转EGFP基因猪胎儿神经干细胞的体外分化  

In vitro Differentiation of EGFP Gene Transfected Porcine Fetal Neural Stem Cells

作　　者：郑月茂[1,2] 赵雪[1,2] 贺小英[1,2] 权富生[1,2] 刘军[1,2] 张涌[1,2] 

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]西北农林科技大学生物工程研究所,陕西杨凌712100

出　　处：《中国农业科学》2009年第9期3305-3313,共9页Scientia Agricultura Sinica

基　　金：国家"863"计划(2004AA213072);西北农林科技大学科研专项基金(07ZR001)

摘　　要：【目的】获得转EGFP基因神经干细胞并监测其自我更新、增殖和多向分化潜能;通过用EGFP对神经干细胞进行标记和体外追踪实验,为EGFP作为示踪标记对神经干细胞进行体内移植研究奠定基础。【方法】利用神经干细胞培养技术体系,从胎龄30d猪胎儿脑组织中分离培养神经干细胞,通过脂质体介导转染技术,将EGFP基因导入神经干细胞,诱导转基因神经干细胞贴壁分化,观察其体外增殖、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】成功分离培养出神经干细胞,获得转EGFP基因神经干细胞,神经干细胞在表达EGFP的同时仍具有多向分化潜能。神经干细胞中Nestin表达强阳性,NogoA、DCX、CyclinD2、CD133、Hes1、Oct4、CD-90、Nanog和Sox2表达阳性;体外诱导的神经干细胞可以分化为星形胶质细胞(表达GFAP)、少突胶质细胞(表达GalC)和神经元细胞(表达NF、NSE和MAP2);能分化为脂肪细胞(表达LPL和PPARγ-D)、成骨细胞(表达Osteonectin和Osteocalcin)、肌细胞(表达myf-5、myf-6和myoD)、内皮细胞(表达CD31、CD34、CD144和eNOS)和软骨细胞(表达COL2A1)。【结论】从猪胎儿大脑组织分离神经干细胞具有可行性和有效性,转EGFP基因神经干细胞具有自我更新、增殖和多向分化潜能,可以用EGFP对神经干细胞进行标记、追踪,作为示踪标记进行神经干细胞体内移植研究。Objective Despite the increasing importance of the pig as a large animal model,little is known about the porcine neural stem cells（NSCs）.Method To evaluate the markers expressed by NSCs,expression of EGFP by the cells and in vitro differentiation of the cells,brains were dissected from 30-day fetuses,enzymatically dissociated,and grown in the presence of epidermal growth factor,basic fibroblast growth factor etc.Porcine NSCs could be grown as suspended spheres.Plasmid containing EGFP gene was transfected into NSCs by lipofection and selected by G418. The positive NSCs were abtained. NSCs were induced to differentiate into cell types representing each embryonic germ layer, including cells of adipogenic, osteogenic, myogenic, endothelial, neuronal and chondrogenic lineages. [Result] Expanded NSCs and differentiated cells were bankedor harvested for analysis using reverse transcription-polymerase chain reaction （RT-PCR）. Cultured porcine NSCs widely expressed Nestin, NogoA, DCX, CyclinD2, CD133, Hes1, Oct4, CD-90, Nanog and Sox2. NSCs were differentiated into astrocyte （GFAP^＋）, oligodendrocyte （GalC^＋）, neuron （NF^＋. NSE^＋ and MAP2^＋）, adipocyte （LPL^＋ and PPARy-D^＋）, osteoblast （Osteonectin^＋ and Osteocalcin^＋）, myocyte （myf-5^＋, myf-6^＋ andmyoD^＋）, endothelium （CD31^＋, CD34^＋, CD144^＋ and eNOS^＋） and chondrogenic cells （COL2A1^＋）. [Conclusion] This study shows that porcine NSCs are broadly multipotent, thus, may be useful in porcine cell transplantation studies potentially leading to the application of this strategy in the setting of nervous system disease and injury.

关 键 词：神经干细胞 EGFP基因 细胞标记 细胞分化 猪胎儿 

分 类 号：Q78[生物学—分子生物学]