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标志物mRNA的表达,并观察其致瘤性。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方法鉴定诱导前后神经干细胞(NSCs)标志物巢蛋白(Nestin)及Musashi-1蛋白mRNA的表达;收集培养后7d的MSCs,接种于裸鼠皮下,观察是否有增生物的出现,并观察细胞组织形态学变化。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和CD11b,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(P<0.05),48h达高峰,然后逐渐减弱;细胞接种后12周,在裸鼠皮下不能形成肿瘤,与对照组相比,细胞形态学未出现恶性变化。结论:脐血中存在MSCs,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。在裸鼠体内不具有成瘤性。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, and to detecte the securily by tumorigenic assay. Methods： Isolated mononuclear cells（MNCs） from heparinezed human cord blood（50-100 ml）samples by centrifugation over lymphoprep（density 1.077/ml） density gradient and then adherently cultivated to obtain MSCs clonies.Observed the shape by microscope and examined the phenotypes of the cells by flow cytometry,then induced with NGF and RA in vitro. Reverse transcriptase polymerase chain reaction （RT-PCR）for nestin and musashi-1 antigen were performed to confirm nestln and musashi-1 antigen expression in MSCs.Then tumorigenic assay was performed in nude mice. Results： After adherent cultivation, most cells became rhombic, and differentiated into neuron-like cells after inductlon,expressing CD29 but no CD34,CDlla and CDllb.RT-PCR demonstrated small amount of nestin expressed in MNCs but no musashi-1 antigen before induction,and both shew the strongest expression in 48 h,then faded away gradually.The cells were unable to be tumorigenic in nude mice. 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,and to be nontumorigenic in nude mlce.Human cord blood can be the new source of neural stem cells.
出处
《中国医药导报》
CAS
2007年第09Z期14-16,22,共4页
China Medical Herald
基金
重庆市科委攻关课题(7830)
