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，视力预后差。角膜病变可归因于感染过程中角膜释放的蛋白水解酶，包括MMP。然而，对MMP在假性角膜炎中的作用和行为知之甚少。因此，我们研究了假单胞毒力因子对培养角化细胞的影响，重点研究了MMP的释放。方法：兔角化细胞在含10% FCS的PRMI-1640中达到融合生长后，将培养基改为不含FCS的rmi -1640，其中含有以下假单胞毒因子之一：弹性酶（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LPS（30、10、3、1 mg/ml）。48小时后，收获培养基，进行明胶和酪蛋白酶谱分析，分析蛋白水解酶（MMP）。我们还观察了这些条件下角质细胞的形态学变化。结果：对照培养基中微量检出明胶酶A （MMP-2）。假单胞弹性蛋白酶显著增强了角质细胞中MMP-2和MMP-9的释放。还观察了这些MMPs的活化形式。碱性蛋白酶表现出类似的效果，但没有激活任何MMPs。LPS也观察到类似的增强作用，外毒素a也观察到类似的增强作用，但程度较轻。结论：从假单胞毒力因子增强角质细胞中MMP的释放的结果来看，可以合理地得出角膜MMP也可能导致假单胞性角膜炎溃疡的结论。

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.