应用逆转录病毒载体构建荧光标记的K562细胞模型
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江苏省卫生厅重点课题基金资助项目(H200131)


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

    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EGFP cDNA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,48 h收集的病毒滴度达1.5 ×106 CFU/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4 h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-

    Abstract:

    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 EGFP-K562 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 1.5×106CFU/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.

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周小玉,费小明,吴雨洁,缪扣荣,汪承亚.应用逆转录病毒载体构建荧光标记的K562细胞模型[J].南京医科大学学报(自然科学版),2007,(7):656-660

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  • 收稿日期:2006-11-24
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