秋水仙碱对人眼Tenon囊成纤维细胞增殖及凋亡的影响

摘要

Objective To investigate the effects of colchicine on the proliferation and apoptosis of human Tenon's capsule fibroblasts (HTCFs) in vitro.Methods HTCFs were cultured in vitro,and the subcultured cells were identified.MTS assay was used to observe the inhibitory actions of colchicine on HTCFs at different concentrations.The apoptotic rates of the cells were detected by flow cytometry.The apoptosisrelated proteins,including PARP,Caspase-9,Caspase-3 and active-Caspase-3,were detected by Western blot.Results MTS assay showed that colchicine inhibited the proliferation of HTCFs in a dose-and time-dependent manner.Flow cytometry showed that colchicine induced a dose-dependent apoptosis of HTCFs for 48 hours,the apoptotic rates of 5 μmol · L-1,10 μmol · L-1 and 20 μmol · L-1 were (18.37 ±4.26)％,(33.80 ±5.02)％ and (52.00 ± 2.00)％,respectively,there were significant differences compared with the control (F =91.59,P ＜ 0.001).Western blot showed the activation of Caspase-3 and PARP followed by colchicine treatment.Conclusion Colchicine significantly inhibits the proliferation of HTCFs in vitro,and induced apoptosis,which may be associated with the activation of mitochondrial apoptotic pathways.%目的 研究秋水仙碱对体外培养的人眼Tenon囊成纤维细胞(human Tenon's capsule fibroblasts,HTCFs)增殖及凋亡的影响.方法 体外培养HTCFs,并对传代培养的细胞进行鉴定;采用MTS法检测不同浓度的秋水仙碱对HTCFs增殖的抑制作用;以Annexin V-FITC/PI双标记流式细胞术检测秋水仙碱对细胞凋亡的影响;使用蛋白质印迹法检测秋水仙碱对凋亡相关蛋白聚腺苷二磷酸-核糖聚合酶(poly ADP-ribose polymerase,PARP)、Caspase-9、Caspase-3及active-Caspase-3表达的影响.结果 MTS检测显示秋水仙碱对HTCFs具有明显抑制作用,并呈剂量及时间依赖性.流式细胞术检测发现秋水仙碱作用48 h可诱导HTCFs发生剂量依赖性凋亡,与对照组相比,5μmol·L-1、10 μmol·L-1、20μmol·L-1的秋水仙碱作用HTCFs的凋亡率分别为(18.37±4.26)％、(33.80±5.02)％及(52.00±2.00)％,差异具有统计学意义(F=91.59,P＜0.00l).蛋白质印迹法检测结果显示,秋水仙碱可诱导细胞内凋亡关键蛋白Caspase-3及PARP的活化.结论 秋水仙碱可以明显抑制HTCFs的体外增殖,其机制可能与线粒体凋亡途径的激活而诱导细胞发生凋亡相关.