绿色荧光蛋白GFP在转化细胞和肿瘤细胞中的表达

The Expression of GFP Gene in Transformed and Tumor Cells

目的在肿瘤基因治疗研究中建立一个用绿色荧光蛋白 (greenfluorescentprotein ,GFP)为标记基因的合适条件。 方法用带有GFP突变体的质粒转染到表达细胞 ,观察GFP瞬时表达的情况 :1 直接测定GFP在COS- 7细胞中的表达并观察表达的稳定性 ;2 比较不同启动子驱动的 pcDNA3 - EGFP和 pSVK3 -S6 5T两种质粒在不同肿瘤细胞中的转染效率 ;3 用LacZ基因与GFP基因共转染 48h后 ,通过FACS分选测定两种基因在同一细胞中的表达情况。 结果在不同条件下的活细胞中有GFP基因表达 ,且表达具有稳定性 ,表达持续约 2周左右 ;CMV启动子和SV4 0 启动子调控的GFP对不同肿瘤细胞株的转染效率有差异。当两种基因共转染后用FACS分选 ,选出GFP细胞带有LacZ基因细胞在 1∶4时可达 85 %以上。 结论通过GFP表达可连续而直接地观察活细胞中的基因表达 ,利用GFP可快速地挑选带有靶基因的细胞。

Objective To search the appropriate experimental conditions for using green fluorescent protein (GFP) as a reporter gene in tumor gene therapy. Methods The plasmids carrying mutated GFP gene were transfected into the eukaryotic cells to observe transient gene expression. These studies were conducted to 1. directly determine GFP expression and express stability in the COS-7 cells, 2. compare the transfection efficiency of two plasmids pcDNA 3-EGFP, pSVK 3-S65T with different promoters in different tumor cell lines, 3. determine two genes expression in a single cell using LacZ cotransfected with GFP by FACS. Results Fluorescence could be detected in intact viable cells under different sets of conditions. The expression of GFP might last two weeks or more and the expressed fluorescence was stable. The transfection rate of pcDNA 3-EGFP expressed was different in three tumor cell lines examined. But pSVK 3-GFP expressed similarly in four tumor cell lines examined. FACS showed the probability of two genes entering a single cell is above >85% at the ratio 1∶4. Conclusion The above data indicate that the GFP can be visualized continuously and directly for gene expression in living cells. GFP may also be used for quicklly selecting cells carrying a target gene.