慢病毒介导绿色荧光蛋白转染人胚胎干细胞及其培养  

Lentiviral vectors mediates GFP transfection and human embryonic stem cell culture

作　　者：李宁[1,2] 朱宝长[2] 朱宛宛[1,3] 王淑艳[1,3] 任萍[1,3] 关云谦[1,3] 张愚[1,3] 

机构地区：[1]首都医科大学宣武医院老年病研究所细胞治疗室 [2]首都师范大学生命科学学院,北京100037 [3]教育部神经变性病重点实验室,北京100053

出　　处：《基础医学与临床》2008年第10期1083-1087,共5页Basic and Clinical Medicine

基　　金：国家自然科学基金(30400148);国家973项目(2006CB0F0603);北京市科委科技计划(H020220010290)

摘　　要：目的稳定培养人胚胎干细胞,并通过慢病毒载体对其进行绿色荧光蛋白标记。方法利用小鼠胚胎成纤维细胞作为饲养层或Matrigel作为基质培养人胚胎干细胞,包装带有GFP序列的慢病毒转染人胚胎干细胞。对转染前后的人胚胎干细胞进行了碱性磷酸酶和SSEA-3免疫组化鉴定。结果在MEF饲养层和Matrigel上均可培养出呈克隆样生长,表达标志抗原的人胚胎干细胞,经慢病毒转染及抗生素筛选后仍可稳定表达GFP。结论成功地培养了人胚胎干细胞系,并进行了GFP标记。Objective To establish a culture system for human embryonic stem cells and to label hES cells with GFP by lentivirus. Methods We cultured the human ES cells in the feeders or matrigel. Furthermore, we characterized the undifferentiated hES by their ability to form compact clones positive for stage specific embryonic antigen-3 and surface alkaline phosphatase staining. Results Human ES cells cultured both on feeder cells and in matirgel ex- pressed specific surface markers like SSEA-3 and alkaline phosphatase. After transfection by lentivirus and hygromycin selection, green fluorescent protein was strongly expressed in human ES cells. Conclusion We succeed in human ES cell culture with feeder cells and in matrigel. Got human ES cells expressing GFP continuously.

关 键 词：人胚胎干细胞 绿色荧光蛋白 慢病毒 

分 类 号：R346[医药卫生—基础医学]