脐血单个核细胞体外分离和定向分化为神经干细胞的实验研究  

Isolation and characterization of neural stem cells from human cord blood cells

作　　者：向静[1] 王昌铭[1] 王景周[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所神经内科,重庆400042

出　　处：《重庆医学》2007年第13期1246-1248,1250,共4页Chongqing medicine

基　　金：重庆市科委攻关课题(7830)

摘　　要：目的探讨人脐血单个核细胞的分离培养和诱导后神经干细胞(NSCs)标志物mRNA的表达。方法使用密度梯度离心法得到脐血单个核细胞,用体外贴壁生长的方法获得脐血间质干细胞(MSCs),观察培养过程中脐血MSCs形态的变化,通过流式细胞仪检测干细胞表面抗原的表达,使用NGF和RA联合诱导后,用RT-PCR方法鉴定诱导前后NSCs标志物巢蛋白(nestin)及Musashi-1蛋白mRNA的表达。结果脐血单个核细胞贴壁生长后大部分细胞呈梭形,诱导后可分化为神经元样细胞。流式细胞仪检测该细胞不表达CD34、CD11a、CD11b和强表达CD29,符合MSCs特征。诱导后,NSCs表面标志Nestin及Musashi-1表达明显增强,48h达高峰,然后逐渐减弱。结论脐血中存在间质干细胞,分离后可以体外扩增,诱导后分化为神经元样细胞,并表达NSCs表面标志。脐血能够成为NSCs的新来源。Objective To investigate the isolation and cultivation of human cord blood menchymal cells（MSCs） and the expression of NSCs marker under induction. Methods Mononuclear cells（MNCs） were isolated from heparinezed human cord blood（50- 100ml）samples by centrifugation over lymphoprep（density 1. 077/ml）density gradient and then adherent cultivated to obtain MSCs clonies. To observe the shape by microscope and to examine the pbenotypes of the cells by flow cytometry,tben induced with NGF and RA in vitro. Then reverse transcriptase polymerase chain reaction（RT-PCR）for nestin and musasbi-1 antigen were performed to confirm nestin and musashi-1 antigen expression in MSCs. Results After adherent cultivation,most cells became rhombic,and differentiated into neuron-like cells after induction,expressing CD29 but no CD34,CD 11 a and CD 11 b. RT-PCR demonstrated small amount of nestin expressed in MNCs but no Musasbi-1 antigen before induction,and both showed the strongest expression in 48b, then faded away gradually. Conclusion MSCs can be isolated from human cord blood,and efficiently expanded under this culture condition,and differentiated into neuron-like cells after induction, expressing neural marker. Human cord blood can be the new source of neural stem cells.

关 键 词：脐血单个核细胞 神经干细胞:诱导分化:巢蛋白 MUSASHI-1 

分 类 号：Q813[生物学—生物工程]