Establishment of rabbit dry eye cells model and evaluation of its biological characteristics

Abstract

AIM: To further explore effective drugs for dry eye treatment by isolating and culturing lacrimal gland epithelial cells in vitro, establishing a dry eye cell model and analyzing relevant inflammatory factors. METHODS: Rabbit lacrimal gland epithelial cells were in vitro isolated and cultured, and the activity and purity of primary cells were identified by cell proliferation experiment and immunofluorescence experiment. In addition, 0.5 times IC50 of lipopolysaccharide LPS and TNF-α were used respectively to stimulate rabbit lacrimal gland epithelial cells and then establish two dry eye cell models. Finally, through cell proliferation experiment, ELISA and flow cytometry, the biological characteristics of these two dry eye cell models were compared. RESULTS:After 12h of culture, the primary cells of lacrimal gland epithelial cells basically adhered to the wall of culture bottles; and 48h later, the cells stretched and almost each of them presented a shape of a long triangle. The activity of primary cells of lacrimal gland epithelium was 92%, and the positive rate of marker Pan-rkeratin was more than 90%, which accorded with the experimental requirements. The IC50 of LPS and TNF-α are 20μg/mL and 4.996ng/mL respectively. After 12h of intervention with LPS(10μg/mL)and TNF-α(2.5ng/mL), the cell activity of the two groups was significantly lower than that of control group(P<0.01); compared between these two groups, the apoptosis rate of TNF-α group is higher than that of LPS group(P<0.01). The levers of IL-1β and IL-6 in the cell supernatants of the two groups were significantly higher than those of the control group(P<0.01); compared between the two groups, IL-1β and IL-6 in TNF-α group were significantly higher than those in LPS group(P<0.01). It was suggested that TNF-α was superior to LPS in simulating inflammatory response of dry eye. CONCLUSION: This study successfully established a relatively simple and rapid rabbit dry eye cell model with high cell purity and stability, which provided a more stable in vitro experimental model for the basic research on the function of rabbit lacrimal gland epithelial cells and dry eye.

Keywords

• lacrimal gland epithelial cells
• primary culture
• dry eye cell model
• lipopolysaccharide
• tnf-α