Liu Chuan-Miao, Li Hong-Jun, Yang Tian-Hua, Yang Xiao-Huai, Li Zheng-Hong, Li Yong-Hai
Department of Infectious Diseases, The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004, Anhui Province, China.
Department of Pathophysiology, Bengbu Medical College, Bengbu 233030, Anhui Province, China.
Zhongguo Shi Yan Xue Ye Xue Za Zhi. 2019 Apr;27(2):606-612. doi: 10.19746/j.cnki.issn1009-2137.2019.02.048.
To establish the STO cell lines expressing green fluorescent protein (GFP) and mouse leukemia inhibitory factor (LIF) , and try to culture the mouse embryonic stem cells (mESCs) by using the established STO-GFP-mLIF cells as the feeder layer.
The lentiviral particles containing GFP and mLIF and puromycin-resistance gene were constructed and transduced into STO cell lines. The cell lines stably expressing GFP and mLIF genes were screened out. The expression level of the inserted exogenous LIF gene was tested by Western blot and ELISA. The STO-GFP-mLIF cells were treated with different concentrations of mitomycin C (5, 10, 15, 20 µg/ml) for different time (1.5, 2.5, 3, 3.5 hours) to prepare feeder layers and the cell proliferation level on feeder layer was observed. Mouse embryonic stem cells were cultured on mitomycin C-treated feeder layer and the growth of cell colonies was observed.
The expression level of LIF protein in STO-GFP-mLIF cells was up-regulated, as compared with STO cells (P＜0.05). It was confirmed that the optimal concentration and time for inhibiting the proliferetion of STO-GFP-mLIF cells by mitomycin C were 10 µg/ml and 3 hours respectively. The observation also found that the embryonic stem cells could develop into typic "birdnest" shaped stem cell colony on mitomycin C-treated feeder layer.
The stable STO cell lines effectively expressing green fluorescent protein and mouse leukemia inhibitory factor have been established successfully, which can maintain the undifferentiated state of mouse embryonic stem cells.
建立表达绿色荧光蛋白（GFP）和小鼠白血病抑制因子（LIF）的STO细胞系，并尝试以建立的STO-GFP-mLIF细胞作为饲养层培养小鼠胚胎干细胞（mESCs）。
构建含有GFP、mLIF和嘌呤霉素抗性基因的慢病毒颗粒，并将其转导至STO细胞系中。筛选出稳定表达GFP和mLIF基因的细胞系。通过蛋白质免疫印迹法（Western blot）和酶联免疫吸附测定（ELISA）检测插入的外源性LIF基因的表达水平。用不同浓度（5、10、15、20μg/ml）的丝裂霉素C对STO-GFP-mLIF细胞处理不同时间（1.5、2.5、3、3.5小时）以制备饲养层，并观察饲养层上的细胞增殖水平。将小鼠胚胎干细胞培养在经丝裂霉素C处理的饲养层上，观察细胞集落的生长情况。
与STO细胞相比，STO-GFP-mLIF细胞中LIF蛋白的表达水平上调（P＜0.05）。证实丝裂霉素C抑制STO-GFP-mLIF细胞增殖的最佳浓度和时间分别为10μg/ml和3小时。观察还发现，胚胎干细胞在经丝裂霉素C处理的饲养层上可发育成典型的“鸟巢”状干细胞集落。
成功建立了有效表达绿色荧光蛋白和小鼠白血病抑制因子的稳定STO细胞系，该细胞系可维持小鼠胚胎干细胞的未分化状态。
