大鼠胚胎肠神经干细胞体外培养的时间选择及鉴定  

Timing of in vitro culturing and identification of rat embryo neural stem cells

作　　者：陈东[1] 弭杰[1] 付冬辉 高红[1] 

机构地区：[1]中国医科大学附属盛京医院小儿外科,沈阳110004 [2]中国医科大学附属盛京医院泌尿外科,沈阳110004

出　　处：《中华小儿外科杂志》2015年第3期226-229,共4页Chinese Journal of Pediatric Surgery

基　　金：沈阳市科学技术计划资助项目（F13-316-1-01）

摘　　要：目的 探讨大鼠胚胎肠神经干细胞（ENSCs）体外培养时间的选择.方法 在严格的条件控制下,从盛京医院SPF级动物实验室筛选的VSD大鼠的胎鼠和幼鼠作为研究对象,分别取孕16、18、20 d的胎鼠和生后1d的幼鼠,每组各5只.以其肠管做原代培养,在碱性成纤维生长因子（bFGF）、N2添加剂和B27添加剂联合作用下使其稳定增殖,观察其神经球形成的时间及生长速度,并用10％的胎牛血清诱导其贴壁分化,应用SABC法免疫细胞化学染色检测巢蛋白（Nestin）、胶质纤维酸性蛋白（GFAP）、髓磷脂碱蛋白（MBP）和神经微丝200 （NF-200）的表达,对ENSCs及其分化的细胞进行鉴定.结果 孕16d和孕18 d提取的ENSCs于原代培养后第3天形成肠神经球,孕20 d和生后1d提取的ENSCs于原代培养后第5天形成肠神经球.形成的神经球可以进行传代,鉴定为Nestin染色阳性细胞,并可诱导分化为神经元细胞（NF-200染色阳性细胞）、星形胶质细胞（GFAP染色阳性细胞）和少突胶质细胞（MBP染色阳性细胞）,说明所培养的细胞为肠神经干细胞.结论 采用无血清培养基加入特定生长因子的培养技术,可培养出在体外稳定增殖并有多向分化潜能的大鼠胚胎ENSCs;对于ENSCs体外培养取材时机,选择孕18 d大鼠胚胎为宜.Objective To explore the timing of culturing in vitro enteric neural stem cells （ENSCs） from embryonic rats.Methods The ENSCs were derived from the intestinal tissue of rat embryo at the fetal ages of 16,18,20 days and postnatal 1 day.In strictly controlled conditions,fetal and neonatal rats （n =5 each） were screened from specific-pathogen free （SPF） animal laboratory of our hospital.The intestine was in vitro cultured with microsurgical device in basic fibroblast growth factor （bFG-F） and N2 plus B27 action.Under stable proliferation,time and growth rate of neural sphere were recorded.And 10% fetal bovine serum was used for inducing adherent differentiation and SABC immunohistochemical staining for detecting the expressions of nestin,glial fibrillary acidic protein （GFAP）,myelin basic protein （MBP） and neurofilament 200 （NF-200）.ENSCs and differentiated cells were identified.Results At gestational days 16 and 18,ENSCs formed enteric nervous ball after a 3-day primary culture.And at Day 29 and postnatal 1 day,there was a formation of intestinal neurosphere after a 5-day primary culture.Formed neurospheres,identified as nestin positive cells,could be passaged and differentiated into neurons （nestin positive ＆amp; NF-200 staining）,astrocytes （GFAP positive） and oligodendrocytes （MBP positive）.Thus it suggested that cultured cells were ENSCs.Conclusions Serum-free culture medium plus certain growth factors may cultivate an in vitro proliferation of embryonic rat ENSCs with potential multi-directional differentiation.And pregnant 18 day rat embryo is ideal for culturing ENSCs in vitro.

关 键 词：干细胞 肠神经系统 细胞分化 大鼠 

分 类 号：R329[医药卫生—人体解剖和组织胚胎学]