人脐血源和骨髓源间充质干细胞神经前体细胞分化的研究

目的 对比研究人脐血和骨髓来源的间充质干细胞(mesenchymal stem cells,MSCs)体外的分离、培养和生物学特性,并观察其分化潜能和形态学变化,为组织工程选取种子细胞提供实验依据.方法 Ficoll密度梯度离心结合贴壁培养法分别分离纯化成人骨髓和脐血源MSCs,体外培养和连续传代,并在含有2%B27的Neurobasal培养基中添加碱性成纤维细胞生长因子、表皮生长因子,将获得的MSCs向神经干细胞定向诱导,利用倒置显微镜连续观察细胞培养、传代和向神经细胞表型转化过程的形态学变化;采用免疫组化和荧光免疫组化法对诱导后细胞进行鉴定.结果 原代分离的骨髓间充质干细胞(BMSCs)在接种后48 h贴壁,7 d细胞呈长梭形,有一定的方向性,并达到90%融合;而脐血间充质干细胞(UMSCs)48 h后贴壁,似乎贴的不牢,持续14d才能形成小丛、小簇、小集落,21 d排列才有一定的方向性.培养基添加神经营养因子诱导后的细胞呈现典型的神经前体细胞样表型,免疫组化和免疫荧光结果显示,诱导后的细胞能特异性表达神经元特异性标志β微管蛋白(β-tubulin)和星形胶质细胞特异性标志神经胶质相关蛋白(GFAP).结论 人脐血和骨髓中含MSCs,且具备其基本恃征,体外培养UMSCs生长速度比BMSCs缓慢10 d～15 d左右,传代以后的各组细胞生长速度与形态无明显差异.骨髓和脐血来源的MSCs在体外可定向诱导分化为神经细胞.

Comparative characterization and differentiation to neurocytes of mesenchyeal stem cells from human umbilical cord blood and bone marrow

Objective To isolate and cultive mesenchymal stem cells(MSCs)derived from human umbilical cord blood and bone marrow in vitro and to investigate its basic biological characteristics,phenotype characteristics and multilineage potential.Methods MSCs derived from human umbilical cord blood and bone marrow were isolated and purified by modified ficoll density gradient centrifugation combined with adherence culture method.After expended and digestion-controlled passage,the subculture were expanded and purified.Subculture differentiation were induced into neuronlike cells by cytokine. Morphology and biological characteristics of bone marrow mesenchymal stem cells were observed under an inverted phase contrast microscope. The surface molecule expressions of cells were examined by immunocytochemistry and immunocyte-fluorescence.Results 48-hour culture,BMSCs had begun to adhere. At 7 days,cell were seen adherent 90%the Wall of the culture flask.After 48-hour culture,UMSCs had begun to adhere,not fastness. At 14 days,Cell were seen accumulated colony.At 21 days,cells were gradually showed bostrychoid or whirlpool arranged parallelly. MSCs were induced to differentiate into neuron-like cells.The induced cells were monitored with immunohistochemistry and immunoeyte-fluorescence.Conclusion Human umbilical cord blood and bone marrow have MSCs,and possess the embryonic stem cell dike biological characteristics.The growth rate of UMSCs is slower than BMSCs between 10 days and 15 days.MSCs derived from human umbilical cord blood and bone marrow in vitro may differentiate into neuron-like cells.