人脐带间充质干细胞的富集及向神经细胞的诱导分化

目的:寻找一种稳定、高效的分离人脐带间充质干细胞(MSCs)的方法,并探讨脐带MSCs向神经细胞方向分化的可能性。方法:分别采用组织块贴壁法和双酶消化法分离人脐带MSCs,对比其培养成功率;BrdU掺入实验检测脐带MSCs的增殖能力;流式细胞仪检测脐带MSCs表面分子标志;采用丹参联合生长因子的方法诱导其向神经细胞分化,免疫荧光方法检测神经元特异性烯醇化酶(NSE)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)的表达。结果:组织块贴壁法获得脐带MSCs成功率高;BrdU阳性标记率达90%以上;流式细胞仪检测显示细胞表达CD29、CD44和CD90,不表达CD34;脐带MSCs经诱导分化,伸出长突起,呈神经元样细胞改变,且表达神经元标志性蛋白NSE、MAP2。神经胶质细胞标志性蛋白GFAP表达较少。结论:成功建立了高效、稳定的脐带MSCs分离培养方法。脐带MSCs经诱导可向神经细胞方向分化,为临床移植治疗神经系统疾病提供了理想的细胞来源。

Enrichment and neural induction of mesenchymal stem cells from the human umbilical cord

Objective:To find a stable and efficient method for separating mesenchymal stem cells(MSCs) from human umbilica1cord,and explore their ability to differentiate into neural cells.Methods:MSCs were isolated from human umbilical cord with two methods:tissue adherent and double-enzyme digestion,and followed by comparing their success rate.The proliferative capacity was detected using BrdU incorporation method,and surface markers for MSCs were tested by flow cytometry.The third passage of MSCs were induced into neuron-like cells by danshen combined growth factor in vitro,then the expressions of neuron specific enolase(NSE),microtubule-associated protein 2(MAP2)and glial fiber acidic protein(GFAP) were detected by immunofluorescence techniques.Results:Tissue adherent method had higher ratio of obtaining MSCs than double digests method,the percentase of cells positive to BrdU was more than 90%.Flow cytometry analysis showed that MSCs expressed CD29,CD44 and CD90 strongly,but negative for CD34.The induced MSCs showed long processes and neuron-like morphology and they expressed MAP2,NSE.GFAP,the glial cell marker,was less expressed.Conclusion:We have established an efficient and steady method to obtain MSCs from human umbilica1 cord.MSCs derived from human umbilica1 cord could differentiate into neural cells which was an ideal source for clinical cell transplantation in treatment of nervous system diseases.