Cyclosporin A Inhibit Human Lens Epithelial Cell Proliferation In Culture

ObjectiveTo investigate the effect of cyclosporine A (CsA) on proliferation of human lens epithelial cells in culture. To calculate the half maximal inhibitory concentration(IC50) of CsA;Quantitative determination of PCNA gene expression, and discusses relationship between CsA and PCNA expression quantity and the effects on human lens epithelial cell proliferation. Provide the basis for CsA modified IOL in vivo.MethodsThe houman lens epithelial cells-B3(HLEB-3) were given from ZHONGSHAN OPHTHALMIC CENTER, SUN YAT-SEN UNIVERSITY. The HLEB-3cells were devided into experimental groups and control group. Both of them were cultured with DMEM (10%FBS) fluid, and with the density of2×104/ml vaccination in96-well plates. Each experiment set up10cell concentration gradient (0.125x0.125x104/ml,104ml/104/ml,1.0x,0.5×104/ml,1.5x104/ml,2.0×104/ml,2.5×104/ml,3.0×104/ml,3.5×104/ml,4.0×104/ml); Five stimulate the concentrations of cyclosporine A mu (1mu g/ml and2g/ml,4mu g/ml,8mu g/ml,16mu g/ml); Each group have3blank control. Cyclosporine A stimulus time of24hours,48hours and72hours respectively. Application of RT-PCR observation PCNA copy, and the expression of proliferation cell nuclear antigen(PCNA) was also examined with immunohistochemical methods. Application of SPSS19.0for statistical analysis.Results(1) At low concentration (1ug/ml)24h CsA role of human lens epithelial cell proliferation inhibition of the experimental group and control group no statistically significant difference (P>0.05), the role of48h cell proliferation inhibition of the experimental group and control group differences statistically significant (P<0.05).24h CsA role, IC50=71.010+1.851μg/ml, Probit=2.964+1.601x; CsA role for48h, IC50=25.529+/-1.407mu g/ml, Probit= 1.363+1.969x;72h CsA, IC50=17.881+1.252u g/ml, Probit=5.915+4.723x. CsA on HLEB-3cell inhibition time dependence and concentration dependent.(2) The RT-PCR assay,24h CsA role1ug/ml PCNA expression quantity of experimental group and control group no statistically significant difference (P>0.05), the role of48h PCNA expression quantity of the experimental group and control group differences statistically significant (P<0.05).(3) Immunohistochemical determination of CsAlug/ml24h,48h, PCNA positive cells in the experimental group and control group, the role of24h difference is not statistically significant (P>0.05); Role of48h difference has statistical significance (P<0.05).Conculsion(1) CsA in low concentration effect24h, to HLE-B3proliferation inhibition of no statistical significance, in48h statistically significant, is time dependent. Under the effective inhibition concentration, CsA was concentration dependent on HLEB-3, half of its inhibition rate increased。(2) cyclosporine A can inhibit the expression of PCNA gene, and time dependence. Cyclosporine A, can inhibit the expression of PCNA protein, and time dependence.(3) PCNA gene expression and inhibiting proliferation of lens epithelial cell division, and intervention of the coma cataract formation.In conclusion, cyclosporine A1ug/ml effect48h, can effectively inhibit HLEB-3cell proliferation, intervention of the coma after cataract formation, the feasibility of cataract drugs for prevention and treatment of later development. The results will provide theoretical value for further studies.