稳定表达荧光素酶BGC823细胞系的构建

Construction of BGC823 Cell Line Stably Expressing Luciferase

目的:建立稳定表达荧光素酶的人胃癌细胞系(BGC823),为建立活体成像的人胃癌裸鼠移植瘤模型及治疗研究奠定基础。方法:含绿色荧光蛋白(GFP)及荧光素酶的质粒用脂质体转染法稳转到胃癌细胞BGC823中。不同浓度的嘌呤霉素用于筛选细胞,单克隆细胞用有限稀释法挑选,用流式细胞仪检验构建的BGC823-FLuc细胞的纯度。细胞的荧光素酶表达量用荧光素酶底物试剂检测。MTS比较改造前后BGC823细胞的增殖。结果:流式检测显示单克隆细胞系阳性率达到99.9%,且多功能酶标仪检测到荧光信号值。MTS法检测发现改造前后细胞增殖基本一致,说明加入荧光素酶基因未对细胞增殖产生明显影响。结论:表达荧光素酶及绿色荧光蛋白的单克隆胃癌细胞系构建成功。此细胞系具有可用于体外及体内实验以研究肿瘤模型的优势的。

Objective:The establishment of a human gastric cancer cell line(BGC823)stably expressing luciferase will lay the foundation for the establishment of an in vivo imaging human gastric cancer xenograft tumor model and treatment research.Methods:Plasmid containing green fluorescent protein(GFP)and luciferase was stably transferred to gastric cancer cell BGC823 by liposome transfection.Puromycin at a different concentration was used to select cells.Monoclonal cells were selected by the limiting dilution method.Finally,the purity of the constructed BGC823-FLuc cells was checked by flow cytometry.The expression level of luciferase in the cells is detected with a luciferase substrate reagent.MTS compares the proliferation of BGC823 cells before and after transformation.Results:Flow cytometry showed that the positive rate of the monoclonal cell line reached 99.9%,and the fluorescence signal value was detected by the multifunctional microplate reader.The MTS method detected the cell proliferation before and after the transformation and found that the proliferation was basically the same,indicating that the addition of luciferase gene did not have a significant effect on cell proliferation.The above results indicate that the construction of a monoclonal gastric cancer cell line expressing luciferase and green fluorescent protein was successful.This cell line has the advantage that it can be used in vitro and in vivo experiments,which is convenient for subsequent tumor model research.