不同培养条件对小鼠诱导性多能干细胞分化为神经元样细胞的影响

Inducing effects of different microenvironments on the differentiation of mouse induced pluripotent stem cells into neuron-like cells

目的研究小鼠诱导性多能干细胞(i PSC)在不同培养条件诱导下向神经元样细胞分化的能力。方法将小鼠i PSC悬浮培养形成拟胚体,然后随机分为全反式维甲酸(ATRA)组、脑片共培养组和脑组织匀浆上清组,将其诱导分化成神经元样细胞。倒置显微镜下观察细胞形态变化;免疫荧光染色技术对其进行鉴定分析;Western blot法检测巢蛋白(nestin)、微管相关蛋白2(MAP2)、胶质纤维酸性蛋白(GFAP)的表达。结果小鼠i PSC在不同培养条件下都能够向神经元样细胞分化,这些神经元样细胞可以被神经细胞标志物nestin、MAP2标记;ATRA组的nestin、MAP2、GFAP蛋白相对表达量明显高于脑片共培养组和脑匀浆上清诱导组,但脑片共培养组和脑匀浆上清诱导组间无显著性差异。结论脑片微环境和脑组织匀浆上清均可诱导小鼠i PS细胞向神经元样细胞分化,但效果不及ATRA组。

Objective To investigate the effects of different microenvironments on the differentiation of mouse induced pluripotent stem cells（ i PSCs） into neuron-like cells. Methods Mouse i PSCs were cultured in suspension and became embryoid bodies（ EBs）,and then the EBs were randomly divided into all-trans-retinoic acid（ ATRA） group,brain slice co-culture group,and brain tissue homogenate supernatant group. The above three groups were induced to differentiate into neuron-like cells. Morphological changes were observed under an inverted microscope. Immunofluorescence staining technology was used for cell identification. The expressions of nestin,microtubule-associated protein 2（ MAP2） and glial fibrillary acidic protein（ GFAP） were detected by Western blotting. Results The three different culture conditions could all induce mouse i PSCs to differentiate into neuron-like cells. These neuron-like cells could be marked by neuron markers like nestin and MAP2. The levels of nestin,MAP2 and GFAP proteins in the ATRA group were significantly higher than those in both the brain slice co-culture group and the brain tissue homogenate supernatant group,but there was no significant difference between the brain slice co-culture group and the brain tissue homogenate supernatant group. Conclusion Both brain slice microenvironment and brain tissue homogenate supernatant can induce the differentiation of mouse i PSCs into neuron-like cells,but the effect is inferior to ATRA.