摘要
目的观察脱氧胆酸(deoxycholate,DC)能否诱导体外培养的人正常食管黏膜上皮细胞凋亡,并探讨其机制。方法采用无血清的角朊细胞培养基体外培养人正常食管黏膜上皮细胞;通过流式细胞仪(FCM)观察DC干预的细胞经碘化丙啶(PI)单染色后凋亡细胞百分比;AnnexinV-FITC结合PI双染色观察早期凋亡细胞比率;TUNEL试验凝胶DNA梯度电泳确定凋亡的发生。通过FCM分析干预细胞激活的Caspase3变化,免疫印迹法观察Fas蛋白、Bcl-2和Bax蛋白的表达情况。结果4种凋亡检测方法确定DC可诱导体外培养的食管黏膜上皮细胞凋亡,并成浓度时间正相关性变化。500μmol/LDC干预30min,含激活状态Caspase-3的细胞达21.3%,明显高于对照组的1.5%(P<0.01)。免疫印记法示细胞经DC干预前后Fas受体蛋白均为阴性表达,但Bcl-2蛋白表达水平下降,而Bax蛋白则上升。结论DC可诱导人正常食管黏膜上皮细胞凋亡;此过程与Fas-L/Fas凋亡信号系统无关;而存在Caspase-3激活、Bcl-2减少和Bax增加,即与线粒体凋亡调节途径密切相关。
Objective To study the effect of deoxycholate in inducing apoptosis of human normal esophageal mucosal epithelial cells in vitro and investigate the molecular mechanism. Methods Cultured normal human esophageal mucosal epithelial cells were treated with deoxycholate, and the cell apoptosis were evaluated with TUNEL, DNA ladder, flow cytometry with PI-staining, Annexin V-FITC conjugated with PI staining, and Western blotting. Results Flow cytometry, TUNEL and DNA ladder demonstrated that deoxycholate could induce apoptosis in normal human esophageal mucosal epithelial cells in a doseand time-dependent manner. A percentage of 21.3% of the cell population treated with deoxycholate at 500 p.mol/L for 30 min exhibited detectable caspase-3 activity shown by flow cytometry, which was significantly higher than the control level （1.5% , P〈0.01）. Western blotting suggested that deoxycholate down-regulated Bcl-2 protein expression and up-regulated Bax expression, but Fas was negative in the cells before and after deoxycholate treatment. Conclusions Deoxycholate could induce apoptosis in cultured human esophageal mucosal epithelial cells. Aaspase-3 activation, Bcl-2 protein down-regulation and Bax up-regulation are involved in deoxycholate-induced apoptosis, which does not involve Fas-L/Fas.
出处
《第一军医大学学报》
CAS
CSCD
北大核心
2005年第10期1240-1243,共4页
Journal of First Military Medical University
基金
卫生部临床学科重点课题(20012130)~~
关键词
脱氧胆酸
食管
黏膜上皮
凋亡
deoxycholate
esophagus
mucosal epithelial cell
apoptosis
