骨髓间充质干细胞分化为神经元样细胞后电生理特性的改变

背景：目前体外实验对骨髓间充质干细胞来源的神经元样细胞的研究多集中于形态学层面和神经标志物方面，对分化后的电生理功能研究较少。 目的：观察脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导Wistar大鼠骨髓间充质干细胞分化为神经元样细胞后电生理特性的变化。 设计、时间及地点：细胞学体外培养，对比观察，于2005-06/2007-10在天津市环湖医院细胞室和南开大学生命科学院完成。 材料：6周龄雄性Wistar大鼠3只，体质量160 g左右。 方法：贴壁培养法体外分离纯化间充质干细胞，用脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸联合诱导间充质干细胞向神经元样细胞分化。诱导前和诱导3 d后分别用膜片钳技术检测细胞膜电流。 主要观察指标：流式细胞仪检测间充质干细胞表型；倒置显微镜观察诱导分化前后细胞形态变化；免疫细胞化学鉴定神经元特异性烯醇化酶的表达，以及全细胞电流测定结果。 结果：①流式细胞仪检测结果显示，CD90阳性率(99±3)%，CD31阳性率(3.4±0.8)%，CD34阳性率(0.3±0.1)%。说明这一细胞群大部分处于未分化的干细胞状态，其纯度可达95%。②光镜下可见未经诱导的间充质干细胞多为扁平形带突起的细胞，似纤维样细胞，诱导3 d后出现神经元样细胞。③免疫细胞化学结果显示，诱导前间充质干细胞的神经元特异性烯醇化酶呈弱阳性，诱导后呈强阳性。诱导72 h时分化率为(24.01±3.76)%。④诱导组神经元样细胞外向电流峰值及最大外向电流密度高于对照组(P < 0.05)，但未发现内向钠电流。 结论：脑源性神经营养因子/碱性成纤维细胞生长因子/全反式维甲酸诱导方法可以诱导间充质干细胞向神经元方向分化，虽未发现具有成熟神经元电生理功能，但有向成熟神经元分化的趋势。

Electrophysiological changes of bone marrow mesenchymal stem cells following the differentiation into neuron-like cells

BACKGROUND: Previous in vitro studies mainly focused on morphological and nerve marker aspects in the study of bone marrow mesenchymal stem cells (BMSCs) -derived neuron-like cells, but less focused on the electrophysiological properties of neuron-like cells following differentiation. OBJECTIVE: To study the electrophysiological changes of differentiation from BMSCs into neuron-like cells after induction by brain-derived neurotrophic factor (BDNF)/ basic fibroblast growth factor (bFGF)/ alltrans-retinoic acid (AT-RA). DESIGN, TIME AND SETTING: The cytological comparative in vitro study was performed at the Department of Neural Cell Laboratory, Tianjin Huanhu Hospital and College of Life Science of Nankai University from June 2005 to October 2007. MATERIALS: Totally 3 male Wistar rats (6-week old, weighing about 160 g) were used in this study. METHODS: BMSCs were cultured and purified by their characteristic of plastic adhesion in vitro, and then induced by BDNF, bFGF and AT-RA, and differentiate into neuron-like cells. Whole-cell patch clamp technique was used to detect cell membrane current prior to and 3 days following induction. MAIN OUTCOME MEASURES: MSC phenotype was determined by flow cytometry. Cell morphology was observed under the inverted microscope before and after differentiation. Neuron specific enolase expression was assessed by immunocytochemistry. Whole-cell current results were measured. RESULTS: Flow cytometry results demonstrated that CD90 positive rate (99±3)%, CD31 (3.4±0.8)%, and CD34 (0.3±0.1)%. This indicated that these cells were undifferentiated stem cells, with purity of 95%. Undifferentiated MSCs under the optical microscope were mostly flat cells with processes, similar to fibroblast-like cells. Neuron-like cells appeared 3 days following induction. Immunocytochemistry results showed that MSCs before induction were weakly positive for neuron specific enolase, but strongly positive for neuron specific enolase. At 72 hours, the differentiated rate was (24.01±3.76)%. The peak currents of outward currents in neuron-like cells were significantly higher in the induction group compared with the control group (P < 0.05), but no inward Na+ current was detected. CONCLUSION: (bFGF& BDNF)/AT-RA could induce the differentiation of MSCs into neuron-like cells. These cells showed the tendency to differentiate into mature neurons, though having no electrophysiological properties of mature neurons.