小鼠MSCs分离体外扩增及向神经样细胞诱导分化的研究

Study on Isolation, in Vitro Expansion and Differentiation into Neuron-like Cells of MMSCs

目的:研究小鼠骨髓间充质干细胞(mMSCs)的分离、体外扩增及向神经样细胞分化的潜能。方法:取 8-9周小鼠骨髓悬浮至全培养基中,接种于纤连蛋白覆盖的培养器皿中,纯化并扩增mMSCs;检测第9-10代细 胞的生长状态、细胞周期、表型,并进行光镜形态学观察;用二甲基亚砜和丁羟茴醚诱导细胞分化,免疫荧光法检测 其诱导分化前后神经细胞和神经干细胞的特异性标记蛋白的表达。结果:mMSCs为贴壁细胞,表现为成纤维细胞 样,具有梭形、多角形等形态,传至15代仍可维持其形态特征;83%的细胞处于G0/G1期;mMSCs不表达CD45而 表达CD44、CD13,与人MSCs表型一致;诱导分化后,大部分细胞变成双极、多极和锥形,并相互交织成网状结构, 呈现神经细胞和神经干细胞表型。结论:mMSCs可被成功分离及体外扩增培养,并具有同人类MSCs相同的形 态、表型、生长特征和向神经样细胞分化的潜力,是研究细胞及基因治疗小鼠病理模型的良好工具细胞。

Objective: To explore the isolation, expansion and differentiation of murine bone marrow-derived mesenchymal stem cells (mMSCs) into neuron-like cells in vitro. Methods:Bone marrow was collected from 8- to 9-week-old KM mouse, suspended in the complete culture medium and planted into FN-coated plates. MMSCs were purified and expanded in vitro. After 9-10 passages, the growth status, cell cycle, phenotype and morphology of mMSCs were analyzed and observed by a microscope. The specific marking proteins of neurons, glia and neural stem cells were detected by immunofluorescence before and after induction. Results: MMSCs were adherent cells with a morphology of fibroblastoid presenting spindle-shaped and polygonal-shaped and the morphology was similar in culture of 15 passages. Cell cycle assay demonstrated 83 % of mMSCs were in G0/G1 phase. Phenotype showed that mMSCs were positive for the surface markers CD44, CD13 and negative for CD45, which was identical to human bone marrow-derived MSCs. After the inducement, most mMSCs turned into bipolar, multipolar and taper, then intersected as network structure. Moreover, they had the phenotypes of neurons, glia and neural stem cells. Conclusion: MMSCs sharing the same morphology, phenotype, growth characteristics, and potentiality of differentiation into neuron-like cells as human MSCs can be successfully isolated from bone marrow and expanded in vitro. Therefore, mMSCs will be an perfect tool to study the cellular and genetic therapy in vivo in mouse pathology models.