文章摘要
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,2007,(7):656~660
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
投稿时间:2006-11-24  
DOI:10.7655
中文关键词: 增强型绿色荧光蛋白  逆转录病毒  流式细胞仪  NK细胞  K562
英文关键词: EGFP  retrovirus  flow cytometry  nature killer cell  K562
基金项目:江苏省卫生厅重点课题基金资助项目(H200131)
作者单位
周小玉 南京医科大学第一附属医院输血科, 江苏 南京210029 
费小明 南京医科大学第一附属医院血液科, 江苏 南京 210029 
吴雨洁 南京医科大学第一附属医院血液科, 江苏 南京 210029 
缪扣荣 南京医科大学第一附属医院血液科, 江苏 南京 210029 
汪承亚 南京医科大学第一附属医院输血科, 江苏 南京210029 
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中文摘要:
      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?
英文摘要:
      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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