摘要：
目的 探讨多西环素对角膜肌成纤维细胞核仁组成区嗜银蛋白(AgNOR)及α平滑肌肌动蛋白(α-SMA)表达的抑制作用.方法 实验研究.分别用基础培养液配制的1.0及2.0 g/LⅠ型胶原酶对牛角膜基质层进行二步消化分离,制成细胞悬液后转入培养瓶中用RPMI-1640培养液(含10％胎牛血清)培养.取生长良好的细胞进行波形蛋白免疫细胞化学染色和免疫荧光细胞化学染色.选用生长良好的牛角膜肌成纤维细胞分成3个组:阴性对照组(无药物干预),地塞米松组(地塞米松120mg/L)及各浓度梯度(10、20、40、60及80mg/L)多西环素组,每组30例.指标测定:采用AgNOR及α-SMA免疫荧光细胞化学染色等技术测定各实验组在对角膜肌成纤维细胞干预24和48h后,对细胞DNA复制及α-SMA合成的影响.各组间比较采用单因素方差分析,同一药物浓度下两组间比较采用两样本均数比较t检验.结果 在体外成功分离培养牛角膜肌成纤维细胞,显微镜下观察及α-SMA免疫荧光细胞化学染色后证实所培养的细胞为角膜肌成纤维细胞.细胞被干预24h后,阴性对照组AgNOR颗粒数为(6.40±0.62)个,AgNOR颗粒面积为(34.80±2.36) μm2；60mg/L多西环素组AgNOR颗粒数(2.23±0.43)个,AgNOR颗粒面积为(19.91 ±2.15) μm2.细胞被干预48h后,阴性对照组AgNOR颗粒数为(7.27±0.64)个,AgNOR颗粒面积为(36.27±1.99) μm2；60 mg/L多西环素组AgNOR颗粒数为(2.80±0.76)个,AgNOR颗粒面积为(13.75±2.09) μm2.差异有统计学意义(干预24 h:F=252.55,202.16;P ＜0.05;干预48 h:F=169.38,853.23;P ＜0.05;在同一浓度干预下:t=6.98,11.62;P ＜0.05).当多西环素浓度达60 mg/L时作用效果与120 mg/L地塞米松相当,差异无统计学意义(t=1.182,0.213;P ＞0.05).α-SMA免疫荧光细胞化学染色后行荧光定量分析,地塞米松对α-SMA的合成无明显作用(t=1.096,P =0.287);而多西环素组可抑制角膜肌成纤维细胞中α-SMA的合成,各浓度多西环素组细胞荧光染色的灰度值分别为189.90±7.48、140.20±7.79、113.20±8.98、98.00±3.50、85.50±4.99,差异有统计学意义(F=761.79,P=0.00).结论 在10 ～ 80 mg/L浓度范围内,多西环素可抑制体外培养的牛角膜肌成纤维细胞的AgNOR增生,呈剂量-时间-效应依赖性；在药物浓度达到60 mg/L时,与120 mg/L地塞米松拥有相当的作用效果.多西环素同时具有抑制细胞合成α-SMA的作用,与地塞米松比较,对抑制角膜瘢痕的形成更具研究价值.%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 and α-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 (AgNOR) staining and the immunohistochemistry for α-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 of α-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 2control group Ag-a was (36.27 ± 1.99) μ m2,60 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-a24 h =202.16,P ＜ 0.05,FAg-c48h =169.38,FAg-a48h =853.23,P ＜0.05),in the same concentrations intervention (tAg-e =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-a 24h =1.182,P =0.242,tAg-a 48 h =0.21,P =0.832).As the concentrations investigated,doxycycline can inhibit the expression of α-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 of α-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 AgNOR count and AgNOR area of bovine corneal myofibroblasts can be significantly reduced in vitro.Compared with dexamethasone,doxycycline significantly suppressed the expression of α-SMA in bovine corneal myofibroblasts in a dose-dependent positive trend.(Chin J Ophthalmol,2013,49:)
