于基质金属蛋白酶在假性角膜炎中的作用和行为，我们知之甚少。因此，我们研究了假单胞菌毒力因子对培养的角质形成细胞释放基质金属蛋白酶的影响。方法：兔角膜基质细胞在含10%胎牛血清的PRMI-1640上融合生长后，换成不含胎牛血清的RPMI-1640，其中含有以下几种假单胞菌毒力因子：弹性蛋白酶(100，30，10，3 ng/ml)、碱性蛋白酶(100，30，10，3 ng/ml)、外毒素A(10，3，1，0.3 mg/ml)和脂多糖(30，10，3，1 mg/ml)。48h后收集培养液，明胶和酪蛋白酶谱分析蛋白水解酶。并观察了在此条件下角膜基质细胞的形态变化。结果：对照培养上清液中有少量明胶酶A(MMP2)的表达。假单胞弹性蛋白酶显著促进角质形成细胞释放基质金属蛋白酶-2和基质金属蛋白酶-9。并观察了这些MMPs的活化形式。碱性蛋白酶表现出类似的作用，但不能激活任何MMPs。脂多糖和外毒素A也有类似的促进作用。结论：根据假单胞菌毒力因子促进角质形成细胞释放基质金属蛋白酶的结果，可以得出结论：角膜基质金属蛋白酶可能也参与了假单胞性角膜炎的溃疡形成。

Purpose : Pseudomonal keratitis often results in severe corneal ulcer and perforation with poor visual prognosis. The corneal lesions could be attributable to proteolytic enzymes including MMP released in the corneas during the infection. However, little is known about the role and behavior of MMP in pseudomonal keratitis. Therefore, we investigated the effects of pseudomonal virulence factors on the cultured keratocyte with emphasis on MMP release. Methods : After rabbit keratocytes reached to confluent growth in PRMI-1640 containing 1o% FCS,the medium was changed to RPMI-1640 without FCS containing one of the following pseudomonal virulence factors : elastase (100,30,10,3 ng/ml), alkaline protease (100,30,10,3 ng/ml), exotoxin A (10,3,1,0.3 mg/ml) and LPS (30,10,3,1 mg/ml). At 48 hours, the culture media were harvested and processed for gelatin and casein zymography to analyze proteolytic enzymes (MMP). Morphologic changes of keratocytes under these conditions were also investigated. Results : Gelatinase A (MMP-2) was detected slightly in the control medium. Pseudomonal elastase enhanced remarkably the release of both MMP-2 and MMP-9 from the keratocytes. The activated forms of these MMPs were also observed. Alkaline protease showed a similar effect but did not activate any MMPs. Similar enhancements were observed also with LPS and to a lesser extent with exotoxin A.Conclusions : From the results that pseudomonal virulence factors enhanced the release of MMP from keratocytes, it is reasonable to conclude that corneal MMP might also contribute to ulceration in pseudomonal keratitis.