Ramsby M L, Kreutzer D L
Department of Pathology, University of Connecticut Health Center, Farmington 06030.
Invest Ophthalmol Vis Sci. 1993 Jan;34(1):165-74.
Fibrin deposition in the anterior chamber of the eye occurs in response to injury or inflammation and can permanently damage the corneal endothelium. Fibrin functions as a mediator of inflammation and wound healing by affecting cell morphology and function in a variety of cells, including corneal endothelial cells. We hypothesized that fibrin can directly induce corneal endothelial cells to express injury-related proteins (eg, thrombospondin [TSP]) necessary for corneal repair processes.
Bovine corneal endothelial cells (BCECs) were pulse- or continuously labeled with 35S-methionine in the presence or absence of in situ polymerized fibrin (2 mg/ml). BCECs were harvested after 3-48 hr, and 35S-labeled proteins were analyzed by SDS-PAGE, autoradiography, and immunochemical techniques.
Fibrin selectively induced BCECs to express a high molecular weight (MW) protein that was present extracellularly in conditioned medium and fibrin matrix. This induction represented a 3-5 fold increase relative to nonfibrin-treated BCECs, was not accompanied by corresponding changes in 35S-labeled intracellular proteins, and was evident at early (3 hr) or late times (24 hr) post-fibrin treatment. The induced protein had an apparent MW of 180 kD (reduced) and > 420 kD (nonreduced), consistent with the characteristics of TSP. A polyclonal antibody to human TSP recognized the reduced form (180 kD) on Western blots and the native form (> 420 kD) in immunoprecipitation studies.
Fibrin induces BCECs to express TSP, a matrix protein involved in cell-cell and cell-matrix interactions and implicated in wound healing.
眼房水中纤维蛋白沉积是对损伤或炎症的反应，可永久性损害角膜内皮。纤维蛋白通过影响包括角膜内皮细胞在内的多种细胞的形态和功能，作为炎症和伤口愈合的介质。我们推测纤维蛋白可直接诱导角膜内皮细胞表达角膜修复过程所需的损伤相关蛋白（如血小板反应蛋白 [TSP]）。
在有或无原位聚合纤维蛋白（2 mg/ml）的情况下，用35S-甲硫氨酸对牛角膜内皮细胞（BCEC）进行脉冲或连续标记。3 - 48小时后收获BCEC，通过SDS-PAGE、放射自显影和免疫化学技术分析35S标记的蛋白质。
纤维蛋白选择性诱导BCEC表达一种高分子量（MW）蛋白，该蛋白存在于条件培养基和纤维蛋白基质的细胞外。这种诱导相对于未用纤维蛋白处理的BCEC增加了3 - 5倍，不伴有35S标记的细胞内蛋白质的相应变化，并且在纤维蛋白处理后的早期（3小时）或晚期（24小时）很明显。诱导的蛋白表观分子量为180 kD（还原型）和> 420 kD（非还原型），与TSP的特征一致。抗人TSP的多克隆抗体在Western印迹中识别还原型（180 kD），在免疫沉淀研究中识别天然型（> 420 kD）。
纤维蛋白诱导BCEC表达TSP，TSP是一种参与细胞间和细胞与基质相互作用并与伤口愈合有关的基质蛋白。
