建立稳定高效表达绿色荧光蛋白和小鼠白血病抑制因子的STO细胞系  

Establishment of STO Cell Lines Expressing Green Fluorescent Protein and Mouse Leukemia Inhibitory Factor

作　　者：刘传苗[1] 李红俊[2] 杨天华 杨小淮[3] 李正红[2] 李永海[2] 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;Department of Urinary Surgery,the First Affiliated Hospital of Bengbu Medical College,Bengbu 233030,Anhui Province,China)

机构地区：[1]蚌埠医学院第一附属医院感染科,安徽蚌埠233004 [2]蚌埠医学院病理生理实验室,安徽蚌埠233030 [3]蚌埠医学院第一附属医院泌尿外科,安徽蚌埠233004

出　　处：《中国实验血液学杂志》2019年第2期606-612,共7页Journal of Experimental Hematology

基　　金：国家自然科学基金(81472656);安徽省教育厅基金项目(NO.KJ2017A247);安徽省教育厅自然科学基金一般项目(KJ2015B059by);蚌埠医学院研究生科研创新项目(Byycx1605)

摘　　要：目的:建立表达绿色荧光蛋白(green fluorescent protein,GFP)和小鼠白血病抑制因子(leukemia inhibitory factor,LIF)的细胞系,并尝试使用其作为饲养层培养小鼠胚胎干细胞(ESC)。方法:构建含有GFP、mLIF和嘌呤霉素抗性基因的慢病毒粒子,并感染STO小鼠成纤维细胞系,筛选得到稳定表达GFP和mLIF基因的细胞系(STO-GFP-mLIF);通过Western blot和ELISA方法检测STO-GFP-mLIF细胞中LIF基因的表达水平;经不同浓度(5,10,15,20μg/ml)丝裂霉素C处理不同时间(1.5,2.5,3,3.5 h)制备饲养层,并观察细胞在饲养层上增殖情况。将小鼠胚胎干细胞在丝裂霉素C处理过的STO-GFP-mLIF上培养,观察细胞集落生长情况。结果:STO-GFP-mLIF细胞中LIF蛋白表达水平上调(P <0.01);丝裂霉素C抑制STO-GFP-mLIF细胞增殖的最佳浓度及作用时间为10μg/ml与3 h,且小鼠胚胎干细胞可在该饲养层上发育成典型的"鸟巢"状干细胞集落。结论:成功获得了稳定表达绿色荧光蛋白和小鼠LIF基因,同时可维持小鼠胚胎干细胞未分化状态的STO-GFP-mLIF细胞系。Objective: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-GFPmLIF cells as the feeder layer.Methods: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 STOGFP-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.Results: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.Conclusion: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.

关 键 词：白血病抑制因子 绿色荧光蛋白 STO细胞系 小鼠ES细胞 

分 类 号：R329.28[医药卫生—人体解剖和组织胚胎学]