Effect of immobilized hyaluronidase and subtilisin enzymes on the ultrastructure of human conjunctival epithelial cells

Abstract

The use of enzyme preparations is a traditional trend in various fields of medicine. The usage of immobilized enzymes hyaluronidase (IG) and subtilisin (IS) seems to be very promising for the treatment of damage to the ocular surface. The aim of the study was to establish the nature of changes in the ultrastructural organization of human conjunctival epithelium under the influence of immobilized hyaluronidase and subtilisin in vitro. Material and methods. In the experiment, a culture of normal cells of the human conjunctiva Chanqconjunctiva, clone 1–5 C-4, was seeded in 96well plates in the amount of 2 × 104 cells/well, after 24 h the medium was removed, Eagle’s medium MEM and fetal calf serum were added and cells were cultured for another 48 h. 5 experimental groups were formed: group 1 – without drugs (except for the composition of the incubation scheme), groups 2 and 3 – with IS at a concentration of 37 and 150 U/ml, respectively, groups 4 and 5 – with IG at a concentration of 37 and 150 U/ml, respectively. After preparation of cell preparations under a JEM 1400 electron microscope (Japan), ultrathin sections of 70–100 nm were studied at ×1000 magnification. Results. The article presents the results of electron microscopy of a culture of normal cells of the human conjunctiva in 5 experimental groups of cells with a description of changes in cytoarchitectonics under the influence of IG and IS. Conclusions. The introduction of immobilized enzymes into human conjunctival cell culture at a low dose (37 U/ml) affects the organization of cells without cytotoxic effects, while increasing the dose directly correlates with the occurrence of a cytotoxic effect.

Keywords

• immobilized subtilisin
• immobilized hyaluronidase
• cytoarchitectonics