绿色荧光蛋白细胞的保存及细胞周期分析

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。

Preservation of Green Fluorescent Protein and Analysis of Cell Cycle

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 0.5% 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.