The Effect Of A Novel Small Molecule Complex On The In Vitro Expansion Of Mouse Corneal Epithelial Cells

Purpose:To explore the effects of six small molecule chemicals(6C)on the proliferation and differentiation phenotype of primary mouse corneal epithelial cells in serum-free and trophoblast-free conditions,and to develop and construct tissue-engineered mouse corneal epithelial grafts.Methods:Different compound combinations were added to the culture medium of primary mouse corneal epithelial cells.After passage/continuous culture,the proliferation activity of primary mouse corneal epithelial cells was evaluated by crystal violet staining,growth curve and clone formation efficiency;Immunofluorescence staining and real-time fluorescent quantitative PCR were used to analyze the expression of genes related to primary mouse corneal epithelial cells and epithelial-mesenchymal transition(EMT);In the model of corneal epithelial injury healing in mice,6C solution was used for eye treatment,and the healing rate of corneal epithelium was evaluated by fluorescein sodium staining;Different small molecule components were added to the air-exposed culture system,and the corneal epithelial grafts formed by the culture were immunostained to evaluate the growth and phenotypic differentiation of epithelial cells.Results:In long-term culture and subculture,6C combination increased the proliferation activity of primary mouse corneal epithelial cells.In vivo experiments show that 6C can accelerate the healing of mouse corneal epithelial wounds.At the same time,this mixture inhibited the increase in the expression levels of EMT-related markers ZEB1/2,Snail,β-catenin and αSMA,and maintained the expression levels of primary mouse corneal epithelial cell function-related markers p63,K14,Pax6 and K12.This protective effect of the 6C combination shows that it can reduce the transdifferentiation of functional stem/progenitor cells into mesenchymal cells by extending the life of functional stem/progenitor cells.In addition,adding 6C mixture to the air exposure system can form tissue engineered mouse corneal epithelial sheets that can be used for transplantation.Conclusions:The 6C cocktail not only inhibited the passage-dependent decrease in the proliferation activity of primary mouse corneal epithelial cells,but also inhibited the increase in EMT.These effects indicate that 6C compounds may help improve the treatment of limbal stem cell deficiency.