妊娠中期猪羊水来源干细胞EGFP基因转化及体外分化  

EGFP gene transfection and in vitro differentiation of amniotic fluid-derived stem cells from pig at intermediate gestational age

作　　者：郑月茂[1] 党永辉[2] 贺小英[1] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]西安交通大学医学院,陕西西安710049

出　　处：《中国兽医学报》2010年第4期540-543,共4页Chinese Journal of Veterinary Science

基　　金：教育部留学回国人员科研启动基金(K314020901);西北农林科技大学科研专项基金资助(07ZR001)

摘　　要：本研究旨在获得妊娠中期猪羊水来源干细胞,并通过用EGFP对干细胞进行标记,为以EGFP作为示踪标记对干细胞进行体内移植研究奠定基础。利用羊水来源干细胞培养技术体系,从胎龄60 d猪胎儿羊水中分离获得干细胞,通过脂质体介导转染将EGFP基因导入干细胞,诱导转基因干细胞向肌细胞和神经细胞分化,观察其分化特点。采用RT-PCR技术检测干细胞和分化细胞表面标志或相关基因。结果成功分离培养出妊娠中期猪羊水来源干细胞,并获得转EGFP基因干细胞,干细胞在表达EGFP的同时仍具有分化潜能。干细胞中Oct4、CD-90和Sox2表达阳性;体外诱导的干细胞能分化为肌细胞(表达myf-6和myoD)、星形胶质细胞(表达GFAP)、少突胶质细胞(表达GalC)和神经元细胞(表达NF、NSE和MAP2)。研究表明,从妊娠中期猪胎儿羊水中分离干细胞具有可行性和有效性,转EGFP基因干细胞具有自我更新、增殖和分化潜能,可以用EGFP对羊水来源干细胞进行标记、追踪,为EGFP作为示踪标记对干细胞用于体内移植研究奠定了基础。The aim of this study was to get amniotic fluid-derived stem（AFS） cells from pig at intermediate gestational age,detect their capability of self-renew and differentiation in vitro,and approve wether or not EGFP can be used as a marker in monitoring AFS cells.The AFS cells were isolated from amniotic fluid of a pig at intermediate gestational age.Plasmid containing EGFP gene was transfected into the AFS cells by lipofection.The EGFP gene transfected stem cells were induced to differentiate into cells of myogenic and neuronal lineages.The expanded AFS cells and differentiated cells were harvested for analysis of markers using reverse transcription-polymerase chain reaction（RT-PCR）.The results showed that the EGFP gene transfected AFS cells were obtained from pig at intermediate gestational age.The AFS cells expressed Oct4,CD-90 and Sox2.The positive AFS cells could differentiate into myocyte（myf-6^＋ and myoD＋）.They could also differentiated into astrocyte（GFAP＋）,oligodendrocyte（GalC^＋） and neuron（NF^＋,NSE^＋ and MAP2^＋）.We have demonstrated that AFS cells could be isolated from amniotic fluid of pig at intermediate gestational age,the EGFP gene transfected AFS cells were capable of self-renewal,broadly multipotent,and EGFP can be used as a marker in monitoring AFS cells.

关 键 词：羊水来源干细胞 EGFP基因 细胞分化 妊娠中期猪 

分 类 号：S828[农业科学—畜牧学]