药物诱导细胞凋亡时DNA检测技术的探讨  

Meditation of methodology for improvement on detecting DNAchange during apoptosis of MGC 803 cell line

作　　者：马靖[1] 庞大本[2] 池旭生 彭文烈[1] 徐安龙[1] 

机构地区：[1]中山大学生命科学学院生化系与新药开发中心,广东广州510275 [2]中医研究院基础理论研究所,北京100700

出　　处：《癌症》2000年第8期828-830,共3页Chinese Journal of Cancer

基　　金：广州市科委重点项目! (97-Z-12-01);国家新药博士基金! (96-901-06-36)

摘　　要：比较检测细胞凋亡时DNA变化的三种方法。方法 :联合采用Hoechst33342和PI荧光双染显微镜观察 ,DNA凝胶电泳和流式细胞仪测定三种方法检测甘草提取物诱导MGC 803细胞凋亡时DNA的变化。结果 :低浓度药物作用时 ,荧光显微镜下观察到DNA凝聚和PI排斥 ,但DNA电泳不出现Ladder断裂片段 ,4℃重悬后FCM才能检测到凋亡峰 ;高浓度药物作用时 ,DNA呈Ladder断裂 ,FCM检测到凋亡峰 ,但荧光显微镜下观察到PI着色和DNA凝聚共存。结论 :(1)应慎重使用依据PI着色区分凋亡和坏死细胞的方法;(2)有些情况下 ,凋亡细胞经乙醇固定后DNA断裂片段的逸出是一个缓慢的过程 。Objective: To compare three different methods of detecting DNA change in apoptotic cells Methods: The change of DNA in apoptotic MGC 803 cells induced by glycyrrhiza uralensis Fisch extract was determined by using fluorescence microscope after Hoechst33342 and PI double staining, DNA agarosegel electrophoresis and flow cytometry Results: After treatment with low concentration of glycyrrhiza uralensis Fisch extract to MGC 803 cells, chromatin condensation and PI exclusion were observed, while “DNA ladder”was not revealed on the agarose gel and the sub G1 peak could only be detected after the apoptotic cells were resuspended at 4℃ After treatment with high concentration of the extract, both the “DNA ladder”and the sub G1 peak were obvious, however, chromatin condensation and PI staining were observed simultaneously in the apoptotic cells Conclusions: 1 Discrimination of apoptosis and necrosis based on PI exclusion should be used with due precaution; 2 Insome cases, since the DNA leakage from apoptotic cells after 70%ethanol fixation is a slow process, the cells should be resuspended at 4℃for appropriate time to improve the accuracy in detecting apoptotic cells by flow cytometry

关 键 词：细胞凋亡 DNA断裂 染色质凝聚 药物诱导 

分 类 号：Q26[生物学—细胞生物学]