全反式维甲酸对甲状腺相关眼病眼眶成纤维细胞增殖和凋亡的影响

Effect of all trans-retinoic acid on the proliferation and apoptosis of cultured thyroid associated ophthalmopathy orbital fibroblasts

目的观察全反式维甲酸对体外培养的眼眶成纤维细胞增殖的影响以及促胞凋亡的作用。方法体外培养甲状腺相关眼病患者眼眶成纤维细胞;将不同浓度的ATRA(0、10-9mmol/L、10-8mmol/L、10-7mmol/L、10-6mmol/L和10-5mmol/L)作用于成纤维细胞细胞分别达24、48和72 h后,用噻唑蓝(MTT)比色法观察ATRA对细胞增殖的影响,采用流式细胞仪检测ATRA处理48 h后细胞周期的改变及细胞凋亡率;检测ATRA处理48 h后细胞内bcl-2蛋白的表达水平。结果 MTT法显示ATRA能降低成纤维细胞的A值(P<0.05),细胞生长抑制率随作用时间和药物浓度的增加而增加。流式细胞仪检测细胞周期阻滞在G1期;凋亡率随着浓度的增加而上升;bcl-2蛋白的表达水平较正常对照组细胞明显下调。结论 ATRA能够抑制成纤维细胞的增殖,抑制作用呈时间和剂量依赖性;ATRA通过将细胞阻滞在G1期而起到抑制作用;ATRA可以诱导成纤维细胞凋亡,诱导细胞凋亡作用与下调细胞内bcl-2蛋白的表达有关。

[ Objective ] To investigate the function of all trans-retinoic acid （ATRA） in influencing the prolifera- tion and the apoptosis of orbital fibroblasts in vitro. [ Methods ] Orbital fibroblasts of thyroid associated ophthal- mopathy were cultivated in vitro. MTr assay were used to detect the effect on proliferation of orbital fibroblasts after incubation for 24 h, 48 h and 72 h in the different concentration of ATRA （0, 10-9 mmol/L, 10-s mmol/L, 10-7 mmol/ L, 10-6 mmol/L andl0-5 mmol/L）. The apoptotic ratio and the change of the cell cycle and the change of the expres- sion levels of bcl-2 of orbital fibroblasts were evaluated by the flow cytometry assay （FCM）. [ Results ] All trans- retinoic acid could decrease A value of and increase the inhibitory rate in a time-and dose-dependent manner. The FCM analysis shows the cell cycle was blocked at G1 phase; The apoptotic rates of orbital fibroblasts showed a in- creasing tendency in ATRA exposure group with the enhance of ATRA concentration. The FCM analysis shows that the level of bcl-2 in the cells declines significantly in experimental group compared with control group after cultured with ATRA for 48 h. [ Conclusions ] ATRA has growth- and proliferation-inhibitory effects on orbital fibroblasts in a time-and dose-dependent manner. ATRA could induce the apoptosis of the orbital fibroblasts and was blocked at the G1 phase of the cell cycle by ATRA. ATRA could induce the apoptosis of the orbital fibroblasts by conduct thedecline of the express of bcl-2.