The Effects Of LiCl On The Proliferation And Differentiation Of Rcecs By Triggering The Canonical Wnt/?-catenin Signaling Pathway

Objective Objective To investigate the effects of Lithium Chloride-activated canonical Wnt/?-catenin signaling pathway on the proliferation and differentiation of rabbit corneal endothelial cells(RCECs) and to find a new way to regulate RCECs' s proliferation and differentiation.Methods Following descemet's membrane was teared and digested using collagenase, purified RCECs were collected and cultured, and then subcultured when the cell dish wall was full of 80%-90%. Different concentrations of lithium chloride treatment groups(Li Cl 2.5,5,10,20,40mmol/L) and the normal control group in vitro culture of RCECs were set, the morphological changes of cells in each group were observated and MTT assays were used to detect cell proliferations at different phases(24h, 48 h, 72h).Cells of all groups were collected at 72 h, the expression level of ?-catenin and ?-SMA were examined by immunofluorescence staining, the growth cycle of CECs was detected using Flow Cytometry, cytoplasmic ?-catenin, nuclear ?-catenin, P-?-catenin, P-GSK-3?, GSK-3? protein expression were detected using Western-Blot.Results 1) Low concentration of Lithium Chloride could induce the change of cell endothelium to spindle shape, high concentration of it could inhibit cell growth and promote cell apoptosis. 2) Immunofluorescence staining showed that Li Cl could upregulate intracellular expression and accumulation of ?-catenin, low concentration of Li Cl could enhance ?-catenin nuclear translocation and high concentration of it could inhibit the nuclear translocation. Expression levels of ?-SMA were gradually upregulated with increasion of Li Cl concentration. 3) Flow Cytometry results showed that Li Cl has no influence on regulation of the cell cycle arrest and could not promote cell proliferation. 4) MTT detection showed that the cell growth were restricted in the low concentration group at 24 h and 48 h, there was a proliferation trend at 72 h when compared with the control group, but the difference was not significant; cells growths in high concentration groups was significantly limited at differet times. 5)Western-Blot test results showed that Li Cl made RCECs the nuclear ?-catenin, P-GSK-3? upregulation and cytoplasmic ?-catenin, P-?-catenin downregulation, and no significant effect on the GSK-3? expression.Conclusions 1.The canonical Wnt/?-catenin signaling pathway may be inactivated in cultured RCECs. 2.Lithium Chloride could activate the canonical Wnt/?-catenin signaling pathway in RCECs, and then promote ?-catenin intracellular accumulation and nuclear translocation, upregulate a-SMA expression and promote the differentiation of RCECs. 3.Lithium Chloride could inhibit the activity of GSK-3?, but had no significant effect on the total protein expression of GSK-3?.