Page 57 - 《南京医科大学学报》自然科学版2026年第2期

第46卷第2期 南京医科大学学报（自然科学版）
2026年2月 Journal of Nanjing Medical University（Natural Sciences） ·213 ·

·基础研究·

微孔板成像系统在高表达荧光蛋白单克隆细胞株筛选中的应用

沈链链 ，管强东 ，丁竞竞 ，熊建平 ，王 丽 ，吴 炜 1，3，4*
2
1
1
1
1
南京医科大学公共卫生学院科研共享平台，医学教育研究所，生殖医学与子代健康全国重点实验室，现代毒理学教育部重
1 2 3 4
点实验室，江苏 南京 211166
［摘 要］ 目的：建立一种基于微孔板成像系统筛选稳定转染后高表达荧光蛋白的单克隆细胞株的筛选流程。方法：利用慢
病毒将绿色荧光蛋白（green fluorescent protein，GFP）基因转至人支气管上皮细胞（human bronchial epithelial cell，HBE），通过流
式细胞术分选荧光信号强的单个细胞入96孔板。按照所建立的筛选流程，通过4轮基于微孔板成像系统的成像与分析功能进
行逐步筛选：分选当天确认含单细胞的孔；待大多数克隆团细胞数>20个时，筛选出可形成克隆团的孔；将筛选孔中的细胞消
化并转移至新孔后，进一步筛选出细胞平均荧光强度较高的孔；待生长最快的细胞增殖4~8倍时，筛选出细胞正常增殖的孔。
稳定转染细胞持续培养，培养条件同单克隆细胞。待单克隆细胞增殖至数量足够，利用流式细胞术与荧光成像比较最终筛选
所得单克隆细胞株与稳定转染细胞在荧光强度方面的差异。结果：依照筛选流程，依次筛选得到89个含单细胞的孔，19个可
形成克隆团的孔，6个细胞平均荧光强度较高的孔，以及3个细胞正常增殖的孔。流式细胞术结果显示：与稳定转染细胞相比，
来源于这3个孔的单克隆细胞株的平均荧光强度提高了近9倍，GFP荧光信号强的细胞占比也显著提高。微孔板成像结果亦
显示单克隆细胞株的GFP荧光强度明显高于稳定转染细胞。结论：应用微孔板成像系统，结合本研究所建立的筛选流程，可逐
步、高效地筛选出稳定转染后高表达荧光蛋白的单克隆细胞株。相比传统的显微镜观察或其他成像方法，该方法操作简单、
方便，具备良好的推广应用价值。
［关键词］ 单克隆细胞株；荧光蛋白；微孔板成像系统；筛选
［中图分类号］ Q503 ［文献标志码］ A ［文章编号］ 1007⁃4368（2026）02⁃213⁃08
doi：10.7655/NYDXBNSN250751

Application of a microplate imaging system in screening monoclonal cell strains with high
fluorescent protein expression
1 1 2 1 1 1，3，4*
SHEN Lianlian ，GUAN Qiangdong ，DING Jingjing ，XIONG Jianping ，WANG Li ，WU Wei
Research Sharing Platform，School of Public Health，Institute of Medical Education Research，State Key Laboratory
1 2 3
of Reproductive Medicine and Offspring Health，Key Laboratory of Modern Toxicology of Ministry of Education，
4
Nanjing Medical University，Nanjing 211166，China

［Abstract］ Objective：To establish a workflow using a microplate imaging system for screening monoclonal cell strains with high
fluorescent protein expression after stable transfection. Methods：Human bronchial epithelial（HBE）cells were transfected with green
fluorescent protein（GFP）gene using a lentiviral vector. Single cells with strong fluorescence were sorted by flow cytometry into a 96⁃
well plate. According to the established screening protocol，four rounds of stepwise screening were performed using the imaging and
analysis functions of the microplate imaging system：on the day of sorting，wells containing a single cell were identified；when the
majority of clonal clusters contained more than 20 cells，wells with clonal clusters were selected；after trypsinization and transfer of
cells from the selected wells to new wells，wells with cells exhibiting higher mean fluorescence intensity were identified；when rapidly
proliferating cells expanded 4-8 fold，wells with cells exhibiting normal proliferation were selected. The stably transfected cells were
continuously maintained under the same culture conditions as the monoclonal cells. When the monoclonal cells proliferated to an
adequate number，flow cytometry and fluorescence imaging were used to compare the fluorescence intensity between the finally
selected monoclonal cell strains and the stably transfected cells. Results：Following the screening protocol，89 wells containing a single

［基金项目］ 国家科技重大专项（2023ZD0507401）；江苏省大型科学仪器开放共享自主研究课题（TC2023A024）
通信作者（Corresponding author），E⁃mail：wwu@njmu.edu.cn（ORCID：0000⁃0002⁃0267⁃1284）
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