Munjal I D, Crawford D R, Blake D A, Sabet M D, Gordon S R
Department of Biochemistry, Meharry Medical College, Nashville, TN 37208.
Eur J Cell Biol. 1990 Aug;52(2):252-63.
Thrombospondin is a cell adhesion molecule which interacts via specific domains with a wide array of extracellular matrix components, including fibrinogen, fibrin, fibronectin, collagen, and heparan sulfate proteoglycan. Although this protein has been localized in several human tissues, its presence in corneal tissues had not been previously established. In the present study, we have demonstrated that cultured bovine corneal endothelial cells synthesize thrombospondin and incorporate it into their extracellular matrix. We have also shown immunofluorescently the presence and distribution of thrombospondin in these cultured cells and in the noninjured and injured corneal endothelium in situ. Ultrastructural immunoperoxidase cytochemistry revealed that thrombospondin could be displaced from the cell surface by heparin, but not by keratan sulfate. Confluent cultures of corneal endothelium synthesize and secrete the three cell adhesion proteins laminin, thrombospondin, and fibronectin in the ratios 1:8.2:51.8. Only the laminin B chains were detected in immunoprecipitates. Immunofluorescent studies of these cultured cells, using a polyclonal antiserum raised against purified thrombospondin, revealed a low level of fluorescence associated with the cell layer but a punctate fluorescent pattern at the level of the extracellular matrix. Noninjured corneal endothelium in situ also demonstrated a low level of fluorescence throughout the cell layer. However, this dramatically changed after a circular freeze injury to the tissue. By 24 h after wounding, cells surrounding the injury zone displayed a prominent fluorescence that was still observed at 48 h post-injury. In addition to its increased intracellular fluorescence, thrombospondin was also localized as migration tracks, oriented in the direction of cellular migration into the wound site. Thus, in corneal endothelium, thrombospondin appears to play a major role in injury-induced cell migration in situ along a natural basement membrane.
血小板反应蛋白是一种细胞黏附分子，它通过特定结构域与多种细胞外基质成分相互作用，这些成分包括纤维蛋白原、纤维蛋白、纤连蛋白、胶原蛋白和硫酸乙酰肝素蛋白聚糖。尽管这种蛋白质已在多种人体组织中定位，但此前尚未确定其在角膜组织中的存在情况。在本研究中，我们证明培养的牛角膜内皮细胞能合成血小板反应蛋白并将其整合到细胞外基质中。我们还通过免疫荧光法显示了血小板反应蛋白在这些培养细胞以及原位未损伤和损伤的角膜内皮中的存在及分布。超微结构免疫过氧化物酶细胞化学显示，肝素可使血小板反应蛋白从细胞表面移位，但硫酸角质素则不能。角膜内皮的汇合培养物以1:8.2:51.8的比例合成并分泌三种细胞黏附蛋白，即层粘连蛋白、血小板反应蛋白和纤连蛋白。免疫沉淀物中仅检测到层粘连蛋白B链。使用针对纯化的血小板反应蛋白产生的多克隆抗血清对这些培养细胞进行免疫荧光研究，结果显示细胞层相关的荧光水平较低，但在细胞外基质水平呈现点状荧光模式。原位未损伤的角膜内皮在整个细胞层也显示出低水平的荧光。然而，在对组织进行环形冷冻损伤后，这种情况发生了显著变化。受伤后24小时，损伤区域周围的细胞显示出明显的荧光，在损伤后48小时仍可观察到。除了细胞内荧光增加外，血小板反应蛋白还定位为迁移轨迹，其方向为细胞迁移到伤口部位的方向。因此，在角膜内皮中，血小板反应蛋白似乎在损伤诱导的原位细胞沿天然基底膜迁移中起主要作用。
