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

本研究的最终目的是寻求角膜溃疡治疗的新模式。为此，我们利用体外培养的角膜基质细胞，从三维角度研究了胶原溶解的调控机制。通过测量酸水解后培养基中羟脯氨酸的量来估计胶原溶解。用抗哺乳动物基质金属蛋白酶（MMP，collagenase）的单克隆抗体ELISA法测定MMP的含量，加入白细胞介素1（IL-1）后，胶原降解呈剂量依赖性。同时，MMP的活性和MMP的量增加。转化生长因子B和白细胞介素1受体拮抗剂以剂量依赖方式抑制IL-1激活的胶原溶解。IL-1诱导的MMP主要是MMP的活性形式，提示IL-1可促进MMP前体和激活MMP的纤溶酶原激活物的合成和分泌，从而推测侵袭性多形核白细胞（PMN）在角膜溃疡的发病中可能起重要作用。然而，角膜基质细胞和PMN的混合培养没有表现出协同增加胶原溶解。因此，发炎角膜处的活化角膜基质细胞可能在角膜的胶原降解中起主要作用。提示角膜基质细胞可能是角膜溃疡形成的主要细胞。

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.