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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。
Neural stem cells were seeded on hyaluronic acid scaffolds for three-dimensional (3D) culture and compared with conventional planar (2D) cultures for inducing differentiation at 1, 7, 14 d in the induction medium. Cell immunohistochemistry and Real-time PCR were used to detect the expression of neural stem cell-specific markers nestin, neuron tubulin and glial fibrillary acidic protein (GFAP) Protein levels and m RNA levels. CCK-8 and viable cell staining were used to detect the proliferative ability of nerve cells and the repair effect of nerve cell membrane damage. The results showed that the expression of tubulin in neural stem cells induced by 3D-2D and 3D-2D medium for 14 d increased obviously, while the expression of GFAP decreased and the 3D effect was more obvious. The results of CCK-8 and viable cell staining showed that the stem cells had a remarkable effect in repairing the damaged and differentiated nerve cell membrane under 3D culture condition compared with 2D culture condition. Three-dimensional culture model can play a better protective effect on the model of drug injury after neural cell differentiation. Therefore, 3D hyaluronic acid - neural cell differentiation model is more advantageous model for the construction of neural drug screening and safety testing in vitro.