摘要
目的探讨人脐血单个核细胞(UBC-MNCs)体外定向诱导分化为神经干细胞(NSCs)的可行性,观察NSCs中Nestin基因mRNA的表达情况。方法采用密度梯度离心法从人脐血中分离出UBC-MNCs,用含人表皮细胞生长因子(hEGF)、碱性成纤维细胞生长因子(bFGF)和B27因子的Neurobasal培养基联合诱导其向NSCs方向分化,观察NSCs增殖分化及形态学特点;免疫组织化学法检测培养细胞中神经细胞标志抗原巢蛋白Nestin、神经元特异性烯醇化酶(NSE)、胶质纤维酸性蛋白(GFAP)的表达情况;RT-PCR法检测诱导前后培养细胞中NestinmRNA的表达变化。结果 UBC-MNCs可定向诱导分化为神经元样细胞,形成典型NSCs克隆团,表达Nestin、NSE、GFAP标记抗原蛋白,诱导前细胞Nestin、NSE、GFAP的表达均非常低,诱导6d时Nestin阳性细胞数达高峰,然后开始下降,NSE、GFAP阳性细胞数逐渐增高。Brdu标记靶细胞阳性率达90%。Nestin mRNA在诱导前细胞中低表达,诱导3d、6d、9d、12d时表达逐渐升高(P<0.05)。结论体外诱导培养可获得UBC-MNCs源性NSCs,Nestin基因是NSCs增殖分化的关键调控因子,在UBC-MNCs诱导分化后表达明显增强。脐血能够成为NSCs的新来源。
Objective To explore the feasibility of human umbilical cord blood mononuclear cells（UBC-MNCs）induced and diffe-rentiated into neural stem cells（NSCs）,and to observe the expression of Nestin gene mRNA that appeared on NSCs.Methods The UBC-MNCs were isolated from human umbilical cord blood with density gradient centrifugation method,cultured in Neurobasal medium added with hEGF,bFGF and B27 factures in vitro,and the UBC-MNCs were induced and differentiated into NSCs.The characteristics of NSCs morphology and prolifferation and differentiation were observed.Cultured cells specific neuron antigen markers of Nestin,NSE,GFAP were detected by immunohistochemistry.The cultured cell,expression of Nestin gene mRNA were performed by reverse transcription polymerase chain reaction（RT-PCR）.Results The UBC-MNCs could be differentiated into neuron-like cells after oriented induction,and cultured cells could grow into typical NSCs cloning balls,expressed labelled antigens of Nestin,NSE and GFAP.Before inducion,cells expression of Nestin,NSE,GFAP were very low,but Nestin positive cells came to its peak on the sixth day after induction,then began to decline,NSE and GFAP positive cells gradually increased after induced.Positive Brdu labeled target cells were 90%.The expression of Nestin gene mRNA was low before inducted,then increased gradually after induction（P0.05）.Conclusions NSCs from UBC-MNCs can be obtained,which is induced and cultured in vitro.Nestin gene is the key proliferation and differentiation regulation factor of NSCs,as it significantly increases after induction and differentiation.Human cord blood can be the new source of NSCs.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2010年第19期1503-1505,共3页
Journal of Applied Clinical Pediatrics
基金
河南省医学科技攻关计划项目(200703066)
关键词
NESTIN
脐血
神经干细胞
诱导分化
脑性瘫痪
Nestin
umbilical cord blood
neural stem cells
induction and differentiation
cerebral palsy
