Effects Of RhNGF And NTX On Proliferation And Migration Of Human Corneal Epithelial Cells

Objective:To investigate the changes of OGF(Opoid growth factor)and NGF(Never growth factor)content changes and it's effect on corneal epithelial proliferation,migration,and apoptosis through observation of human corneal endothelial cells in steady high glucose and fluctuant glucose.To explore the optimum doses of rhNGF(Recombination human nerve)and OGF by vitro cultivation.So as to provide new ideas and new methods to treat diabetic corneral diseases.Methods:1.The cultured HCECs in vitro were as the object to investigate the protection and promotion effct of rhNGF and NTX,They were treated with 2 different glucose concentration conditions group:Steady normal glucose(5.5mmol/l);Fluctuation glucose concentration(5.5mmol/l glucose DMEM complete medium was cultured for 8 hours and then changed into 50mmol/l DMEM complete medium);Cell cultured for 1week,and cell culture supernatant was collected,Through ELISA method for detecting NGF,OGF content changes.2.Select the appropriate concentration and treatment time of NTX and rhNGF: the FHG group were added with different doses of rhNGFand different dose of NTX,cultured 24 h,36h,48 h,60h,72 h,using(Cell Counting kit,CCK8)measured cell proliferation.after statistical analysis to determine the appropriate concentration of rhNGF and NTX.3.Take rhNGF and NTX concentration of suitable concentration and treatment time,the stability of normal glucose concentration group(SNG)and fluctuating high glucose concentration group(FHG)were given different treatments: negative control group(Control C group),rhNGF group,NTX group and rhNGF+NTX group.(1)cell proliferation was measured by cell plate cloning.(2)cell scratch test was used to detect the migration ability of HCEC cells.(3)flow cytometry were used to detect apoptosis of HCEC cells.Result:1.ELISA: compared with the Steady Normal Glucose concentration group(SNG)Fluctuant High Glucose group(FHG)were measured the content of rhNGF was significantly decreased(P<0.05),the concentration of OGF was significantly increased(P< 0.05).2.(1)Compared with SNG,the proliferation activity of HCEC cells in FHG group was decreased,the apoptosis rate was increased,and the cell migration ability was decreased(P<0.05).(2)the proliferation activity of FHG group was higher than that of HCEC cells before the addition of rhNGFand NTX,the apoptosis rate was decreased and the cell migration ability was increased(P<0.05).(3)rh NGF and(or)NTX cells of FHG group proliferative activity were lower than those in SNG group(P< 0.05),the apoptosis rate was higher than that of SNG group(P < 0.05),and the cell migration ability was higher than SNG(P <0.05).(4)compared to the effects of rhNGF and NTX on the proliferation,migration and apoptosis of HCEC were not significantly different(P > 0.05).3.the appropriate concentration of rhNGF and NTX to promote HCEC proliferation in group FHG was 125ng/ml(P<0.05)and 250?mol/l(P<0.05)respectively.And the rhNGF and NTX combination group was better than rhNGF or NTX group(P < 0.05).Conclusion:1.The steady high glucose and fluctuant high glucose could inhibit the proliferation of HCEC,induce the apoptosis of HCEC,decrease the expression of rhNGF and increase the expression of OGF.2.rhNGF and NTX can enhance the proliferation activity of HCEC cells and reduce the apoptosis of HCEC cells in fluctuant high glucose environment.125ng/mlrhNGF and 250?mol/l NTX can obviously promote the proliferation of HCEC.Moreover,rhNGF and NTX may have synergistic effects on the proliferation of HCEC.