Bevacizumab对人Tenon囊成纤维细胞转分化的抑制作用

Inhibitive effects of Bevacizumab on transdifferentiation of human Tenon capsule fibroblasts

目的观察Bevacizumab(贝伐单抗)对转化生长因子(transforming growth factor,TGF)-β2诱导下的人Tenon囊成纤维细胞转分化的抑制作用,探讨抗新生血管药物抑制青光眼术后滤过泡瘢痕化的机制。方法用含体积分数10%胎牛血清的DMEM高糖型培养基对人Tenon囊成纤维细胞株进行体外常规培养和传代。实验分为空白对照组、TGF-β2处理组(加入终浓度为10μg·L-1的TGF-β2DMEM完全培养基)及Bevacizumab干预组(加入10μg·L-1的TGF-β2和1.0 g·L-1的Bevacizumab DMEM完全培养基),置于37℃、体积分数5%二氧化碳培养箱中培养48 h,并分别应用细胞免疫荧光染色技术和Western Blot实验检测Bevacizumab对TGF-β2刺激下人Tenon囊成纤维细胞α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)表达的影响。结果α-SMA蛋白主要表达在细胞质中,免疫荧光染色结果显示TGF-β2处理组可以见到α-SMA的荧光表达,而Bevacizumab干预组α-SMA蛋白表达受到抑制。Western Blot结果显示空白对照组、TGF-β2处理组及Bevacizumab干预组α-SMA蛋白相对表达量分别为0.630±0.038、1.130±0.071和0.340±0.033,Bevacizumab干预组α-SMA蛋白相对表达量低于空白对照组和TGF-β2处理组(均为P<0.05)。结论 Bevacizumab可明显抑制TGF-β2诱导下人Tenon囊成纤维细胞α-SMA蛋白的表达,抑制成纤维细胞表型转化。

Objective To investigate the inhibitive effects of Bevacizumab on transdifferentiation of human Tenon＇s capsule fibroblasts induced by transforming growth factor-β2（ TGF-β2）in vitro,and explore the mechanism of anti-angiogenesis drugs inhibiting bleb scar after glaucoma filtering surgery. Methods Human Tenon＇s capsule fibroblasts cell line were cultured and passaged in DMEM / High glucose medium with 10% fetal bovine serum. The cells were divided into blank control group,TGF-β2group and TGF-β2＋ Bevacizumab group. Medium containing a final concentration of 10 μg·L- 1TGF-β2was added into TGF-β2group and medium containing a final concentration of 10 μg·L- 1TGF-β2plus 1. 0 g·L- 1Bevacizumab was added into TGF-β2＋ Bevacizumab group which were used into the 37 ℃,5% carbon dioxide cultured 48 hours. Immunofluorescence staining and Western Blot were used to measure the expression of α-smooth muscle actin（ α-SMA） protein after culture for 48 hours with Bevacizumab. Results The expression of α-SMA protein mainly located in human Tenon fibroblast cytoplasm. Immunofluorescence staining showed that TGF-β2up-regulated the expression of α-SMA protein,while Bevacizumab significantly inhibited the expression of α-SMA protein. Western Blot results showed that the relative α-SMA protein expression of blank control group,TGF-β2treatment group and TGF-β2＋ Bevacizumab treatment group were 0. 630 ± 0. 038,1. 130 ± 0. 071 and 0. 340 ± 0. 033,TGF-β2＋ Bevacizumab treatment group was lower than blank control group and TGF-β2treatment group（ all P〈0. 05）. Conclusion Bevacizumab can obviously inhibit the expression of α-SMA protein of human Tenon capsule fibroblasts,and inhibit fibroblast phenotype transformation.