摘要
目的研究环巴胺(cyclopamine)对人食管癌EC9706细胞生长抑制作用,并初步探讨其作用机制。方法培养EC9706细胞,加入不同浓度环巴胺,作用不同时间,MTT法检测细胞生长抑制率;倒置显微镜下观察细胞形态变化;流式细胞仪、AO/EB双荧光染色观察药物作用于细胞后的细胞凋亡情况;Western blot观察不同浓度的药物作用于细胞后对Gli-1蛋白表达的影响。结果 MTT法显示,与空白对照组和DMSO对照组相比,环巴胺对EC9706细胞有明显的生长抑制作用,且环巴胺对EC9706细胞增殖的抑制作用呈剂量依赖性;AO/EB双荧光染色显示,细胞体积缩小,皱缩变圆,边缘毛糙,形成不规则突起,细胞核凝集、固缩;环巴胺通过促进细胞凋亡而达到对细胞的生长抑制作用,其调亡率明显高于空白对照组及DMSO组,且呈剂量依赖性。Western blot显示,环巴胺可能是通过影响Gli-1蛋白的表达来调控细胞的生长。其表达率低于空白对照组及DMSO组,呈剂量依赖性。结论环巴胺对人食管癌细胞系EC9706的生长有明显抑制作用,提示食管鳞状细胞癌的发生和发展与Hedgehog信号转导通路的异常激活有关,且Gli-1对肿瘤的发生发展起重要作用,这对食管癌的诊断、治疗以及新药开发提供新的思路。
Objective To research the inhibitory effect of cyclopamine on the human esophageal carcinoma cells EC9706 and make the preliminary discussion of its mechanism. Methods EC9706 cells were cultured in vitro. After adding different concentrations of cyclopamine and with different time ,the cells growth inhibition rate was detected by MTY method, then the morphological change of cells was observed by inverted microscope. The apoptosis of cells after adding different concentrations of drugs was observed by flow cytometry and AO/EB double fluorescence staining. The expression of Gli-1 protein after adding different concentrations of drugs was observed by Western blot. Results MTY method showed that compared with blank control group and DMSO control group, cyclopamine had significant inhibitory effect on the growth of the EC9706, and the inhibitory effect on proliferation of cells was in dose-dependent manner. AO/EB double fluorescence staining showed that the morphological changes of cells included ： cell volume shrunk and turned round ; edge was coarse and formed irregular process ; karyopyknosis and karyorrhexis. The inhibitory effect of cyclopamine on cell growth may be via promoting cell apoptosis ;its apoptosis rate was higher than those in blank control group and DMSO control group in dose-dependent manner. Wesern blot showed that cyclopamine regulated growth of cells may be through influencing the expression of Gli-1 ;its expression rate was lower than those in blank control group and DMSO control group in dose-dependent manner also. Conclusions Cyclopamine can effectually inhibit the growth and proliferation of EC9706 cells ,which points out that the genesis and development of esophageal squamous cell carcinoma might be associated with abnormal acting of hedgehog signal transduction path-way, and Gli-1 may play an important role in genesis and development of tumor. This may provide new idea for diagnosis and treatment of esophageal carcinoma and development of new drugs.
出处
《中国临床研究》
CAS
2012年第11期1046-1049,F0002,共5页
Chinese Journal of Clinical Research
