秋水仙碱对甲状腺相关眼病患者眼眶成纤维细胞增生的影响  

The effect of colchicine on the proliferation of human orbital fibroblast in thyroid associated ophthalmopathy in vitro

作　　者：刘燕[1] 许蓓[1] 许雪亮[1] 

机构地区：[1]中南大学湘雅医院眼科,长沙410008

出　　处：《中华实验眼科杂志》2013年第7期630-635,共6页Chinese Journal Of Experimental Ophthalmology

基　　金：湖南省科技厅基金项目（2008JT3002）

摘　　要：背景甲状腺相关眼病（TAO）患者的眼眶成纤维细胞（OFs）在眼部的增生性和炎症性反应中发挥重要作用，寻求抑制OFs增生的药物对TAO的防治具有重要意义。已有研究表明，秋水仙碱具有抗组织纤维化的作用，但其对TAO患者OFs的作用及其机制研究较少。目的研究秋水仙碱对体外培养的OFs增生的影响。方法将3例TAO患者行开眶减压术中获取的球后结缔组织用组织块培养法进行培养和鉴定，取3-8代的传代细胞进行体外实验。分别将1×10-8、1×10-7、1×10-6、1×10-5、1×10-4mol／L秋水仙碱加入含质量分数10％小牛血清的RPMI1640培养基中孵育OFs24、48和72h，用无秋水仙碱的RPMI1640培养基孵育细胞作为对照，采用细胞计数试剂盒．8（CCK-8）检测各组细胞450nm处的吸光度（A450）值，评估OFs的生长情况和各浓度组秋水仙碱对OFs的抑制率。此外，分别在培养基中加入1×10-6、1×10-6、1×10-4mol／L秋水仙碱作用48h，采用流式细胞术及应用MCYCLE软件进行分析，用百分率记录亚二倍体细胞数据及细胞周期各时期数据，检测各浓度药物组及无药物培养基组OFs的凋亡率和细胞周期的分布；采用免疫组织化学染色法测定转化生长因子-β（TGF-β）在OFs中的表达，以评价不同浓度秋水仙碱对OFs分泌TGF-β功能的影响。结果培养和传代的OFs呈梭形，波形蛋白免疫化学染色为阳性反应。1×10-4、1×10-5、1×10-6、1×10-7、1×10-8mol／L秋水仙碱作用24、48和72h，随着秋水仙碱浓度的增加，OFs的增生值（A450）逐渐下降，差异有统计学意义（F浓度=62．004，P〈0．05）；此外，随着秋水仙碱作用时间的增加，A450值逐渐下降，差异有统计学意义（F时间=459．582，P〈0．05）；随着秋水仙碱浓度的增加，其对OFs生长的抑制率逐渐升高。对照组、1×10-6、1×10-5、1×10-4mol／L秋水仙碱组OFs凋亡率分别为（1．73±0�Background The orbital fibroblasts （OFs） in thyroid associated ophthalmopathy （ TAO ） play important roles in the proliferative and inflammatory response. Seeking the drug which inhibit OFs growth is of a vital significance for the prevention and treatment of TAO. Research documented that eolchicine has an anti-fibrosis effect. But its influence on OFs of TAO patient is few known. Objective This study was to investigate the effect of eolchicine on growth and apoptosis of OFs in vitro. Methods The retroobital connective tissue was obtained form 3 TAO patients and cultured using explant method. OFs were passaged and identified by immunochemistry, and 3-8 genetaions of cells were used in the study. Colchicine at the concentrations of 1×10-8,1×10-7,1×10-6,1×10-5,1×10-4 mol/L was added into the RPMI 1640 with 10% fetal bovine serum（FBS） to incubated the cells for 24,48 and 72 hours respectively, and only RPMI 1640 was used to culture the cells as the control group. Cell counting kit-8 （CCK-8） was used to detect the absorbance value（A450 ） of OFs for the evaluatuion of OFs and the inhibitory rate of colchicine to OFs. The colchicine of 1×10-6,1×10-5,1×10-4 mol/L was added into the culture medium for 48 hours, and then the apoptotic rate of the cells and the cell percentage in various cellular cycle was assayed by flow cytometry（FCM）. The expression of transforming growth factor-β （TGF-β）in the ceils was detected by immunochemistry to assess the influence of colchicine on the serection of the ceils. Results Cultured cells showed the spindle-like in shape and the cell number was significantly increased with the incubation time. After incubated with 1×10-4,1×10-5,1×10-6,1×10-7,1×10-8mol/L colchicines, the A450values were gradually reduced with the increase of the concentrations of colchicine（F ,ratio, = 62. 004, P〈0.05 ）, and significant differences were found between different contrations of colchicine groups（all P〈0. 05）. Aslo,gradually declined A450values of the ce

关 键 词：眼眶成纤维细胞 甲状腺相关眼病 秋水仙碱 增生 凋亡 转化生长因子-Β 

分 类 号：R771.3[医药卫生—眼科] R581[医药卫生—临床医学]