稳定表达红色荧光蛋白的人胚胎干细胞系的建立  

Establishment of human embryonic stem cell line stably expressing red fluorescent protein

作　　者：胡菲菲[1] 张纬[1] 欧阳琦[2] 王荣根[1] 李泽[1] 王冠[1] 王盈[1] 杨海元[1] 戴一凡[1] 

机构地区：[1]南京医科大学江苏省异种移植重点实验室,江苏南京211166 [2]中南大学生殖与干细胞工程研究所,湖南长沙410078

出　　处：《南京医科大学学报（自然科学版）》2016年第7期868-873,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：江苏省异种移植重点实验室科研经费(BM2012116)

摘　　要：目的 :建立可以稳定表达红色荧光蛋白(red fluorescent protein,RFP)的人胚胎干细胞(human embryonic stem cells,h ESCs)系。方法:优化核转染方法后,将RFP表达载体p EF1α-Ds Red-Express2导入h ESCs中。利用碱性磷酸酶染色、RT-PCR、免疫荧光染色和类胚体形等方法,比较核转染前后h ESCs的干细胞特性。结果:核转染后h ESCs的碱性磷酸酶染色结果为阳性。核转染前后h ESCs的Oct4、Sox2、Klf4、Nanog基因的表达未出现明显差异。RFP-h ESCs具有三胚层分化潜能,并且可稳定传30代以上。结论:成功建立稳定表达红色荧光蛋白的h ESCs,干细胞特性不受RFP的影响,可用于后续的实验研究。Objective：To establish human embryonic stem cell（hESC）lines labeled with red fluorescent protein（RFP）. Methods： The RFP expression vector pEFlcL-DsRed-Express2 was transfected into hESCs by optimized nucleofection method. Alkaline phosphatase （AP） staining,RT-PCR,immunofluorescent staining and embryoid body（EB） formation were performed to compare the characteristics of hESCs before and after nucleofection. Results：AP staining of RFP-hESCs was positive. The expression of Oct4, Sox2,Klf4 and Nanog of hESCs were no difference before and after nucleofection （P 〉 0.05）. RFP-hESCs could differentiate into the three germ layers and could stably passage over 30 generations. Conclusion：hESC cell lines stably expressing RFP has been successfully established in this study. RFP does not affect the pluripotency properties of hESCs. The cell lines can be used as seed cells for subsequent research.

关 键 词：人胚胎干细胞 核转染 红色荧光蛋白 类胚体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]