六个转录因子将人脐带间充质干细胞高效重编程为诱导性多能干细胞的实验研究  

Induced pluripotent stem cells can be reprogrammed from human umbilical cord mesenchymal cells by six transcription factors

作　　者：杨汉华[1] 陈元国[1] 赖秀蓝 谢丽春[1] 郑泽鑫[1] 蒋学武 马廉 

机构地区：[1]汕头大学医学院第二附属医院儿科,515041 [2]转化医学研究中心

出　　处：《中华实用儿科临床杂志》2014年第17期1331-1336,共6页Chinese Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金（31040057,81070478）

摘　　要：目的建立人脐带间充质干细胞（HuMSCs）来源的诱导性多能干细胞（iPSC）系。方法以慢病毒载体将OCT4、SOX2、KLF4、c—Myc、NANOG、LIN-286个转录因子转入HuMSCs使其重编程为iPSC。通过形态学观察、多能细胞特异性标志检测、碱性磷酸酶（AKP）染色、核型分析、拟胚体形成、体内成瘤实验鉴定获得的iPSC。结果慢病毒感染后第4天，HuMSCs逐渐变成类圆形，第10天开始出现不规则细胞团，第14天出现较大的细胞团，约1．25％的细胞重编程为iPSC。建系后iPSC呈典型的克隆状生长，边缘清晰，内部细胞细小，排列紧密；AKP染色阳性；表达多能性相关基因及胚胎干细胞特异性蛋白；细胞核型正常（46，XY）；体内外均能向3个胚层方向分化。结论慢病毒携带OCT4、SOX2、KLF4、c-Myc、NANOG、LIN-286个因子能将HuMSCs高效诱导为完全重编程的iPSC。Objective To establish and identify the induced pluripotent stem cell（iPSC） line reprogrammed from human umbilical cord mesenchymal cells（HuMSCs）. Methods HuMSCs were cultured by adhesion method,and OCT4, SOX2,KLF4, c-Myc, NANOG, LIN-28 were transfected into HuMSCs with lentiviral victor to reprogramme HuMSCs into iPSC. Morphological observation, pluripotency genes （ SOX2, TDGF1, THY-1, OCT4, REX1 and TERF1 ） expression, alkaline phosphatase detection, karyotype analysis, embryonic stem ceils（ESC） specific proteins（ NANOG, OCT4, SSEA-4, TRA-1-81 ） immunofluorescence staining, differentiated into teratomas in vivo （ inject the iPSC into SCID mice ） and embryniod bodies in vitro were performed to exam the pluripotency of the iPSC. Results Four days after being infected by lentivirus,the HuMSCs became round-shape; 10 days after infection, some embryonic stem （ESC）-like colonies appeared. Fourteen days after infection, picked up the regularly shaped colonies and cultured several passages. About 1.25% HuMSCs were reprogrammed into iPSC. The iPSC presented clone-like growth like ESC. All the cells were positive to alkaline phosphatase staining and expressed the pluripotency genes. The iPSC also expressed the ESC specific proteins, and karyotype analysis showed normal chromosome caryotype （46, XY）. Furthermore, the iPSC could form embryoid bodies in vitro, expressed alpha fetoprotein （ AFP）, smooth muscle actin （ SMA ） and β-tubulin. The iPSC could alsoform teratomas in vivo. Conclusion OCT4, SOX2, KLF4, c-Myc, NANOG, LIN-28 can reprogram HuMSCs into iPSC efficiently.

关 键 词：脐带间充质干细胞 诱导性多能干细胞 转录因子 慢病毒载体 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]