[Inhibitory effects of doxycycline on argyrophilic nucleolar organizing regions and a-smooth muscle actin expression in proliferative bovine corneal myofibroblasts in vitro].

Objective To investigate the effect of doxycycline on the nucleolar organizing regions and a-smooth muscle actin expression in bovine corneal myofibroblasts in vitro and assess its contribution to ocular surface repair mechanisms. Methods Cell culture and identification: bovine corneal fibroblasts were cultured after the stroma was incubated in 1.0 and 2.0 g/L type I collagenase in two stages.Isolated cells were plated at mantaryay culture flask in 10% of BSA RPMI-1640.Vimentin and alpha-smooth muscle actin (α-SMA) organization were evaluated by immunocytochemistry.The cells staining positive for Vimentin andα-SMA indicated the presence of corneal myofibroblasts.Bovine corneal myofibroblasts were treated with different concentrations of doxycycline (10,20,40,60,80 mg/L),a bland control group and the dexamethasone group (120 mg/ L) were set up,each group had 30 cases.The argyrophilic nucleolar organizing regions (Ag NOR) staining and the immunohistochemistry forα-SMA were performed when the cells were treated for 24 hours and 48 hours.The AgNOR count (Ag-c),AgNOR area (Ag-a) and the expression ofα-SMA in the bovine corneal myofibroblasts among each experiment group and control group were compared using one-way ANOVA,further pairwise comparisons using Independent-Samples t test. Results Cell culture techniques were successfully used to establish a method for the isolation and culture of bovine corneal myofibroblasts.Microscopic examination and immunohistochemical staining confirmed that the cells cultured were bovine corneal myofibroblasts.The Ag-c and Ag-a of bovine corneal myofibroblasts progressively decreased as the concentrations of doxycycline was increase.24 h:bland control group Ag-c was 6.40±0.6,60 mg/L doxycycline group Ag-c was 2.23±0.43;bland control group Ag-a was (34.80±2.36) μm2,60 mg/L doxycycline hormone group Ag-a was (19.91±2.15) μm2.48 h:bland control group Ag-c was 7.27±0.6,60 mg/L doxycycline hormone group Ag-c was 2.80±0.76,bland control group Ag-a was (36.27±1.99) μm2,60 mg/ L doxycycline group Ag-a was (13.75±2.09) μm2.The differences were statistically significant:in the same time intervention (FAg-c 24 h=252.55,FAg-a 24 h=202.16,P<0.05,FAg-c 48 h=169.38,FAg-a 48 h=853.23,P<0.05),in the same concentrations intervention (tAg-c=6.98,tAg-a=11.62,P<0.05).And 60 mg/L of doxycycline had an obviously inhibitory action as 120 mg/L dexamethasone in the same treated hours (dexamethasone group Ag-a 24 h=30.56±3.66,dexamethasone group Ag-a 48 h=28.35±1.23),the differences were not statistically significant (tAg-a24h=1.182,P=0.242,tAg-a48h=0.21,P=0.832).As the concentrations investigated,doxycycline can inhibit the expression ofα-SMA in the bovine corneal myofibroblasts (189.90±7.48,140.20±7.79,113.20±8.98,98.00±3.50,85.50±4.99),the difference was statistically significant (F=761.79,P=0.00).While dexamethasone had no significant role in the expression ofα-SMA (bland control group was 225.10±6.74,the dexamethasone group was 228.50±7.12),and the statistically difference was not obvious (t=1.096,P=0.287). Conclusions As the concentrations of doxycycline was increased from 10 mg/L to 80 mg/L,the Ag NOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro.Compared with dexamethasone,doxycycline significantly suppressed the expression ofα-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend. (Chin J Ophthalmol,2013,49:) Key words: Cornea; Myofibroblasts; Doxycycline; Nucleolus organizer regions; Staining and labeling; Actins; Cells,cultured