绿色荧光蛋白细胞的保存及细胞周期分析  

Preservation of Green Fluorescent Protein and Analysis of Cell Cycle

作　　者：刘双又[1] 李小兰[1] 肖薇[1] 陶德定[1] 罗学来[1] 高霞[1] 胡俊波[1] 龚建平[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院分子医学中心,武汉430030

出　　处：《华中科技大学学报（医学版）》2007年第6期798-800,820,共4页Acta Medicinae Universitatis Scientiae et Technologiae Huazhong

基　　金：国家自然科学基金(No.30300338;No.30471673);国家"973"计划(No.2002CB513100-2);教育部留学回国人员启动基金[教外司(2003)14]资助项目

摘　　要：目的探讨在绿色荧光蛋白（GFP）质粒转染后的细胞中，既能最大限度地保存GFP阳性细胞又有利于细胞周期分析的固定方法。方法以胃癌MKN-28和乳腺癌MCF-7细胞为模型，脂质体Lipofectamine2000转染细胞，转染后的细胞分别用：①80％乙醇固定；②0．1％、0．25％、0．5％和1．0％的多聚甲醛固定；③0．1％、0．25％、0．5％、1．0％多聚甲醛4℃固定30min后再用80％乙醇固定等3种方法固定细胞，然后用含0．1％triton的PI染色，在流式细胞仪上检测GFP的自发绿色荧光及行细胞周期分析。结果单纯乙醇固定的细胞中GFP均消失。单纯0．25％～1．0％的多聚甲醛固定后的细胞不能得到有效的DNA直方图，单纯0．1％的甲醛固定的细胞虽可得到良好的直方图，但PI染液中的triton破坏了大部分的GFP。先甲醛再乙醇固定的细胞中，0．5％～1．0％的甲醛可较好地防止乙醇和triton对绿色荧光蛋白的破坏作用，保留下2／3的GFP阳性细胞，0．5％的甲醛固定的细胞可得到CV值〈8％的G0／G1期峰，而1．0％甲醛固定后的细胞CV值〉8％。结论在GFP质粒转染后的细胞中，先用0．5％的多聚甲醛固定细胞30min后再用乙醇固定，既能最大限度地保存GFP，又可得到CV值良好的DNA直方图。若不考虑保留GFP，或普通细胞作细胞周期分析时，单纯0．1％的多聚甲醛固定细胞，即可得到与传统的单纯乙醇固定细胞相似的CV值很小的DNA直方图。Objective To investigate an easy and useful fixation in transfected adherent cells with a GFP-encoding plasmid, which allowed the cells not only to maintain most of GFP but also to acquire adequate DNA histogram. Methods The gastric tumor MKN-28 and breast cancer MCF-7 cell lines were used as the models. Cells were transfected with lipofectamine2000. The transfected cells were fixed with A （80% ethanol）, B （0. 1%, 0. 25%, 0.5%, 1.0% formaldehyde）, C （0.1%, 0.25%, 0.5%, 1.0% formaldehyde for 30 min followed by 80% ethanol）, respectively. Fixed cells were stained with PI and analyzed by flow cytometry for GFP fluorescence and DNA content. Results All GFP disappeared in cells with ethanol only. In group B, cells fixed with 0. 25%-1. 0% formaldehyde didn＇t obtain adequate DNA histogram to analyze, cells in 0. 1% formaldehyde had good DNA histogram but 0.1% triton in PI destroyed most of GFP. In group C, 0.5%-1.0% formaldehyde could prevent damage to GFP from ethanol and triton, preserving most of them （2/3）, the CV of G0/G1 peak in DNA histogram was less than 8% in 0.5% formaldehyde cells but more than 8% in 1.0% formaldehyde cells. Conclusion Adherent cells transfected with a GFP-encoding plasmid, after fixed with 0.5G formaldehyde before ethanol, could maintain most of GFP and acquire adequate DNA histogram in flow cytometry. It was also found that cells fixed with 0. 1% formaldehyde only could obtain the good DNA histogram like ethanol only did when don＇t care if preserving GFP.

关 键 词：绿色荧光蛋白 流式细胞术 细胞周期 细胞固定 多聚甲醛 

分 类 号：R34-33[医药卫生—基础医学]