应用逆转录病毒载体构建荧光标记的K562细胞模型

Establishment of auto-fluorescent K562 cell model using recombinant retrovirus vector

目的:采用逆转录病毒载体系统构建荧光标记的K562细胞模型。方法:将增强绿色荧光蛋白(EGFP)cDNA插入载体pCMV-hyg,构建重组质粒pCMV-EGFP-hyg,并与逆转录病毒载体质粒pCMV-G和pCMV-GP共转染293T细胞生产重组逆转录病毒,感染K562细胞;通过Hyg筛选建立EGFP自身标记的K562细胞模型。应用流式细胞仪及荧光显微镜观察绿色荧光蛋白表达。比较EGFP-K562细胞与K562细胞生长曲线。通过外周血淋巴细胞的NK活性试验观察EGFP-K562能否用于有关实验研究。结果:应用EGFPcDNA成功构建pCMV-EGFP-hyg,与逆转录病毒载体系统质粒共转染293T细胞后产生含EGFP的重组逆转录病毒,48h收集的病毒滴度达1.5×106CFU/ml。重组逆转录病毒感染K562细胞后,经Hyg筛选,98%以上稳定表达EGFP。长期培养20代,EGFP表达稳定,对K562细胞生长无影响。以EGFP-K562为靶细胞的NK活性试验显示40∶1效靶比,孵育4h最敏感。结论:应用逆转录病毒载体系统成功构建稳定表达绿色荧光蛋白的EGFP-K562细胞模型,NK活性试验提示EGFP-K562可用作实验研究的新型靶细胞模型。

objective：To establish an auto-fluorescent K562 cell model using recombinant retroviral vector system. Methods：The full length cDNA of EGFP gene was inserted into the pCMV-hyg to construct recombinant retroviral plasmid pCMV-EGFP-hyg. The retrovirus containing EGFP was produced by co-transfection of T293 cell line with pCMV-EGFP-hyg, pCMV-G and pCMV-GP. The K562 cells expressing EGFP （EGFP-K562） were obtained by co-culture with recombinant retrovirus-producing T293 cells and Hyg selection. EGFP expression was examined with flow cytometry（FCM） and fluorescence microscopy. To show the application of EGFPK562 in laboratory research, NK activity assay using EGFP-K562 as target cells was carried out in a series of effector （E） to target （T） cell ratio. Results：Recombined retrovirus containing EGFP was successfully constructed with high titers through co-transfection of T293 cells with pCMV-EGFP-hyg, pCMV-G and pCMV-GP. The highest titer of recombinant virus was 1.5×10^6CFU/mL when harvested at time point of 48 hours. Strong fluorescent in EGFP-K562 cells were developed and retained without any change during up to 20 passages of cell culture. More than 98% EGFP-K562 was achieved after selection with Hyg. NK activity assay employing EGFP-K562 as target cells showed peripheral blood mononuclear cells （PMNC） exhibited a different cytotoxicity with different E ： T ratio and incubation time. The optimal condition of NK activity assay was at a ratio of 40 ： 1 between E and T with 4 h incubation. Conclusion：Auto-fluorescent K562 cells was stably established which might provide a novel cell models in the experimental studies.