三维神经干细胞分化模型在神经药物检测中的应用

Development of A 3D Stem Cell-derived Neuron Model for Neuronal Drug Testing

将神经干细胞接种在透明质酸支架进行三维(3D)培养,使用传统平面(2D)培养做对比,经诱导培养基进行1,7,14 d诱导分化。采用细胞免疫组织化学和Real-time PCR技术检测神经干细胞特异性标记物巢蛋白(nestin)、神经元微管蛋白(tubulin)及胶质细胞胶原纤维酸性蛋白(glial fi brillary acidic protein,GFAP)在蛋白水平和m RNA水平上的变化;CCK-8和活细胞染色技术检测神经细胞的增殖能力及神经细胞膜损伤修复效果。结果显示,神经干细胞在3D和2D培养条件下经诱导培养基诱导14 d后,tubulin表达量明显增加,而GFAP表达量降低,3D效果更加明显。CCK-8和活细胞染色结果显示,干细胞在3D培养条件下较2D培养条件下其分化和分化后的神经细胞膜损伤修复效果显著。三维培养模型能够对神经细胞分化后的药物损伤模型起到更好的保护作用。因此认为,3D透明质酸–神经细胞分化模型是更适合于构建体外神经药物筛选及安全性检测的优势模型。

The neural stem cells were cultured in hyaluronic acid-based three-dimensional （3D） scaffold and induced differentiation for 1, 7 and 14 days, besides the conventional two-dimensional （2D） culture was set as a control. The expressions of sternness marker （nestin）, neuronal marker （tubulin） and glial cell marker （glial fibrillary acidic protein, GFAP） were detected using immunocytochemistry staining and Real-time PCR; The cell survival and proliferation ability during differentiation culture and drug testing were assayed using the CCK-8 Kit and Live- Dead Cell Staining Kit. During culture period, tubulin expression was significantly increased and glial cell marker GFAP expression reduced, whereas the changes observed in 3D model were more obvious. Ceils cultured in the 3D model were shown a significant protective effect on drug-induced nerve damage post differentiation. Neural stem cells cultured in 3D hydrogel retained a high differentiation potential to form neuron cells compared to conventional 2D culture. The 3D culture model could provide an additional protection on drug-induced nerve damage post dif- ferentiation, also the developed 3D stem cell-derived neuronal model could be an advanced in-vitro model for drug toxicity testing and for efficacy and safety assessments in stem cell therapy to treat neurodegenerative diseases.