人脐血间充质干细胞的分离培养与向神经元样细胞诱导分化的实验研究

Study on the Isolation Cultured of Human Cord Blood Cells MSCs and Its Induction Differentiation Into Neuro-like Cells

目的 探讨来源于人脐血的间充质干细胞(mesenchyma／stem cells,MSCs)分化成神经元样细胞的可行性,以及在体外分离、纯化和扩增的条件。方法 无菌条件下收集正常足月剖腹产胎儿的脐带血,经肝素抗凝,用淋巴细胞分离液分离脐血的单个核细胞,以偏酸性的MesencultTM作为培养基进行培养和纯化,获得贴壁细胞,取扩增第三代后的MSCs向神经元样细胞诱导分化。用免疫荧光标记方法检测神经元特异性标记物。结果 来源于脐血的单个核细胞种植于特定的培养基中后,可产生贴壁细胞,主要表现为破骨样和间充质样细胞;传3代后,这些细胞可得到纯化、扩增;免疫组化标记显示经诱导后的MSCs表达神经丝蛋白(NF)和神经元特异性烯醇化酶(NSE)。结论 源于脐血的MSCs在体外可以培养、扩增,并向神经元样细胞分化,可作为神经干细胞的来源而用于实验研究和临床。

Objective To investigate the feasibility of using MSCs(mesenchymal stem cells) derived from HUCB(human umbilical cord blood) to regenerate neuron like cells and to explore the optimal condition of isolation, purification and amplification in vitro. Methods HUCB was collected from full term deliveries scheduled for cesarean section, all specimens were obtained sterilelly with preservative-free heparin, the cord blood mononuclear cell were isolated by lymphocyte separation medium, purified and expanded with MesencultTM medium and acidic environment to produce adherent layer, after the passage 3 of the expanded of MSC were induced to differentiate to neuron like cells. Specific marks of neuron were detected by fluorescent Immunohistochemical staining method. Results The HUCB-derived mononuclear cells, when seeded in specific medium, gave rise to adherent cells, which exhibited either an osteoclast-or mesenchymal-like phenotype. After passage 3, these cells were able to be purified and expanded. Immunohistochemistry staining showed that the induced MSCs expressed neurofilament(NF) and neuron-specific enolase(NSE). Conclusion This study shows that MSCs in HUCB can be cultured and expanded in vitro, and could be a source of stem cells which can be differentiated into neural like cells for experimental and clinical application.