The Expression Of Integrins In Cultured Human Lens Epithelial Cells In Vitro And The Inhibition Of Verapamil On It

Objects: To study the expression of various integrin subunits in cultured human lens epithelial cells(HLECs) in vitro and the influence of Verapamil(Ver), a calcium channel blockers (CCB), on integrins expression in HLECs.Methods: The human lens epithelial cells line, SRA01/04, was cultured in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) supplemented with 10% fetal bovine serum (FBS) and penicillin (100 IU/ml)/ streptomycin (50 mg/ml). Cells were maintained at 37℃in 5% CO₂ atmosphere. Medium were changed three times weekly. Cells were subcultured following trypsinisation. We use two different ways to detect the expression of various integrin subunits In this HLECs line by using five monoclonal antibodies specific for integrin subunitsα₂,α₃,α₅,β₁ andβ₂. Fristly, Immunofluorescence analysis were performed to determine whetherα₂,α₃,α₅,β₁ andβ₂ integrins express In this HLECs line. In brief, HLECs were grown on coverslips and slides were washed with phosphate buffered solution (PBS) and then blocked with 5% goat serum for 30 minutes at room temperature. After rinsing, the specimens were stained with monoclonal antibodies against the integrin subunits at 4℃overnight, slides were washed with PBS, then secondary antibody were applied for 30 minutes at room temperature. Negative control slides were incubated in PBS instead of the primary antibodies. Secondly, flow cytometry (FCM) were used to detect the positive percentages of different integrin subunits In this HLECs line. Briefly, cells for flow cytometric analysis were resuspended in PBS and sedimented cells (5×10⁵/L) were incubated with monoclonal antibodies against the integrins, isotype control or negative control. After washing, cells were incubated with FITC-conjugated goat anti-mouse secondary antibodies, washed again and resuspended in PBS. Cytometry was performed on a fluorescence-activated cell sorting (FACS) scan with 488nm argon laser. Results are presented as arbitrary units of fluorescence intensity. Futhermore, SRA01/04 cells were incubated with 0.02, 0.04, 0.08 mmol/L Verapamil for 24 hours or 48 hours, respectively, then the positive percentages of different integrin subunits in this HLECs line were analyzed by flow cytometry.Results: Our study showed that the HLECs line, SRA01/04 expressedα₂,α₃,α₅,β₁,β₂ integrin subunits. The positive percentages were 85.3%, 97.6%, 74.0%, 97.7%, 3.65%, respectively. There were significant differences of positive expression percentages between each two integrin subunits except forα₃ andβ₁. Verapamil dose, time-dependenly inhibitted the expression of integrin subunits in SRA01/04.Conclusion: The positive expression ofα₂,α₃,α₅,β₁,β₂ integrin subunits in the HLECs line, SRA01/04 suggests that integrins may promote the residual LECs proliferation, differentiation, and migration after cataract surgery, which may lead to posterior capsule opacification(PCO). Verapamil, a calcium channel blockers, inhibits integrin subunits expression in SRA01/04, which seems to be a suitable drug to prevent and treat PCO.