脐血混合培养纯化人间充质干细胞及生物学特性研究

目的探讨体外分离、混合培养纯化人脐血间充质干细胞的适宜体系,观察该人脐血间充质干细胞生物学特性。方法收集孕足月新生儿脐血,每3份脐血[(70—100)ml/份]混合;采用1.077 g/ml的淋巴细胞分离液(Ficoll)以1 500 r/min密度梯度离心法分离脐血中的单个核细胞,在Mesencult培养基中贴壁培养筛选人脐血间充质干细胞,显微镜下观察细胞生长形态变化,细胞计数并绘制细胞生长曲线,流式细胞仪检测细胞抗原表达,分析细胞周期;体外诱导脐血间充质干细胞成脂、成骨分化。结果密度梯度离心法所获得的单个核细胞,在培养基中培养约3—5 h开始出现贴壁生长,24 h后贴壁细胞增多,呈明显纺锤状,10 d后开始出现细胞克隆,3周后呈漩涡状生长。原代培养时间为15 d,P1代倍增时间为26 h。流式细胞仪鉴定:贴壁生长的细胞表达CD29、CD44和CD105,不表达CD34、CD45。分化潜能鉴定体外培养细胞可以成脂、成骨分化。结论所贴壁培养出的细胞的生长形态、流式表形、分化潜能具有人脐血间充质干细胞生物学特征,混合培养可以提高间充质干细胞培养成功率,实验中采用的培养体系适宜人脐血间充质干细胞的分离培养。

Mixed culture of human umbilic cord blood mesenchymal stem cells and their biological characteristics

Objective To establish a feasible isolation /cultivation system for human umbilical cord blood mesenchymal stem cells(hUCBMSC),and to observe their biological characteristic.Methods Human umbilical cord blood(70—100ml) were collected and each of three umbilical cord blood were mixed.Mononuclear cells were isolated by density-gradient centrifugation,hUCBMSC was selected by attachment culture in Mesencult culture medium.Growth curve was portrayed following observation of growth pattern by microscope.Antigen expression and cell cycle were detected by flow cytometry.The potential for osteocyte formation or lipocyte formation of hUCBMSC was studied by ex-vivo induction.Results The primary cells can be procured by density gradient centrifugation.Adherence growth can be observed after 3—5 h cultured in Mesencult culture medium,more and more adherent cells appeared by 24 h,and cell clone appeared in 10 d.Primery culture took 15 d and the doubling time for P1 was 26 h.The cells with adherent growth expressed CD29,CD44,CD105,and didn't express CD34,CD45.Differentiation study showed that the cells could be induced into adipocytes or osteocytes.Conclusion Growth morphology,phenotype,potential for differentiation of the cells consist with the characteristics of MSCs.Mixed culture could improve the success rates of culture.The culture system is appropriate for hUCBMSC culture.