摘要
目的探索体外分离培养新生大鼠肠神经嵴干细胞(ENCSCs)的方法,观察肠神经嵴干细胞神经球(NLBs)的原代及分化情况。方法取新生SD大鼠,无菌环境下解剖分离从小肠至直肠的肠管,胰酶消化成单细胞悬液后,接种于培养瓶行原代培养,并传代;传到第3代时,加入分化培养基进行分化培养。观察ENCSCs原代培养及分化培养的情况,免疫荧光染色观察NLBs巢蛋白(nestin)的表达以及分化后ENCSCs Hu D蛋白、胶质纤维酸性蛋白(GFAP)的表达情况。结果 ENCSCs原代培养第1天,可见细胞贴壁成长,第2天,贴壁细胞减少,出现大量悬浮的细胞团,第4天,悬浮细胞团增大,形成体积稍小的NLBs,7 d后,大量NLBs漂浮于培养液中。未分化的NLBs行免疫荧光染色可见nestin阳性表达;NLBs分化后贴壁的细胞可见Hu D、GFAP阳性表达。结论新生大鼠肠道可提取出ENCSCs,培育出ENCSCs神经球,并能够分化成神经元和神经胶质细胞。
Objective To explore the method of ENCSCs＇ culture invitro,observe the primary culture cells and the differentiated cells.Methods Gut tissues of the neonatal rats within 24 h after birth were collected,digested into single cell suspension by trypsin,and transfered into culture bottles for primary cultures,subcultures and differentiation cultures at the 3rd generations.Observed and recorded the each period of cells,the expression of nestin in NLBs and the expression of Hu D protein,GFAP in ENCSCs after differentiation was examined through immunofluorescence（IF） staining.Results The primary cultures：few cells attached to the bottom on the first day;the adherent cells were decreased and small cell clusters suspension formed on the second day;the suspension cell clusters increased in size and formed little NLBs after 4 days;a large number of NLBs floating in the medium at 1 week.The immunofluorescence staining：positive expression of nestin in NLBs and positive expression of Hud and GFAP in differentiated cells.Conclusion The intestine of postnatal rats could be used to extract ENCSCs successfully,and generate NLBs in vitro,which could differentiate into neurons and glial cells.
出处
《遵义医学院学报》
2016年第3期314-318,共5页
Journal of Zunyi Medical University
基金
贵州省科科学技术基金资助项目(NO:黔科合J字[2012]2364)
关键词
肠神经嵴干细胞
细胞培养
细胞分化
enteric neural crest stemcells
cell culture
cell differentiation
