人脐血及脐带间充质干细胞的分离培养及表面标记分析

目的探讨从人脐带血及脐带分离和培养间充质干细胞(MSCs)的方法,并分析MSCs的表面标记。方法人脐带血按常规方法制备单个核细胞,利用MSCs贴壁生长的特性,经培养、换液、传代纯化MSCs;分离脐带华尔通胶(Wharton’s jelly),采用组织块贴壁法获得脐带MSCs并传代。将传代的MSCs冻存,1个月后再复苏,观察复苏后MSCs的生长情况。利用FACScan流式细胞仪检测脐带血及脐带细胞表面抗原。结果经过传代后,贴壁细胞形态趋于同一。人脐血及脐带MSCs体外生长形态相似,类似成纤维细胞,可以稳定增殖和传代。经冷冻保存,复苏后仍能较好生长。人脐血及脐带来源的MSCs表面标记CD29、CD44、CD59高表达,而表面标记CD14、CD33、CD34和CD45低表达。结论人脐带血及脐带均可分离出MSCs,在体外能扩增纯化及冻存复苏,为组织工程提供丰富的细胞来源。

Isolation,culture and detection of molecular marker of mesenchymal stem cells from human umbilical cord blood and umbilical cord

Objective To investigate the isolation,purification,expansion,cryopreservation and resuscitation of mesenchymal stem cells(MSCs)from human umbilical cord blood(UCB)and umbilical cord,and identify molecular markers of MSCs.Methods Normal fresh human UCB cells were cultured in Dulbecco’s modified Eagle’s medium(DMEM)containing 10% fetal bovine serum(FBS)and MSCs were separated by their adherence to plastic.Umbilical cord was cut into little tissue block and the tissue attachment culture method was used to obtained MSCs.All these MSCs were passed 3 times,then cryopreserved.The frozen cells were resuscitated after one month.Surface markers of MSCs were identified by flow cytometry.Results After several passages,the adherent cells became more uniformly spindle-shaped in appearance.The shapes of two kinds of cells were same,they were processed to obtain fibroblast-like cells,and they were expanded as undifferentiated cells in culture for more than 10 passages,indicating their proliferative capacity.The frozen cells were resuscitated successfully.The UCB and umbilical cord derived MSCs highly express CD29、CD44、CD59 but weakly express CD14,CD33,CD34 and CD45.Conclusions MSCs can be obtained from UCB and umbilical cord.They maintained good self-renewal capacity and their cryopreservation and resuscitation could meet the need of tissue engineering.