Investigation on the pathobiology of corneal ulcer and the development of new drugs.

本研究的最终目的是寻求治疗角膜溃疡的新模式。为此，我们利用培养的角膜基质细胞在胶原基质中三维研究了胶原酶降解的调节机制。通过测定酸解后培养上清液中羟脯氨酸的含量来判断胶原酶的降解情况。用抗哺乳动物MMP的单抗，用酶联免疫吸附试验测定基质金属蛋白酶(MMPs，胶原酶)的含量。与之相对应的是，基质金属蛋白酶的活性和基质金属蛋白酶的含量均增加。转化生长因子b和白介素1受体拮抗剂对IL-1激活的胶原酶活性有剂量依赖性抑制作用。IL-1诱导的基质金属蛋白酶是基质金属蛋白酶的主要活性形式，提示IL-1可促进基质金属蛋白酶原和纤溶酶原激活剂的合成和分泌，从而激活基质金属蛋白酶。而角膜基质细胞与中性粒细胞混合培养并未显示协同促进胶原分解的作用。因此，炎症角膜上活化的角膜基质细胞可能在角膜胶原降解中起主要作用。这些结果提示，角膜溃疡的主要细胞可能是角膜基质细胞，而不是中性粒细胞。

The ultimate purpose of the present study is to seek for the new mode of treatment for the corneal ulceration. For this end, we investigated the regulatory mechanisms of the collagenolysis using the cultured corneal keratocytes in the collagen matrix three dimensionally. Collagenolysis was estimated by measuring the amounts of hydroxyproline in the culture media after acid hydrolysis. The amounts of matrix metalloproteinase (MMP,collagenase) was measured by ELISA assay using the monoclonal antibody against the mammalian MMP.The addition of interleukin 1 (IL-1) stimulated the collagen degradation in a dose dependent manner. Concomitantly, the activities of MMP and the amounts of MMP were increased. Transforming growth factor b and interleukin 1 receptor antagonist inhibited the IL-1 activated collagenolysis in a dose dependent manner. MMP induced by IL-1 was mostly active forms of MMP.These results suggested that IL-1 increased the synthesis and secretion of pro-MMP and plasminogen activator which activates the MMP.It was believed that invaded polymorphonuclear leukocytes (PMN) might play important role in the pathogenesis of corneal ulceration. However, the mixed culture of corneal keratocytes and PMN did not demonstrate the synergistic increase of collagenolysis. Thus, the activated corneal keratocytes at the inflamed cornea may play the major role in collagen degradation of the cornea. These results suggest that the principle cells playd in the corneal ulceration might be the cornea keratocytes compared to the PMN.