脐血间充质干细胞诱导向神经元样细胞分化的实验研究

Experimental Research on Differentiation of Umbilical Cord Blood-Derived Mesenchymal Stem Cells Into Neuron-Like Cells

目的:研究脐血间充质干细胞生物学特性及向神经元样细胞分化的潜能。方法:采用密度梯度离心结合贴壁培养法自脐血中分离间充质干细胞,观察细胞生长情况,描绘生长曲线,流式细胞仪检测细胞表面标志物,分别向成骨细胞、脂肪细胞、神经元样细胞进行诱导分化,通过茜素红染色、油红O染色检测脐血间充质干细胞成骨、成脂肪细胞诱导分化能力,而以免疫组织化学检测诱导后细胞表面神经标志物的表达。结果:纯化的脐血间充质干细胞贴壁生长,呈均一梭形,生长曲线呈S型,并以P3代增殖能力最强,细胞表面不表达或弱表达CD34、CD35、CD106,高表达CD29、CD44、CD105。成骨诱导2周后,可检测到钙化基质的形成,成脂肪诱导3周后,可检测到脂滴的形成。向神经元样细胞诱导分化后,可观察到典型的神经元样形态改变,且NSE、NF、GFAP阳性表达。结论:分离纯化的脐血间充质干细胞具有较强的增殖能力与分化潜能,并在体外诱导条件下可以向神经元样细胞定向分化。

Objective： To investigate the biological characteristics of umbilical cord blood-derived mesenchymal stem cells（UCB-MSCs）,and the differentiation potential of UCB-MSCs into neuron-like cells.Methods： By density gradient centrifugation and ad-herent culture,MSCs cells were harvested from human umbilical cord blood.The morphology of MSCs and their growth characteristics were observed.The surface antigen phenotype was detected by flow cytometry.MSCs was respectively induced to differentiate into os-teoblasts,adipocytes and neuron-like cells,subsequently analyzed by alizarin red staining,oil-red O staining and immunohistochemistry.Results： UCB-MSCs were adherent to a fibroblast like morphology.All the growth curves presented ＂S＂ type,and the third generation of MSCs showed the greatest reproductive activity.By flow cytometry,UCB-MSCs were found positive for CD29,CD44 and CD105 ex-pression,but failed to express CD34,CD35 and CD106.After induced to osteogenic differentiation for 2 weeks and adipogenic for 3 weeks,the MSCs were detected the formation of mineral extracellular matrix and neutral lipid vacuoles.When MSCs were induced to neuron-like cells,morphology change was evident and the induced MSCs became positive for NSE,NF and GFAP expression.Conclusion： The obtained UCB-MSCs have great capability of proliferation and differentiation,and can be induced to differentiate to neuron-like cells.