小肠上皮细胞迁移过程中膜电位检测方法的建立及应用  

Establishment and application of membrane potential detection method for intestinal epithelial cell migration

作　　者：宋厚盼[1] 李茹柳[1] 王一寓 陈炜璇[1] 陈昫[1] 徐颂芬[1] 陈蔚文[1] 

机构地区：[1]广州中医药大学脾胃研究所,广东广州510405

出　　处：《中国药理学通报》2013年第12期1758-1761,共4页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金项目资助(No 81173254);广州中医药大学中医内科学特色重点学科建设项目

摘　　要：目的用流式细胞仪建立检测小肠上皮细胞(IEC-6)膜电位(membrane potential,Em)的方法,为细胞迁移过程病理生理及药理研究检测膜电位提供参考。方法在划痕法细胞迁移模型上,采用细胞膜电位荧光探针DiBAC4(3)预先负载到IEC-6细胞,应用流式细胞仪测定细胞膜电位。结果①细胞迁移实验显示,外源性多胺(精脒,SPD)可促进细胞迁移,多胺合成抑制剂DFMO可抑制细胞迁移;②当负载到细胞的DiBAC4(3)浓度为1μmol·L-1,孵育时间为10min时,IEC-6细胞在流式细胞仪上呈现最大荧光强度;③空白组细胞平均荧光强度为712.05±19.73,SPD组和DFMO组平均荧光强度分别为406.07±46.54和1030.15±87.63,与空白组比较差异均有显著性,表明SPD组Em增加,DFMO组Em降低。结论建立的流式细胞仪检测细胞膜电位方法,适用于多胺介导的IEC-6细胞迁移过程细胞膜电位的检测,也可为其它单层贴壁细胞在生理和病理状态下检测细胞膜电位变化提供参考。Aim To establish a method for membrane potential （Em） detection during intestinal epithelial cell migration, there- by providing a reference for detecting membrane potential of IEC- 6 cells in pathophysiological and pharmacological studies. Meth- ods A wounding model of IEC-6 cells was created in a stand- ardized fashion by a single-edge razor blade; IEC-6 cells were preloaded with membrane potential fluorescent probe DiBAC4 （3）, FACSCalibur flow cytometry was used to determine the mean fluorescence intensity of IEC--6 cells. Results @ Spermi- dine （SPD） promoted IEC-6 cell migration, α- difluoromethylor- nithine（DFMO） inhibited IEC-6 cell migration; （2） When the loaded concentration of DiBAC4 （3） was 1 μmol· L^-1 ,the incu-bation time was 10 min, IEC-6 cells showed maximum fluores- cence intensity in the flow cytometry; （3） The mean fluorescence intensity of control, SPD, and DFMO groups were 712. 05 ± 19. 73, 406. 07 ± 46. 54, and 1030. 15 ± 87.63, respectively, which indicated that the Em of IEC-6 cells was increased when treated with SPD, and decreased when treated with DFMO. Conclusion The method created to determine the Em by flow cytometry is suitable for Em detection of polyamine-mediated IEC-6 cell migration as well as other single-layer adherent cells in physiological or pathological state.

关 键 词：小肠上皮细胞 细胞迁移 多胺 膜电位 荧光探针 流式细胞仪 

分 类 号：R322.45[医药卫生—人体解剖和组织胚胎学] R329.24[医药卫生—基础医学]