成年SD鼠骨髓基质干细胞向神经元样细胞分化过程中分化与凋亡关系的研究

The correlation between differentiation and apoptosis in the process of differentiation from adult rat mesenchymal stem cells into neuron-like cells

目的 :探讨体外诱导骨髓基质干细胞向神经元样细胞分化过程中分化和凋亡间的关系 ,为进一步延长神经元样细胞的存活提供理论和实验依据。方法 :采用全骨髓法分离培养成年SD鼠骨髓基质干细胞至第 5代 ,以 β-巯基乙醇 (1mmol/L)及维甲酸 (RA ,1 0 - 6 mol/L)诱导分化 ,于诱导后 1h、3h、5h分别行神经元特异性抗原MAP- 2免疫组化鉴定 ,并计数神经元样细胞数 ,计算分化率。核荧光染料DAPI检测诱导后 5h和 2 4h细胞核的凋亡特异性形态学改变 ,流式细胞仪对细胞凋亡峰的分析检测诱导后 1h、3h、5h细胞凋亡率。结果 :成年鼠骨髓基质干细胞经 β -巯基乙醇 (1mmol/L)及维甲酸 (RA ,1 0 - 6 mol/L)诱导 30min后可见神经元样细胞出现 ,经MAP - 2免疫组化鉴定 ,此类细胞呈阳性 ,诱导后 1h、3h、5h神经元样细胞分化率分别为 41 %、44 %、73 %和 2 4 %、37%、61 %。但诱导后 5h见细胞漂浮并死亡 ,此时DAPI染色显示部分细胞核浓缩 ,出现核碎片。流式细胞仪检测经 β -巯基乙醇诱导后 1h、3h、5h细胞的凋亡率分别为 1 .5 %、6 .1 %、2 1 .5 % ,经RA诱导后的凋亡率分别为 1 .5 %、1 .7%、1 2 .3 %。结论 :①β -巯基乙醇及RA诱导骨髓基质干细胞分化成为神经元样细胞的能力不同。

AIM:To supply the theoretic evidences of elongating the lifetime of neuron-like cells differentiated from adult rat mesenchymal stem cells, we investigated the relationship between the differentiation and apoptosis in the process of induction. METHODS: The mesenchymal stem cells(MSCs) were isolated primarily from rat bone marrow, and purified by passage culture. The 5th passage of MSCs was induced by β-mercaptoethanol and all-trans-retinoic acid (ATRA). After 1 h, 3 h and 5 h of induction, the cells were stained immunocytochemically with anti-MAP-2 and anti-GFAP antibodies, respectively. In addition to counting the ratio of neuron-like cells in MSCs, DAPI staining was employed to identify whether the differentiated cells have an apoptotic morphological changes. The ratio of apoptotic cells at 1 h, 3 h and 5 h after induction were detected by flow cytometry (FCM). RESULTS:Neuron-like cells appeared among MSCs and could be identified by MAP-2 staining 30 min after MSCs being treated by β-mercaptoethanol and ATRA. However, 5 h after induction, some of these neuron-like cells began to float and die. With DAPI staining, the nuclei of some dying cells were shown to be condensed and later fragmented. The ratios of cells undergoing apoptosis at 1 h,3 h and 5 h after induction by β-mercaptoethanol and ATRA were 1.5%?6.1%?21.5% and 1.5%?1.7%?12.3%, respectively . CONCLUSIONS:1. β-mercaptoethanol and ATRA had the different ability that induced MSCs to differentiate to neuron-like cells. 2. Apoptosis was also initiated in the process of differentiation, and there is positive correlation between the ratio of differentiation and apoptosis.